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ImageMaster 2D Platinum6 User Manual

1. F F Figure 4 4 The MatchSets folder At each level the matches are propagated This means that once all match sets have been effectively matched indicated by a red tick mark the spots of gel A_T1_Gell for instance can be directly compared with those of Gel B_T2_Gel3 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace 47 48 A_T1_Gel3 A_T2_Gel3 B_T1_Gel3 B_T2_Gel3 A_T1_Gel2 An _Gel2 aT _Gel2 B_T2_Gel2 A_T1_Gell A_T2_Gell B_T1_Gell B_T2_Gell lj Figure 4 5 Example of a hierarchical match set structure Case 1 Alternatively you could directly put all gels of population A in a match set and create a similar match set for the gels in population B Populations A and B can subsequently be matched together Figure 4 6 Bacteria 2 ofa A DD N B_T1_Gel3 B_T1_Gel2 B_T1_Gell Figure 4 6 Example of a hierarchical match set structure Case 2 A_T1_Gel3 A_T1_Gel2 A_T1_Gell Aa _Gel2 A_T2_Gell It is not possible to recommend an ideal matching scheme Depending on how you carried out your experiment what you would like to show in your study or how similar your gels are you may want to opt for a particular match set structure You may also desire to carry out several analyses using different matching schemes You can copy match sets to use them in another configuration In Figure 4 7 for example the match sets A_T1 A_T2 B
2. 4 6 8 Matei onsu A S 4 6 9 Mat h set pr pertie Ses E E A 47 Working WIth Clas SeSe san 4 7 1 Cr amp ating a Class err a EEEE REEE 4 7 2 Adding g ls t d Clas Snai an 4 7 3 Moving gels or classes 4 7 4 Displaying CIOSSES anra ea EE Ran 4 7 5 Statistical analysis essseeeessssssssscccssssssesccccccsssssssssssssssssssunssssseseeseseeeeeseeesssessessa 4 7 6 Class prop rti S Sn er nn e E 48 Working WITH PED OMS u neennigeessere 4 8 1 Creating a subfolder 4 8 2 Adding repor Senan a E a E 4 8 3 OBERING FEDORS are a a E 4 8 4 Moving reports or folders nn 60 49 Working with documents on eececsssssscssssssssssseneesssssccssssssssssnsessssseeeesssssssssees 60 4 9 1 Creating a subfolder 4 9 2 Adding fil Saros i a E E EE E E 4 9 3 OPENING GOCUMENTS iesistie ar R A a 4 9 4 Moving documents or folders eeessessssssssscssssseccecessssssssssssssssssssssnunnessssseeseee 61 ATO Geland report identifie Snai e UR 61 4 11 Archiving d worksp ce sisiztecsnsicrnsessnis icecdeseerseurswpeosehtsandMearnssteieeecls 62 4 11 1 Backup Restore workspace nnnnnnnsennssssssnsennunnunnnnnennennennennnnenn 62 4 11 2 Backup Restore project 63 5 Reports 5 1 lNtroduetion een 5 11 Reports MENU sasani NE ERAEN 5 1 2 Analyze menu 52 Report toolbars enueia A O ER 5 2 1 Standard tool Sienne n a a 5 2 2 Adding comments yea i iaee E a E ee Gy 5 2 3 Selecting and navigating esanian 5 2 4 Displaying related reports 53 Editi
3. Gek B Spots jo Annotations o Figure 6 9 Grid lines displayed over gels The left gel shows grid lines in Image coordinates Fixed mode the middle gel uses Image coordinates Adaptive mode and the right gel displays a grid in pl MW units Adaptive mode NOTE Displaying grid lines is a helpful way to visualize deformations in aligned gels see below for details about aligning images because the grid lines are warped in the same way as the image ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 91 E 92 ER L N ES ZA T 1 SEITE ve Na a 6 3 6 Aligning gels The Align feature facilitates the visual comparison of images stacked images in particular that demonstrate large variations in protein migration In fact gel alignment essentially warps a gel so that it superimposes better with another one thus allowing easy identification of corresponding spots Figure 6 10 When aligning gels in a MatchSet or Classes worksheet the selected gels are automatically aligned to the Master see Chapter 9 to learn more about master images When aligning gels in a Gels worksheet a reference gel must be specified Gel alignment is purely a visual tool It is not used in and will not improve the matching process Therefore the only reason to align your gels is to ease their visual comparison possibly in combination with the Transparency mode To align a selected gel to a master image or s
4. ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace 61 When you delete rename or move your gels and associated reports outside ImageMaster you will get a message at program startup indicating that the file s can not be found The corresponding gels and reports will also have a red cross on them in the Workspace project To solve this problem ImageMaster lets you search for the files at program startup or with the Search option in the Workspace contextual menus You must indicate the folder in which the files are located If the software still can not find a file when it has been overwritten for instance the only option is to delete it from the workspace and insert it again 4 11 Archiving a workspace It is important to realize that the files included in your workspace can be located anywhere on your hard disk or network They are not necessarily all found in the same directory Gel and report files are usually shared by different users on one or several network folders whereas the workspace files project files and matching data are by default saved in the ImageMaster folder of the user s My Documents directory access to this data should generally be restricted In order to make a backup of the entire experiment or to share the files with a person that cannot access your network you can save your workspace or just a single project with all its respective files in a single location NOTE It is not
5. Studied spot value type Regression line Regression line equation a 0 95 x 1513 P Corr 0 949 Count 300 Correlation coefficient Number of matches Figure 10 1 Scatter graphs for matched spots ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis The linear dependence is defined as the best fit line through the data points The best fit line is described by a slope and its offset from the equation y slope x x offset The goodness of fit for this approximation is given by the correlation coefficient Corr This coefficient can vary between 1 and 1 where an absolute value near 1 indicates a good fit the spot values of one gel can be predicted to some extent by the values of the other gel while a low value indicates that the data could not be approximated by a straight line The types of conclusions that can be drawn from the regression line equations and the correlation coefficients 10xx 0 andCorr 1 indicates that the spot values for all matched spots are the same in the two gels 12xx 0 and Corr 0 95 indicates that almost all spot values are about 20 higher in the reference gel 1 0 x x 0 2 and Corr 0 95 indicates that almost all spot values are shifted by 0 2 with respect to the reference gel In general when the data are highly correlated Corr close to 1 but the best fit line is far from identity 1 0 x x 0 you shoul
6. 7 6 2 Spot color By default unselected spots are displayed in red selected spots in green and overlapped spots in blue You can change these default colors in the Display tab of the Options window accessible by choosing Tools gt Options in the menu Click on the colored box you wish to change and the Color dialog box opens Choose the preferred color from the spectrum and click OK You can also change the color of a selection of spots as you go To change the color of selected spots 1 Select the spots you want to change the color 2 Choose Show gt Spots gt Set Color in the menu and pick one of the three proposed colors Please note that the Default option corresponds to the spot color defined in the ImageMaster Options accessible by choosing Tools gt Options in the menu 3 ImageMaster displays the selected spots with the chosen color 4 The colors remain displayed this way until you choose Show gt Spots gt Set Color gt Reset All to revert back to the default spot colors 7 6 3 Show hide spots You may want to hide some spots temporarily for clarity or illustration purposes or perhaps to concentrate only on those spots you consider important Hiding spots also corresponds to filtering the spots because you cannot select hidden spots and they therefore do not appear in generated reports Hiding spots has the additional effect of displaying images faster ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 1
7. The concepts of match sets and population matching can best be illustrated with an example Imagine a set of samples from bacteria that were cultivated with either substrate A or with substrate B In both growing conditions two treatments have been tested Therefore 4 different populations exist A gel was run for each of the 3 samples in a population giving a total of 12 gel images Different possibilities exist to construct your match sets You could first match the three gels of each population Figure 4 5 thus giving the match sets i e populations A_T1 A_T2 B_T1 B_T2 You could then match the populations A_T1 and A_T2 to give population A and do the same to construct population B Finally the match sets A and B can be matched to give match set Bacteria 1 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Example Workspace 1 projects 3 Bacteria Gels CA MatchSet J Bacteria 1 a A p ATI BY A_T1_Gel3 mel fy A_T1_Gel2 mel PR A_T1_Gell mel of AT2 B A B_T1 M B_T2 S Bacteria 2 Abis FA A_T2_Gel3 mel FA A_T2_Gel2 mel FA A_T2_Gell mel FA A_TI_Gel3 mel FA A_TI_Gel2 mel FR A_T1_Gell mel H a Bhis An Bacteria 3 TI AR B_T1_copy PR B_T1_Gell mel E B_TI_Gel2 mel E B_TI_Gel3 mel A_TI_copy H a T2 Gi Classes Ea Reports Documents z fe
8. 2 Choose View gt Rename Worksheet in the menu ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 25 Getting started 3 Inthe Rename Active Worksheet box input the name you want Click OK 4 The new worksheet name appears in the banner To close a worksheet 1 Select the worksheet If all of its contents are not already selected press Ctri A 2 Choose File gt Close Images in the menu 3 The worksheet is removed from the Display Zone 4 In order to close all worksheets at one time choose File gt Close All in the menu Panes When an image or folder of images is opened in a worksheet it is automatically placed in a pane The pane banner is labeled with the same name that was given to the folder of images in the Workspace Random images are opened in a pane with the generic label Gels The color of the pane banner indicates whether it is selected green or not gray In order to select a pane click on its banner To close a pane 1 Select the pane If all of its contents are not already selected then click on its banner 2 Choose File gt Close Images in the menu 3 The pane is removed from the worksheet A worksheet can contain one or more panes Panes can be laid out so that they do not overlap tiled panes or so they do overlap stacked panes Stacked panes resemble a pile of papers lying one on top of another only the topmost pane is displayed in full You can move a pane to the top of the stack by cl
9. AA Composition file AA P10413 xls 4 2 lt D lt UP AD TO NW 16 L 8 1 F 14141 PN Sheeti Sheet Sheet3 Jal 2 Draw amp Autosnapes N OR 4G o e 2 TI a a ae a Figure 8 3 The annotation on the spot containing protein P10413 includes linked labels such as a link to a Web site top a link to a file bottom right and a text link bottom left 160 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Note that you can set a different Annotation folder for each workspace You can also create subfolders in the Annotation folder to arrange your files The file names indicated in the labels must then contain the name of the subfolder for example AA composition AA P10413 xls For example you can link a protein spot to an Excel file containing the amino acid composition of a protein Figure 8 3 The label should then consist of the string file followed by the file name with its extension Text link In some cases you might want to associate a long text comment with a specific protein spot but without overloading the display The solution is to create a text link rather than a very long annotation label Double clicking on the linked label is sufficient to display a dialog box containing the entire text Figure 8 3 Text links are particularly useful to attach the bibliographic references for a spot for instance or any other comment such as the one in Figure 8 3 P
10. Data analysis 204 Normalization By default the histograms display the raw spot values Nevertheless to simplify comparisons across matches it can be convenient to normalize the spot values relative to their intra class statistics The following normalizations can be chosen from the Displayed Value list Figure 10 5 Value Raw spot value Relative Spot value Central tendency This normalization places the central tendency values to 0 and if the Adaptive Gradations option see below is deactivated you have a good overview of the dispersion and therefore of the homogeneity of the matches This normalization is sensitive to high spot values Ratio Spot value Central tendency This normalization divides all values by the central tendency and thus gives a ratio for all data If you deactivate the Adaptive gradations option see below all histograms have the same scale and thus it becomes easier to detect matches that do not have homogenous values This normalization is more sensitive to low spot values Normalized Spot value Central tendency Dispersion This normalization is a compromise between the two normalizations described above and is sensitive to all values Adapt gradations To highlight spot variations inside a match the histogram gradations are adjusted according to the spot values in each match However in order to display an identical gradation in all histograms you can
11. Heuristic Clustering Plot 7 x Heuristic Clustering Plot Vol Non developed branch Developed branch A A A Ja JA Ja B B B B T tE E FE fRA mE mrm ke i 1 1 2 2 2 1 1 1 2 2 2 1 e e je e e le le fe je e le le e e e e e e e e e l I l l l l l l 2 3 2 3 1 1 2 1 3 1 2 3 m m m m m m m m m m m m e e e e e e e e e e l l l l l l Figure 10 16 Dendrogram with two classification levels representing the classes found and their corresponding gels In the example gels were run on samples from bacteria grown on two different substrates A and B and for which two different treatments T1 and T2 were tested The substrate populations were found by the clustering algorithm But the different treatments were not correctly recognized through heuristic clustering each time a gel from T1 has been clustered with the gels from T2 This could be due to the way the match sets were defined though ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 217 Data analysis 218 In the Inter Class Histograms and Reports the displayed statistical descriptor or overlapping measure needs to be selected from the Displayed value list in the toolbar Center Raw central tendency of the current class Dispersion Raw dispersion value of the current class NOTE The center and dispersion values define the interval that characterizes the protein sample of e
12. 5 Inthe next Import Gels box specify the new names and destination folder for saving the gels in ImageMaster format You can also add an extension to all file names or change an existing one 6 Inthe Gel Properties box specify the Staining the stain that was applied to the gel Click OK ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 7 The Gel Name appears in the Gels folder or subfolder that was selected in step 1 The image file is saved with the mel extension 4 5 3 Importing a new DIGE gel DIGE gels must be placed in the designated DIGE Gels folder This feature is only available in ImageMaster 2D Platinum DIGE To import DIGE gel images 1 Right click on the DIGE Gels folder 2 Choose Import DIGE Gel from the contextual menu 3 Inthe Import Image box specify the Reduction Factor and File Format Click OK 4 Inthe Import Gels box browse the directory where the image files are located select the two or three images belonging to the same DIGE gel and click Open Use the Shift or Ctrl keys to make multiple selections 5 Inthe next Import Gels box specify the new names and destination folder for saving the gels in ImageMaster format You can also add an extension to all file names or change an existing one 6 Inthe Create DIGE box specify a DIGE Gel Name the Dye Chemistry DIGE Min or DIGE Sat and a Comment Click OK By default the common part of the image names is suggested as gel name 7 Inthe DIGE
13. Alt B Select gt Annotations gt In Region Alt C Edit gt Annotations gt Copy Alt D Edit gt Annotations gt Delete by Category Alt E Edit gt Annotations gt Modify Alt F Edit gt Annotations gt Add Label Alt H Show gt Annotations gt Hide All Alt I Show gt Annotations gt Hide Selected Alt J Show gt Annotations gt Visible Categories Alt K Show gt Annotations gt Packed Categories Alt L Edit gt Annotations gt Link with Spot Alt N Select gt Annotations gt Inverse Selection Alt O Show gt Annotations gt For Spots Alt P Edit gt Annotations gt Paste Annotations Alt R Show gt Annotations gt In Region Alt T Show gt Annotations gt Only Selected Alt U Edit gt Annotations gt Unlink from Spot Alt V Edit gt Annotations gt Paste Labels Alt X Edit gt Annotations gt Delete Alt Y Show gt Annotations gt Show All Alt 4 Show gt Annotations gt Set Color gt Purple Alt 5 Show gt Annotations gt Set Color gt Orange Alt 6 Show gt Annotations gt Set Color gt Brown Alt 7 Show gt Annotations gt Set Color gt Default Alt 8 Select gt Annotations gt By Color gt Purple Alt 9 Select gt Annotations gt By Color gt Orange Alt 0 Select gt Annotations gt By Color gt Brown SEE 272 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA B 5 Match shortcuts Shortcut Menu Command Ctrl Shift A Select gt Matches gt
14. Central tendency The central tendency gives the general location of a variable This is commonly calculated by the arithmetic mean also known as the average or center of gravity of a distribution the median the middle value which divides the sample in two equal parts or the midrange middle location between the two sample extremes Mean or midrange values are very sensitive to extreme values outliers and can be seriously contaminated by a single observation On the other hand the median is highly resistant to outliers A compromise between mean or midrange and median is given by the trimmed mean or midrange where a predefined number of outliers are removed from the sample The trimmed measures are more robust than the mean or midrange but are more sensitive than the median All of the above central tendencies can be obtained through the selections and the percentage slider available in the dialog box The slider allows you to remove outliers A 100 value means that all the spot values available in a match will be used to calculate the statistics no outliers are suppressed With a value of 80 for example 10 of the minimum values and 10 of the maximum values are discarded from the sample and the trimmed measure is calculated with the remaining values Therefore ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 199 Data analysis 200 e by just selecting Mean the arithmetic mean is calculated that is the su
15. Information on selected matches Master gel Master_A Master_A A mi cen A_T1_Gel2 A m cea A t2 cen A t2 cez A tT2 cea 4 3428 3780 3799 4811 4787 Figure 9 6 Report on selected matches for six matched gels ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 189 9 Matches 190 9 6 2 Match statistics report Another way to evaluate matching results is to generate a Match Statistics Report Figure 9 7 This report lists the number of matches NbMatches found between each of the selected gels in the match set and the master image as well as the percentage of spots that were matched PercentMatches calculated as follows 2m Ng tp PercentMatches where m is the total number of matches between the gel and the master image Ng is the number of spots in the gel and Nm is the number of spots in the Master Match Statistics Report x la e Report on matching results between the selected gels and the Master gel GelName1 GelName2 NbMatches PercentMatches Master A A_T1_Gelt 576 100 000 MasterA A_T1_Gel2 371 76 4161 Mastera A_T1_Gel3 343 71 5328 master A A_T2_Gelt 435 69 5444 Master A A_T2_Gel2 378 69 0411 Master_ A A T2_Gel3 65 1076 Figure 9 7 Report on the match statistics between selected gels and the master image To display a Match Statistics Report 1 Make sure you opened a match set from the workspace 2 Choose Repo
16. Matches can be manually defined by the user or automatically determined by ImageMaster s powerful gel matching algorithm The match is the basic element for searching and investigating protein expression changes across gels through the use of reports histograms and statistical methods Match sets To be able to initiate the matching process in ImageMaster 2D Platinum 6 0 gels must be part of a Match set A match set includes gels or populations of gels in sub match sets that should be compared and therefore matched together Every match set is represented by a Master image This master image is created by ImageMaster based on the Reference for the match set chosen by the user Classes A class is a set of gels or gel populations that you can compare with other such entities In the Classes folder you state your biological questions For example a class of gels obtained from the infected tissue samples of different patients can be compared to a class of gels representing healthy tissue from other patients A class is the basic element for searching and investigating protein expression changes across gels through the use of intra class reports histograms and scatter plots You can also compare different classes through inter class analyses To compare gels in a class or within different classes the gels must be matched together This means that they must belong to the same match set ImageMaster 2D Platinum User Manual 11 0034 38 Edit
17. Saas ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 57 58 2 Left click on the gels a Hand cursor appears 3 Drag and drop the gels into the destination class 4 The gels are added to the class 4 7 3 Moving gels or classes You can rearrange your gels or classes in the Classes folder to change their position in the list or move them into another class Just drag your gel or class to the desired position It is generally inserted after the item you drop it on Whenever there is a possibility to insert it inside or after a class you will be asked for your choice 4 7 4 Displaying classes To carry out statistical analysis on the gels in your classes they must be opened in a Classes worksheet You can select several classes from the workspace and open them simultaneously Each selected class will be opened in a separate pane carrying the name of the class Note that the gels will be opened as part of the highest level class selected This means that if you select the classes A with subclasses A_T1 and A_T2 and B with subclasses B_T1 and B_T2 the gels will be opened in two panes as part of the classes A and B If you select the four subclasses and open them the gels will be opened in four panes as part of the classes A_T1 A_T2 B_T1and B_T2 To open a class 1 Select one or more classes Use the Shift or Ctrl keys to make multiple selections Right click on one of the classes 2 Choose Open from the contex
18. 0 101097 0 150307 0 779736 ost4i0a 0 306517 Figure 10 11 Factor Analysis Report The number of factors equals the number of gels being analyzed Factor analysis cannot be performed with less than two gels and so at least two factors are always calculated Of course the more gels you use the more reliable the results are likely to be and the more factors will be calculated Since the first factors are generally far more important for characterizing gels and matches than any subsequent ones the factors are ranked in order of importance 210 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Factor Projection Plot Data analysis x ula oe Projection onto Axes 1 2 Vol The chosen statistics are Mean 100 and M S D Factor 2 Te Sib 4679 A_T Gel2 as a920 Shea 4898 4484 aye E 4477 Factor 1 Matches Gels bd Figure 10 12 Factor Projection Plot Note that the options in the lower left corner allow you to display only matches or only gels The blue curve represents a part of the correlation circle its form is linked to the scale of the axes Points corresponding to matches can be selected on the plot in green and highlighted on the gels or any open reports In the current context of gel image analysis it has been noted that the first two axes are usually the most significant ones for finding gels and matches that behave similarly Fig
19. Displays the file path A 3 Display zone contextual menus A 3 1 Worksheet pane or legend e Close Images Closes one or more selected gel images This operation can also be carried out by selecting the applicable images and going to File gt Close Images or press Ctrl W e Close All Closes all worksheets available in the Display Zone This operation can also be carried out by going to File gt Close All e Save Worksheet Saves all the changes made to the gel images displayed in the active worksheet e Pane Show Vectors Hide Vectors Stacked pane Tiled pane e Select All Aligned Inverse Selection A 3 2 Image Gels e File Close Images Close All Save Worksheet e Select All Aligned Inverse Selection e Report Generates a Gel Report Spots e Edit Detect Edit Enabled Copy from Image Delete e Show Shape Show All Hide All In Region Only Selected Hide Selected Show ID Hide All ID Set Color ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Appendix SSS e Select By ID By Color In Region From Report File All Refine by Value Inverse Selection e Report Generates a Spot Report Annotations e Edit Add Label Modify Copy Paste Annotations Paste Labels Duplicate Labels Delete Delete by Category Copy Matched Labels Link with Spot Unlink from Spot e Show Show All Hide All In Region For Spots Only Selected Hide Selected Visible Categories Packed Categories Linke
20. Figure 2 1 Simple scheme for DIGE Three protein extracts are labeled combined on one 2 DE gel and then separated according to the fluorescence spectra ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 9 Two dimensional gel analysis Depending on the amount of sample available different dye to protein ratios can be used Minimal labeling with Cy2 Cy3 and Cy5 is most straightforward to use When only a small amount of sample is available saturation dyes might be the best choice The Cy3 and Cy5 saturation dyes are also mass and charge matched except that they are maleimide fluors that react with thiols found on cysteine residues to form a thioether Keep in mind that if no lysine or cysteine amino acid which are targeted by the minimal and saturation dyes respectively is present in a protein the protein is undetectable 2 2 2 Internal standard This type of methodology allows an internal standard to be included Ideally the internal standard consists of a subset taken from all of the samples within the experiment The internal standard is labeled with one of the cyanine dyes Typically Cy2 for minimal dyes and Cy3 for saturation dyes and run on each gel in the experiment This creates an image that is the average of all experimental samples with all proteins in the experiment represented The presence of the internal standard in every DIGE gel provides an inherent link between samples It is the use of an internal stand
21. History to the Script windows without ever needing any programming knowledge Figure 12 3 Several features in ImageMaster make it possible to modify a script to best suit your immediate requirements io x we S AES amp Actions list from file Differential nalysis2 gt gt Select All Groups Groups Report Set Displayed value Refine Objects Selection from Report By Value H Select Gel Objects from Report gt Set Spots Color 1 gt Select Rows from Report EB Window H E Sort B E Selection First index 1 Last_index 1000000 Refine Objects Selection from Report By Value Select Gel Objects from Report Set Spots Color 2 Select Spots by Color Default Hide Selected Spots Figure 12 3 Script window ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 255 Safety control and automation 256 To create a script 1 4 Carry out the desired sequence of operations on a selected set of images Choose Edit gt History gt Show to display the History window Copy the required actions to a new script by selecting the actions and clicking the New Script icon in the History window toolbar A Script window appears on your screen The Script window contains a toolbar and a list of actions The functionality of each of the icons is as follows 5 Open an existing script Browse the directory where the script file srt is located and select the file name The drop down menu gives you access
22. ImageMaster enables the creation of multiple matches see Section 9 5 for details about adding matches In contrast to a single match where one spot in a master gel is matched with exactly one spot in the other gels a multiple match implies that a single spot in the master gel can be matched to several spots in another gel Figure 9 3 The opposite case several spots in the Master matched with a single spot in another gel is not possible The spots from such multiple matches on a given gel image are considered as a single spot in the subsequent data analysis The calculated quantification values reflect the size intensity and abundance of the combined spots ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Matches 9 185 9 Matches EN imageMaster 2D Platinum B x File View Edit Show Select Analyze Reports Tools Window Help Spot Report 4 x Intra Class Report alal ale a lele eli 2 gt B ul e S lele 5 wl m al a Information on selected Spots Intra Class Report Vol The chosen statistics are Mean 100 and M S D FileName SpotiD Intensity Area Match ID Mean 100 M S D Variation 94 0005 1 94 0005 3944 329 579 3 58313 533 026 0 3815 903 778 0 o 903 778 2 94 0005 3951 261 040 3 15438 370 752 0 7 4 Cal jE Figure 9 3 Multiple matches One spot in the master gel spot 3815 is matched with two spots spots 3944 a
23. and to enter the description content This option is particularly useful when you want to add identical descriptions to a series of gels because you can create many descriptions at a time instead of having to define each one individually Similarly you can delete all gel descriptions of a certain category for the selected gels Select the gels and choose Edit gt Gels gt Delete Description 6 7 3 Gel calibration report If your gels were calibrated you can find related information in the Gel Calibration Report Figure 6 21 which is available from the Reports menu It displays the gel name Gel ID and the full file path of the image the calibration function the name of the step tablet used the name of the person thot did the calibration and the calibration date ula le 3 slel 1 Information on selected Gels FileName TestCalibration1 Calibration Name Creator Created 0 185551 log X 2 1 02243 lag X 0 175989 Kodak step tablet no 2 sebastien 12 11 2003 08e0f172 34f1 TestCalibration2 0 185551 log X 2 1 02243 log X 0 175989 Kodak step tablet no 2 sebastien 122 Figure 6 21 Gel Calibration Report ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 6 8 Saving exporting or printing images 6 8 1 Saving gels In order to be stored in the gel file and thus to be available for future work any changes made to a gel have to be saved before exiting the prog
24. as well as any other images matched to it The principle is rather simple You just have to define pl_MW annotations for a certain number of spots pixels in the gel The calculated pl and MW values for all spots in this gel and any matched gels automatically become available from a Spot Report DIGE Report or the Cursor Information window see below In addition pl and MW grid lines can be displayed over the images see Section 6 3 5 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA To define pl_MW annotations on an image 1 Select the image for which you know the pl and MW values for several protein spots Spots may or may not have already been detected in this image 2 Click on the Annotation tool 3 Double click on a spot pixel for which you know the pl and or MW values 4 Select the pl_MW category in the Create Annotation by Click window 5 Enter the known pl and MW values respectively separated by a space Replacing one of the values with 1 means that no value is set 6 Dothisfor a sufficient number of protein spots that are distributed over the whole image area Obviously the more spots and annotations the better the approximated pl and MW values will be ImageMaster does the following to calculate approximate pl and MW values In the case of pl it looks up the two closest annotations to the left and to the right of the spot for which the pl will be determined and then interpolates between these 2 po
25. gt Inter Class gt Histograms ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis ss 4 Inthe pop up list select the value type Intensity Area Vol to be displayed 5 Choose the desired statistics central tendency and dispersion values see Section 10 2 2 in the subsequent dialog box 6 Inthe toolbar of the displayed Inter Class Histograms window click on the Inter Class Intra Class Histograms icon Reports Inter Class Intra Class Histograms x alal 5 gt s 2 ul Intra Class Histograms Yol The chosen statistics are Mean 100 and M S D Dispersion interval Central tendency Spot values Class separation abcdefghijkl abedefghijkl abedefghijkl 4894 4895 4896 Adaptive gradations KEETE 22724 F Sorted values Figure 10 21 Inter Class Intra Class Histograms As shown in Figure 10 21 the Inter Class Intra Class Histograms display all the individual spot values in each match separated for each class by vertical gray lines The classes are characterized by their central tendency blue horizontal line and dispersion interval bounded by the outer red lines The information icons and functions enclosed in these specific histograms are similar to those described in the Intra Class Histograms Section 10 2 3 and Inter Class Histograms Section 10 3 3 The only point to be emphasized here is that selecting the Sorted Values check box at the bottom
26. gt Show All F3 Show gt Hide All F4 Select gt Select All F5 Select gt Unselect All E N 270 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA B 3 Spot shortcuts Shortcut Menu Command Shift A Select gt Spots gt All Shift B Select gt Spots gt In Region Shift E Edit gt Spots gt Edit Enabled Shift H Show gt Spots gt Hide All Shift I Show gt Spots gt Hide Selected Shift J Show gt Spots gt Show ID Shift K Show gt Spots gt Hide All ID Shift N Select gt Spots gt Inverse Selection Shift R Show gt Spots gt In Region Shift T Show gt Spots gt Only Selected Shift X Edit gt Spots gt Delete Shift Y Show gt Spots gt Show All Shift 1 Show gt Spots gt Shape gt Crossed Shift 2 Show gt Spots gt Shape gt Outlined Shift 3 Show gt Spots gt Shape gt Filled Shift 4 Show gt Spots gt Shape gt Outlined Filled Shift 5 Show gt Spots gt Set Color gt Purple Shift 6 Show gt Spots gt Set Color gt Orange Shift 7 Show gt Spots gt Set Color gt Brown Shift 8 Show gt Spots gt Set Color gt Default ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Appendix B 271 Bd Appendix B 4 Annotation shortcuts Shortcut Menu Command Alt A Select gt Annotations gt All
27. ss moving the cursor over the guides a shaded blue box appears showing where the window will reside if the left mouse button is released If you move the dockable window to a non predefined location it becomes a floating window Moving a docked window may affect the location and size of other docked windows El imageMaster 2D Platinum lolx Eile View Edit Show Select Analyze Reports Tools Window Help alali el plela Project Bacteria 2 8 Example Workspace 2 p b Bacteria 2 8 Gels C DIGE Gels E Synthetic GA MatchSets a Classes Reports in foe n Gels 6 Spots 0 Annotations 0 Figure 3 5 The Gel Report window previously docked at the bottom edge will now be docked at the right edge of the ImageMaster window Auto hide un pinned A visible window in Auto Hide un pinned mode ial automatically collapses when not in use to become a tab on the edge of the ImageMaster window When you click on a docked tab the window slides back into view and is ready for use You can also click on Collapse g to minimize a window in Auto Hide mode Floating A dockable window in Floating mode will always appear on top It can be dragged to any position within the program or even outside the main program window ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 29 Getting started 30 You can switch in and out of floating mode by double clicking on the title bar of a dockable wi
28. to adjust to the local gray levels The effect is a local increase in contrast that is very useful for viewing very faint spots This mode can also be combined with the Gray Saturation palette in the Colors list Pseudo colors You may want to use a particular color palette instead of the standard gray levels in your gel ImageMaster offers color palettes such as Coomassie Blue Silver Stain Hot lron Spectrum etc A palette that is especially useful is the Gray Saturation palette It corresponds to the standard Gray option except for the maximum value that is displayed in red and the minimum value that appears in blue It is helpful when you want to visualize saturated spots red or the background blue Of course you can modify the minimum and maximum gray levels as explained above to decrease the stringency on what is considered as saturation or background Finally you can just inverse the gray levels by checking the Invert box Please note that any color adjustments and the inversion of the gray levels using the Adjust Contrast function are applied to all selected gels and are not saved with the gel images ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA a To display gels with pseudo colors 1 Select one or more gels Draw a region to get a preview of the color adjustment or inverted gray level modifications 2 Choose Show gt Gels gt Adjust Contrast in the menu 3 Inthe Image Display Settings box selec
29. will not be able to discern which one poses a problem e When a spot is missing on one gel sometimes happens to border spots you should not put a landmark in a hypothetical spot position Missing landmarks are not a problem for ImageMaster as the program just aligns the landmarks that have the same name and ignores the rest To align two or more gels 1 Bearing in mind the rules enumerated above define a few annotations containing labels from the Landmark category on the gels The master reference gel for alignment and other gels should contain the same landmarks that is an identical label inside the same spot ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 93 ED 94 2 Select the gels to be aligned include the reference gel when working in a Gels worksheet 3 Choose one of the options in the Show gt Gels gt Align Images menu Global Exact 4 Specify the reference gel if asked for only in a Gels worksheet 5 The selected gels are aligned relative to the master reference gel 6 If some parts of your gel are still not sufficiently aligned you can add extra landmarks to your gels The alignment is automatically updated 7 Torestore to the original images choose Show gt Gels gt Align gt None 8 The original images are restored in place of the aligned ones In the event that gels are not aligned but have common landmarks operations such as simultaneous gel moving gel matching etc are don
30. 1 000 000 for increases in spot volumes and 1 to 1 000 000 for decreases in spot volumes Values between 1 and 1 are not represented hence a two fold increase and decrease is represented by 2 and 2 respectively and not 2 and 0 5 as might have been guessed NOTE In the DIGE Report the Vol Ratio shown corresponds to Volume of the spot on the image mentioned in the FileName Volume of the spot on the other selected image Measure Histogram DIGE Report ay x ulsl Blo alle E a ul FileName SpotID Intensity Area Vol intensity VYol Vol Ratio Slope GelD1 Spike1 Cy3 2537 207 000 6 30000 697 350 0 0157646 0 0109829 2 42060 0 183077 Gel01 Spike1 Cy3 2577 989 000 4 97000 2429 69 0 0753200 0 0382665 2 48552 0 152470 GelD1 Spike1 Cy3 2583 2207 00 7 47000 5920 05 0 168080 0 0932380 2 11761 0 305566 GelD1 Spike1 Cy3 2592 4416 00 10 2400 17275 0 0 336312 0 272074 2 50321 0 338455 GelD1 Spike1 Cy3 2599 9229 00 12 7600 33661 8 0 702859 0 530157 2 23677 0 277866 Gel01 Spike1 Cy3 2616 223 000 7 34000 340 950 0 0169832 0 00536980 2 33457 0 240933 s olos je lo wm Gel01 Spike1 Cy3 2617 479 000 6 46000 600 740 0 0364795 0 00946138 2 42058 0 352705 Figure 7 13 DIGE Report Depending on how you generate the DIGE Report all the detected spots or only selected spots appear in the repor
31. 12 DIGE Histogram The DIGE Histogram is interactive You can click on the data points representing spots and select them on the gels and any other open reports Just select the spots by clicking on the corresponding points while using the Ctrl or Shift keys for multiple selections To select all spots in a region hold down the mouse button ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA SSS and then drag the cursor to define an area Use the options from the Select on Gels drop down menu in the toolbar to identify the corresponding spots on the gels and open reports The pull down menu next to the Reports icon in the toolbar can be used to display information related to the DIGE Histogram e The Gel Report option displays a Gel Report for the two selected gels e The DIGE Report item show the numerical values corresponding to the spots in the DIGE histogram see below There are two ways of displaying a DIGE Histogram Depending on which one you use all detected spots or only selected spots are included To display a DIGE histogram containing all spots 1 Select two co detected images in the DIGE Gels folder of the workspace Right click on one of the images 2 Choose Histogram from the contextual menu 3 ADIGE Histogram containing all spots from the selected gels is displayed To display a DIGE histogram containing selected spots 1 Select two co detected images in a Gels or Classes worksheet 2
32. 4079 4906 7 32000 1315 50 3458 17 0 0915651 0 0773186 167 652 D 66 A_T1_Gel1 4905 4905 13 2100 1765 50 8074 32 0 0733336 0 104583 233 900 67 A_TI_Geli 4904 4904 15 7500 3066 00 16530 2 0 127353 0 214109 356 296 D A_T1_Gel2 3722 4904 12 1000 2241 00 11596 7 0 152178 0 227187 307 995 20 2900 3097 50 22156 3 0 128661 0 286982 400 979 146 Figure 7 10 Report on selected spots in selected images Measure histogram By clicking on a numerical column header in the Spot Report and then pressing the Measure Histogram icon you can display a histogram showing the distribution ofthe values in the selected column Figure 7 11 The purpose of such histogram is to graphically summarize the distribution of a univariate data set It shows the following e Center i e the location of the data e Spread li e the scale of the data e Skewness of the data e Presence of outliers and e Presence of multiple modes in the data The Measure icon at the top of the Measure Histogram window allows you to choose between one of two options ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA ss e The Histogram is obtained by splitting the range of the data into equal sized bins intervals Then for each bin the number of points from the data set that fall into each bin are counted The horizontal axis represents the response variabl
33. 64 In the Restore Project box choose one of the two options for restoring a project By restoring the files using the original file paths you return to your project by automatically rewriting the previous file and folder structure By restoring the files in a single folder the project is reproduced in a new directory for which you have to enter a file name and location Click Restore The project is restored in your workspace ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 5 Reports 5 1 Introduction The reports available in ImageMaster not only provide options for displaying information on selected objects but also are useful for sifting through data Reports can be accessed from the Reports menu the Analyze menu or sometimes through displayed reports Reports are divided into two different menus for the stated reasons e Reports menu DISPLAYS only quantitative or qualitative information on selected objects gels spots labels annotations categories and matches e Analyze menu COMPUTES differences and similarities between gels allowing data analysis based on robust statistics factor analysis statistical tests and clustering techniques reports as well NOTE Reports are not necessarily in table format Graphical representations of data such as histograms scatter plots and 3D views are regarded as You can display as many reports simultaneously as you wish Reports are Dockable W
34. All Ctrl Shift G Edit gt Matches gt Add Match Ctrl Shift H Show gt Matches gt Hide Vectors Ctrl Shift J Show gt Matches gt Show ID Ctrl Shift K Show gt Matches gt Hide All ID Ctrl Shift M Select gt Matches gt For Spots Ctrl Shift N Select gt Matches gt Inverse Selection Ctrl Shift U Edit gt Matches gt Delete Match Ctrl Shift Y Show gt Matches gt Show Vectors ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Appendix 3 273 Bd Appendix 274 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA C Appendix C 1 Software references Appel RD Hochstrasser DF Roch C Funk M Muller AF and Pellegrini C 1988 Automatic classification of two dimensional gel electrophoresis pictures by heuristic clustering analysis A step toward machine learning Electrophoresis 9 136 142 Appel RD Hochstrasser DF Funk M Vargas JR Pellegrini C Muller AF and Scherrer J R 1991 The MELANIE project from a biopsy to automatic protein map interpretation by computer Electrophoresis 12 722 735 Appel RD Palagi PM Walther D Vargas JR Sanchez J C Ravier F Pasquali C and Hochstrasser DF 1997 Melanie II a third generation software package for analysis of two dimensional electrophoresis images I Features and user interface Electrophoresis 18 2724 2734 Appel RD Vargas JR Palagi PM Walther D and Hochstrasser DF 1997 Melanie II a third generation software package for ana
35. CW 1978 Introduction to factor analysis What is and how to do it Newbury Park Sage Publications Tabachnick B and Fidell LS 1996 Using multivariate statistics 3 edition New York Harper Collins College Publishers C 3 Further reading Appel RD Bairoch A Sanchez J C Vargas JR Golaz O Pasquali C and Hochstrasser DH 1996 Federated 2 DE database a simple means of publishing 2 DE data Electrophoresis 17 540 546 Appel RD Sanchez J C Bairoch A Golaz O Ravier F Pasquali C Hughes GJ and Hochstrasser DF 1996 The SWISS 2DPAGE database of two dimensional polyacrylamide gel electrophoresis Nucleic Acids Research 22 3581 3582 Binz PA Mueller M Walther D Bienvenut WV Gras R Hoogland C Bouchet G Gasteiger E Fabbretti R Gay S Palagi P Wilkins MR Rouge V Tonella L Paesano S Rossellat G Karmime A Bairoch A Sanchez JC Appel RD and Hochstrasser DF 1999 A molecular scanner to automate proteomic research and to display proteome images Analytical Chemistry 71 4981 4988 Binz PA Wilkins MR Gasteiger E Bairoch A Appel RD and Hochstrasser DF 1999 Internet resources for protein identification and characterization In Kellner R Lottspeich F Meyer HE eds Microcharacterization of Proteins 24 ed pp 277 300 Weinheim Wiley VCH ExPASy Molecular Biology Server 2003 Online http www expasu org Hoogland C Baujard V Sanchez J C Hochstrasser DF and Appel RD 1997 Make2ddb a simple package to
36. Calibration Tablet File At the left of the Create Calibration window you see the theoretical optical density OD values of the different steps in the tablet the values you entered in the Calibration Tablet File ImageMaster may automatically deselect some of the steps grayed out because of their unreliable values and you can deselect additional ones if you estimate that they should be excluded from the calibration process At the right of the Create Calibration window you see the calibration curve between the logarithmic transmittance values on the X axis and the OD intensities on the Y axis Note that the measured intensity values for each step are calculated as median intensities over all the pixels in the small rectangle area for each step on the step overlay The horizontal dispersion intervals in blue or gray for deselected spots represent the intensity ranges when 10 of the less intense and 10 of the most intense pixel values are removed The calibration formula and error are given below the graph You can additionally display some reports to judge the quality of your scanner calibration see below for more details Once you are satisfied with the calibration close the Create Calibration window The software asks whether you want to apply this new calibration If you answer Yes ImageMaster applies the calibration to the image ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA EX imageMaster 2D Platinum lol
37. Data Type F Is Unique External Engine u Exe Display properties Color I caresi Figure 8 2 Create Category dialog box Data type By default the labels in ImageMaster can contain any character However to ensure coherent annotation data the label contents can be constrained to only numerical values or Boolean entities This is done by fixing the Data Type to one of the following e Text Can contain any character e Number Can only contain numerical values e Boolean Can only take the values 0 and 1 Notice that categories using the Boolean Data Type are displayed in the form of check boxes in most reports except for the Label and Category Reports Editing these labels corresponds to checking the box to indicate whether the item should take the value 0 empty check box or 1 checked box Label categories of the Boolean Data Type are useful to indicate that you checked your data for specific properties and that based on these properties you decide to classify your data points into two populations Imagine that you found 50 interesting proteins significant expression variations from a preliminary differential analysis You can add a label of the Boolean type to each of these 50 protein spots and designate whether yes value 1 or no value 0 you want to export them to a spot excision robot Thus you can easily select spots to be picked but you also have a record of which protein spots were carefully scrutinized Image
38. Gel Properties box specify the Staining the dyes that were applied to each gel Click OK 8 Inthe DIGE Reference box specify the Standard Gel typically Cy2 if using minimal dyes or Cy3 if using saturation dyes Click OK 9 The DIGE Gel Name appears in the DIGE Gels folder The DIGE Gel Image Names are found below The gel image files are saved with the mel extension 4 5 4 Adding an existing gel You can add images that are already in ImageMaster 2D Platinum Melanie format version 4 or higher to the Gels folder of a project The Gels folder can only enclose one copy of each gel file To add an existing image 1 Right click on the Gels folder or a subfolder Workspace ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 43 44 2 Choose Add Gels from the contextual menu 3 Inthe Add Gels box browse the directory where the image file is located select its name and click Open Use the Shift or Ctrl keys to make multiple selections 4 Inthe Gel Properties box verify the Gel Name and Staining the stain that was applied to the gel Click OK 5 The Gel Name appears in the Gels folder or subfolder 4 5 5 Adding an existing DIGE gel You can add DIGE gels that are already in ImageMaster 2D Platinum format to the DIGE Gels folder of a project This feature is only available in ImageMaster 2D Platinum DIGE To add an existing DIGE gel 1 Right click on the DIGE Gels folder 2 Choose Add DIGE Gel from
39. Inter Class Histograms 1 Select the matches to be studied on the gels or on a report 2 Select the gels to be included in the analysis 3 Choose Analyze gt Inter Class gt Histograms 4 Inthe pop up list select the value type Intensity Area Vol to be displayed 5 Choose the desired statistics central tendency and dispersion values see Section 10 2 2 in the subsequent dialog box By default the displayed histograms Figure 10 18 show the raw central tendency blue horizontal line and the dispersion interval red vertical line for each class Visualizing the various overlapping measures is possible by changing the Displayed value in the list at the top of the Inter Class Histograms window The Inter Class Histograms can be selected as with any other ImageMaster object and saved printed or exported to other software using the icons in the toolbar Figure 10 18 You can also re select the corresponding matches on the gels by using the Select on Gels drop down menu and systematically navigate through matches with the Select Next and Select Previous icons Finally you can display more complete Inter Class Intra Class Histograms see below for 220 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis E selected histograms These mixed histograms display the individual spot values in addition to the statistical descriptors for each class Inter Class Intra Class Histograms Reports Displayed v
40. Project Name in the Navigator 2 Choose Properties from the contextual menu 3 In the Project Properties box add or modify the Project Name or Comment Click OK 4 The changes to Properties are saved ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace EEE 4 5 Working with gels The Gels 4th folder manages your gel images Figure 4 3 You can easily e Create folders and subfolders in the default structure e Import gel images with tif png gel img or scan extensions e Add ImageMaster 2D Platinum Melanie format gels version 4 or higher with a mel extension e Import and define DIGE gels e Open images in the Display Zone in the active worksheet or a new one e See which images have already spots detected on them Example Workspace 1 projects 2 8 Bacteria 2 0 Gels 5 DIGE Gels 2a GelD1 FA Gell1 Spike1 Oy3 mel FA Gell Spike2 Cy5 mel PR Gelll Standard Cy2 mel o Gell2 o Gel03 a Bacteria OA EB A_T1_Gell mel EB A_T1_Gel2 mel EB A_T1_Gel3 mel FA A_T2_Gell mel EB A_T2_Gel2 mel FA A_T2_Gel3 mel HOB H A MatchSet a Classes H E Reports 3 Documents Figure 4 3 The Gels folder 4 5 1 Creating a subfolder You can create folders and subfolders in the Gels folder in order to customize the hierarchical structure and to organize your images EN ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 41 42 DIGE gels must be p
41. Select spots Use the Shift or Ctrl keys to make multiple selections 3 Choose Analyze gt DIGE gt Histogram in the menu 4 ADIGE Histogram containing only the selected spots is displayed NOTE In the DIGE Histogram the Volume Ratio used to calculate the log Volume Ratio on the X axis corresponds to Volume of the spot on the first selected image Volume of the spot on the second selected image To obtain the inversed spot ratios change the order of the gels in the worksheet Measure parameter e Max Slope Largest gradient associated with co detected spots e Area Number of pixels within the spot boundary e Max Intensity Largest pixel value associated with co detected spots e Max Volume Volume of the largest of the co detected spots A ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 149 7 10 3 DIGE report A DIGE report is a special case of a spot report see Section 7 10 1 enabling you to display NORMALIZED Vol Ratios see the note below for specific combinations of DIGE images not only ratios compared to the DIGE reference image Two images must be selected to generate a DIGE report For each Spot ID in the report generally two lines will show one for each gel Figure 7 13 NOTE The reported Vol Ratios in the DIGE report are NORMALIZED so that the modal peak of volume ratios is zero since the majority of proteins are not up or down regulated This means that the Vol Ratio is expressed in the range of 1 to
42. Shift or Ctrl keys to make multiple selections 4 The files are added to the Documents folder or subfolder 4 9 3 Opening documents You can open a file from the Documents folder by double clicking on it or by selecting Open from the contextual menu The file will be opened in the associated application 4 9 4 Moving documents or folders Move folders subfolders and files within the Documents folder by dragging and dropping or copying and pasting 4 10 Gel and report identifiers ImageMaster allocates a unique identifier ID to each gel and report in order to assure data consistency allow reliable identification of gels and reports across a computer network and enable database integration The gel and report IDs in ImageMaster are UUIDs Universal Unique Dentifiers which are 128 bit numbers that are guaranteed to be unique through combinations of hardware addresses time stamps and random seeds These IDs allow your gels and associated reports to be uniquely recognized This is because the gel or report ID always overrides the file name or file location Thus ImageMaster detects for example if you delete a gel and replace it with another one with the same name NOTE This also means that duplicating a gel outside ImageMaster may lead to confusion as you will end up with two gels bearing the same ID For that reason you should always try to manipulate delete from disk save as rename your gels from within ImageMaster
43. Spot ID the name of the image on which they were detected the coordinates of their center of gravity X and Y the quantification values Intensity Area Vol lntensity and Vol pl MW and all linked labels Depending on the spot detection algorithm used the Saliency Vol Ratio and Slope will be available When working in a MatchSets or Classes worksheet you can also display information on the corresponding spot in the master gel Figure 7 10 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 145 Spot Report NOTE When displaying a Spot Report for DIGE images the Vol Ratio given corresponds to Volume of the spot on the image mentioned in the FileName Volume of the spot on the DIGE reference image The Spot Report can be saved printed exported and used to sort through data Please see Chapter 5 for more details on these functionalities available as icons in a report toolbar and the possibility of customizing the Spot Report Measure Histogram alg mo a lele als m B wl Information on selected Spots FileName Area Vol M SpotilD a Intensity lntensity vol Saliency M Set Verified 63 AT1_Gell 4906 4906 7 568000 2010 00 5586 40 0 0834894 0 0723583 329 152 C A_T1_Gel2 4906 8 19000 1525 50 4723 67 0 103591 0 0925399 227 337 A_T1_Gelt 65 A _T1_Gel3
44. User Manual 11 0034 38 Edition AA Index Numerics 2 DE gel ele trophoresis zunanessessnse nannte 7 3D view display axes synchronize all A Ac category accession number action descriptors activate ImageMaster adapt gradations inter class histograms intra class histograms adjust contrast analyze menu annotation add add labels combine sets copy create create label categories delete display flag position flagpole color hide import link with spot modify pack categories report select select by category select by content show tool auto hide automatic spot detection B backup workspace batch files conversion Bruker Proteineer SP spot picker o eeeecssssssssssssssssssssessssssssssssssssssesesceeseceeseeseeseesstmtstunssnsnsssssssssssseseeeeeeeseee 241 C calculated calibration ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Index 279 Index 280 control export graph open print graph report save strip category create create sets data type external engine is unique rename report central tendency class add gels in workspace create in workspace heuristic clustering open properties comment category contextual menus gel image legend pane banner worksheet banner contrast MOPPING usage a t
45. Vol to be displayed 5 Choose one or more of the statistical tests for which you would like to see the results displayed 230 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis ss Reports Histograms Statistical Tests xj S e m gt u Statistical Tests Vol Match ID Student T Wilcoxon Kolmogorov 1 4545 26 0334 0 1 00000 2 4580 23 0761 0 1 00000 3 4371 21 6115 0 1 00000 4 4617 19 8816 0 1 00000 5 4533 18 9957 0 1 00000 6 4509 16 5454 0 1 00000 7 4603 15 7084 0 1 00000 Figure 10 26 Statistical tests report The icons in the Statistical Tests report toolbar allow you to save print or export your report and navigate through the data on the gels The Histograms icon is used to produce Inter Class Histograms Related reports can be selected from the Reports drop down menu e The Gel Report item can be used as a legend for the classes and their gels e The Report from Selection function creates a new report that only contains the lines that are selected in the active report NOTE In order to focus your analysis on the most significant spot differences between classes you can sort the values in the report by clicking on the column headers This way you can re select the match that meet your own criteria of statistical value NOTE The given statistical values are useless when the samples classes do not cons
46. a factor analysis The following explanations about how to interpret a factor analysis are illustrated with an example of six gels run to compare the effect of two treatments T1 A_T1_Gel1 A_T1_Gel2 A_T1_Gel3 and T2 A_T2_Gel1 A_T2_Gel2 A_T2_Gel3 on bacteria cultivated on substrate A The gels were detected matched and added to a class The class was opened in a Classes worksheet all the gels and matches were selected and an Intra Class Report was displayed The Factor Analysis Report Figure 10 11 was obtained by clicking on the Factor Analysis icon in the Intra Class Report toolbar It summarizes the variance accounted for by successive axes or factors expressed as a percent of the total variance Thus factor 1 accounts for 83 7 of the variance factor 2 for 7 5 and so on The report also lists the coordinates for each gel projected on these axes Factor Analysis Report p x BIS o mlel Factor Analysis Report Vol The chosen statistics are Mean 100 and M S D variance A_T1_Gel1 A_T1_Gel2 A T1_Gel3 A_T2_Gel1 A_T2_Gel2 A_T2_Gel3 89 7890 0 303765 0409168 0415745 0418760 0414466 0 469385 6 95377 0 281310 0453094 0531991 0415717 0 302973 0409809 1 05348 0 597602 0 172084 0 182881 0 151681 0528276 0 527011 1 01026 0 629445 0 487757 0 0302305 0 0284789 0434899 0418366 ase7se7 0272622 0591234 o 697990 0 141463 0 0624916 0 254974 0 325602 0 0306434
47. and annotations en 175 8 7 Annotations and labels in reports an 176 8 7 1 Label repitan nieu AREA AR 176 8 7 2 Annotation report a 8 7 3 GOLCGOFY reports aE aT span 177 8 7 4 Intra Class Repor sirungna kaan lhnsnuishe 177 8 7 5 Inter Class Report 178 9 Matches 9 1 lAtredueti n nesnenin a a A a aa 179 9 2 Matching 9 2 1 AutomaticMatching siressa 181 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA vii Contents viii 10 11 9 2 2 9 3 9 3 1 9 3 2 9 4 9 4 1 9 4 2 9 5 9 5 1 9 6 9 6 1 9 6 2 9 6 3 9 7 9 7 1 9 7 2 Specifying starting matches 182 Seleeting matches essen seen EnA 183 Menl optionsan EEE lesen 183 REPOS nn nenne aan O ARGE EN 186 Displaying MAtEh eS assessorn n ARAR 186 Show hide matches 187 Show hide match IDs Adding deleting matches 188 Adding deleting matches 188 Reports OM MACES uud 189 Match report 189 Match statistics report 190 Intra class report 190 S nthetic9elsa unannesensasen sense 191 Sp t filteriA g screaam s 192 Creating syntheticgelsanunanaanninnaewai ai 193 Data analysis 10 1 10 2 10 2 1 10 2 2 10 2 3 10 2 4 10 2 5 10 2 6 10 3 10 3 1 10 3 2 10 3 3 10 3 4 10 3 5 IHtroduetion ae san naneral near Intra class statistics SCO 0 0 Pe Descriptive statistics een 198 Intra Class Histograms Intra Class Repott Factor analysis 209 Heurist
48. any analysis meaningful it is important to start with good quality image files The following paragraphs give some helpful tips on what image format resolution and depth should be used to obtain the best results 6 1 1 Image format Input formats Your gels must first be digitized by an imaging device such as a flatbed document scanner camera system densitometer phosphor imager or fluorescence scanner and then saved in an appropriate file format The recommended formats are TIFF Tag Image File Format PNG Portable Network Graphics GEL Molecular Dynamics IMG Fuji and SCAN Bio Rad It is important to remember that ImageMaster uses the calibration information contained in these image files ImageMaster format After opening your gels for the first time in ImageMaster they are saved in the ImageMaster 2D Platinum file format unless otherwise stated ImageMaster gel files use the extension mel 6 1 2 Image resolution The scanning resolution of the gel is critical as it influences the amount of visible detail in the image A low resolution corresponds to a large pixel size or a small number of pixels or dots per inch When the image resolution is too low individual spots cannot be distinguished On the other hand when the scan resolution is too high the image file becomes very large and this slows down the gel analysis significantly A resolution between 150 and 300 dpi is generally sufficient for gel analysis Pleas
49. as to what causes the difference ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis 229 Data analysis Empirical Distribution Plot Class X 0 Class Y Fraction of data points to the left of Spot value z 0 02 0 04 0 06 0 08 0 1 0 12 0 14 Spot value z Figure 10 25 The empirical distribution plot for the spot values of match 613 Figure 10 24 in Classes X and Y The Kolmogorov Smirnov statistic D corresponds to the maximum distance between the two empirical distribution functions Statistical tests report The Statistical Tests report Figure 10 26 contains for each selected match the desired statistical values which quantify the differences between the means of two classes These values should be considered as qualitative indications of the variations in protein expression between two populations and only rarely in ideal cases should be used to calculate probabilities and draw quantitative conclusions In addition one should always check the results by visual inspection of the spots since the conclusions may be erroneous due to inaccuracies in the detection or matching steps To obtain a report of statistical test results 1 Select the matches to be studied on the gels or on a report 2 Select the gels to be included in the analysis 3 Choose Analyze gt Inter Class gt Statistical Tests 4 Inthe pop up list select the value type Intensity Area
50. between the mean values X of the two classes normalized by the standard deviation s For this test the number of degrees of freedom equals the total sample size n4 nz minus 2 The t ratio is calculated as follows _ X Xz ny nz 2 eee 2 2 ast adersi nas ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA If the t ratio is larger than a certain threshold chosen according to a confidence level and the sample size you can conclude that the difference between the two populations is statistically significant NOTE Only the t ratio is given and not the probability p of not being wrong when concluding that the two populations have significantly different means This is for reasons of consistency and to make the user more aware of the implications of his or her analysis In numerous cases the restrictive assumptions are not met and the statistics will not be valid Nevertheless this in no way restricts the utility of the t test results notably for classifying the spot differences by their potential significance You should always check your hypotheses by visual inspection of the spots since results may be due to artifacts in the spot detection and or matching steps If the data for the samples to be analyzed come from populations whose distribution violate the assumption of normality then nonparametric tests like the Mann Whitney or Kolmogorov Smirnov tests can provide a better analysis Mann Whitney
51. carry out a second automatic matching cycle while keeping the existing matches Input matches When you manually define starting matches see Section 9 5 1 the matching algorithm uses these matches as matching seeds These seeds are not permanent however No difference is made between user defined matches and those found by the matching algorithm If you restart the matching procedure without taking into account the previously found matches the manually input matches are also not considered ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Matches 9 Landmark annotations If two or more gels have annotations with identical labels for example landmark annotations you can use the landmarks to initiate the matching process on the condition that the annotations are linked to spots The matching algorithm superimposes those annotations that bear the same labels in the master gel and the matched gel and uses them as matching seeds Please see Chapter 8 to find out how to create annotations The following rules should be considered when defining landmark annotations e Keep the number of landmarks to a minimum e Only define landmarks on clearly corresponding spots Protein variants should definitely not be used as landmarks e Landmarks should be placed on small sharp spots rather than on large diffuse ones to reduce the error in the position When the annotations are of the landmark category ImageMaster au
52. copy to other gels 2 Choose Edit gt Annotations gt Copy in the menu The selected annotations are copied into memory 3 Select the gels into which you want to copy the annotations 4 Choose Edit gt Annotations gt Paste Annotations in the menu 5 The copied annotations are pasted to the corresponding locations in the selected gels These locations are estimated by interpolation between the two nearest common landmarks landmarks with the same name When no such landmarks exist the annotations are placed at the same image location X and Y coordinates 6 Adjust the annotation positions using the Annotation tool Copy paste labels Instead of copying entire annotations you can also copy distinct labels from one gel to selected spots or annotations in a series of gels To copy labels from one gel to a set of other gels 1 Select the labels you would like to copy 2 Choose Edit gt Annotations gt Copy in the menu The selected labels are copied into memory 3 Select the spots or existing annotations into which you want to copy the labels 4 Choose Edit gt Annotations gt Paste Labels in the menu 5 The copied labels are pasted to the selected spots and annotations Copy matched labels When gels have been matched labels existing in one gel can be copied to their corresponding spots in other gels This is particularly useful when you have annotated your master gel for instance by adding and modifying annotations to
53. creation of new annotations on this master gel Exhaustive information about annotations categories and labels is given in Chapter 8 However at this point it is useful to know that you can create different label categories which often correspond to columns in your report and that you can apply special constraints to the label content in each category These include the Data Type Text Number or Boolean and whether each label in the category should be Unique las is the case for landmarks or not ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA The Annotate icon allows you to insert a category as an extra A column by selecting its name from the category list You can create a new category by typing its name in the upper field of the displayed window In the latter case you are asked for the category constraints Once this is done you can directly add new labels to the appropriate cells or edit existing ones Just double click in a cell to start typing your label When finished a single click in any cell quits the editing mode The cell corresponding to the modified label appears in dark green when the corresponding line is selected or in gray when it is not selected You can quickly see that modifications were made to your report when an asterisk follows the window title a Subsequently the Update Gel icon should be used to make the modifications definitive and to propagate the information to your gels wh
54. customizable This means that you can choose the data columns to build your personalized report and the order in which they should be displayed This is particularly useful when you only want the essential information to appear for clarity or for printing purposes To customize your report 1 Click on the Settings icon in the report toolbar 2 Inthe displayed window select the attributes that you would like to add to the report from the left column Gels or Master gel list in the case of Figure 5 3 and transfer them to the right column called Visible attributes list in the case of Figure 5 3 by pressing the right pointing arrow button 3 Tohidecolumns in the report select their names in the right column and transfer the chosen items to the hidden list s at the left by pressing the left pointing arrow button 4 Click OK to confirm your choice and view the customized report Please note that the order of the items in the list of visible attributes determines the order of the columns in your report although the column order can later be changed as explained below Thus you can manipulate the column order by adding your attributes in a specific sequence to the list of visible attributes Your adapted settings can be saved and used as report templates For this purpose the following options are available from the Settings window e By clicking Save and typing a file name you can save the new report template e By cl
55. displayed in a separate pane To open a gel or gel folder 1 Select one or more gels or folders Use the Shift or Ctrl keys to make multiple selections Right click on one of the gels or folders 2 Choose Open gt In Worksheet from the contextual menu to open the gels in the active worksheet or Open gt In New Worksheet to open them in a new worksheet 3 The selected images are opened in the active worksheet or a new worksheet in the Display Zone 4 5 8 Spot detection Once gels are added to the workspace and opened in the Display Zone you are ready to detect spots on them See Chapter 7 to learn more about working with spots and carrying out spot detection Note that the gel image icons change when spots have been detected 4 5 9 Folder properties The Name and Comment are properties of subfolders of the Gels folder The Comment describes and or explains the content of the folder It serves as a useful source of information to which a coworker is referred or as a reminder when reviewing old work You can add or modify the Comment or Name while viewing the Properties of a folder Note that you cannot modify the name of a subfolder while its gels are opened To edit the Comment or Name of a subfolder 1 Right click on a subfolder of the Gels folder 2 Choose Properties from the contextual menu 3 Inthe Gel Folder Properties box add or modify the Name or Comment Click OK 4 The changes to Properties are saved 4 5 10 G
56. exiting The open workspace will be saved automatically 3 4 ImageMaster window By reading through the following pages you will become acquainted with the graphical user interface It is divided into four main parts shown in Figure 3 3 which are the Menu Bar the Toolbar the Display Zone and Dockable Windows Each of them will be discussed in more detail below E imageMaster 2D Platinum File View Edit Show Select Analyze Reports Tools Window Help a Measure Max Intensity Gels Gell1 Spikel Cy3 Gel01 alal lol a lsle Bl Gels used in DIGE Histogram d FileName FilePath Id 11a GelO1 Spike1 Cy3 H DATA NMS TESTS Spike ne 9619ab657 91a6 4627 9a1f 2 b GelD1 Spike2 Cy5 N DATA NME TESTS Spike ne che48e1a fc93 478f Be7e dr Figure 3 3 The ImageMaster window a Menu Bar b Toolbar c Display Zone and d Dockable Windows 22 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Getting started fe 3 4 1 Menu bar If gels spots annotations or matches are selected then you can choose actions from the menu bar to be performed on them This is done by choosing the appropriate menu and moving the cursor to the desired option while holding down the left mouse button Certain menu options have sub options You can choose one of these by moving the mouse over the small arrow on the
57. get identical values for corresponding spots that are in fact widely divergent This type of normalization should essentially be restricted to gels that belong to the same population or that represent the same sample For gels with very different protein patterns that present significant disparities in protein loading and or staining you may be able to carry out normalization on the condition that both gels contain a series of internal standards proteins for which the abundance is known to be identical in the two gels In this case you can perform scatter plot normalization as long as only the spots corresponding to the internal standards were selected when displaying the plot 6 6 Processing gels A certain number of functionalities in ImageMaster not only imply the processing of existing 2 DE images on screen but also affect the image files on the hard disk These options are grouped in the Tools gt Gels menu and lead to the creation of new gels except for the Delete from Disk and Rename option that are automatically opened and displayed in the ImageMaster window The newly created gels are also inserted in the Gels folder of the current project in the Workspace Some features such as cropping gels are not provided in the Tools gt Gels menu because they must be carried out in other ways Nevertheless the processing operations are described below 6 6 1 Delete from disk With this function you not only remove your gels from the sc
58. illustrate the use of reports and navigation through data The possibilities are endless The Select on Gels drop down menu gives access to several related options e Select on Gels has the same function as the Select on Gels button e Select on Gels Reports not only synchronizes the selection from your report to the gels but also to any other open reports including open histogram windows or 3D views Imagine for example that you have an open Spot Report and an open Intra Class Report If you select a spot in your Spot Report and then choose the Select on Gels Reports option ImageMaster not only selects and displays the corresponding spot on your gel but also selects the corresponding line in the Intra Class Report Of course this is only true if your spot is part of a match and if that match was selected for the creation of the Intra Class Report e Refine Selection allows you to refine your selection by using an additional search criterion applied only to the highlighted items in your table In an Intra Class Report for example you could first sort your data based on the Separability see Section 5 4 4 for more information on sorting data select the 10 matches with highest Separability and then refine the selection based on the Ratio Thus by keeping matches that have a Ratio higher than 3 one would end up with only 7 selected matches in the example from Figure 5 2 e Select from Gels will select the lines in your report tha
59. in the match set are matched with the Master it is possible to copy additional spots from the matched gels to the Master As briefly described above a match set can include gels but also populations of gels represented by a sub match set The principle can be illustrated with Figure 9 1 The gels A1 A2 and A3 are part of Match Set A This match set is represented by the master image Master A based on the selected reference image Al in red Since Master A is a copy of A1 it contains the same spots The same is true for Master B which represents match set B Once all the gels in match set A are properly matched with Master A and the gels in match set B are matched with Master B the match sets A and B can be compared To do so they are included in a higher level match set AB for which match set A was chosen as a reference In practice comparing match sets A and B amounts to comparing their master gels Master A and Master B The new representative Master of this match set Master AB is an automatically created copy of the master image from the selected reference match set The interesting thing about this procedure is that matches are propagated at each level This means that once all match sets in this case A B and AB are effectively matched spots from gel A2 for instance can be directly compared with those from gel B3 through the existence of a common node Master AB in the matching tree ImageMaster 2D Platinum User Manual
60. indicate their dimensions in pixels Image coordinates Alternatively you can show grid lines with pl MW units provided such information is available in the annotation category pl_MW The pI MW grid can also be displayed over gels that do not contain this information but that were matched against a master gel having pl and MW values The grid values are then calculated relative to the master gel The software will try to partition the visible area in the Number of subdivisions entered by the user The graduations can just be regular subdivisions of the visible space in Fixed mode or a subdivision in terms of real coordinates at whole multiples in Adaptive mode Figure 6 9 shows various grid types drawn as gray lines on the gels To display grid lines 1 Select the gels 2 Choose Show gt Gels gt Grid Lines gt Show in the menu 3 Inthe Grid Settings box specify the Type of grid lines to be drawn Adaptive or Fixed 4 Specify the desired grid Units pl MW or Image coordinates 90 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 5 Enter the number of horizontal and vertical subdivisions using the scrollbars 6 The grid lines display over your gels Figure 6 9 7 Tohide the grid lines again select the gels and choose Show gt Gels gt Grid Lines gt Hide EN ImageMaster 2D Platinum Be lol x Fie View Edit Show Select Analyze Reports Tools Window Help ce gk 2 7 20 Pe s al TA
61. keep in mind that actions can only be undone for images that were selected when the operation was originally performed even if the gel selection subsequently changed Each Action Descriptor in the Undo Redb list is preceded by the time at which the action was carried out This information helps you to identify the particular sequence of actions that you would like to cancel ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 251 Safety control and automation Select commands to undo 21 52 36 Select Gel Objects from Report 198 objects in 1 gels 21 50 53 Select All Spots 2 objects in 1 gels 21 50 44 Split Spat 2 pared in 1 gels j t bj 1 21 49 06 Se ots amp objects in 1 gels 21 48 43 Set Region i gels 21 48 24 Set Region 1 gels 21 48 18 Set Region 1 gels 21 48 15 Set Region 1 gels 21 47 49 Auto Detect Spots 204 objects in 1 gels 21 47 13 Contrast Mapping 1 gels 21 47 00 Set Region 1 gels Displayed Actions without Display x T Only Gels Action con Figure 12 1 Undo window To undo the last action performed 1 Select the images for which you would like to cancel the most recent operation 2 Choose Edit gt Undo from the menu 3 The last action is selected by default 4 Click OK To undo several successive actions 1 Select the images for which you would like to cancel a certain number of operations 2 Choose Edit gt Undo from the menu 3 Sele
62. names must be unique 4 Right click on a copied match set 5 Choose Properties from the contextual menu 6 If necessary change the match set Name in the MatchSet Properties box Click OK Note that you cannot modify the name of an open match set 4 6 5 Setting the match set reference For each match set a reference gel or reference match set should be defined It is used to create the master image that will represent the match set If your match set contains gels right click on the gel to be used as Reference and choose Set Gel as Reference from the contextual menu If your match set contains submatch sets right click on the submatch set to be used as Reference and choose Set MatchSet as Reference from the contextual menu Icons of reference gels and reference match sets have a red component NOTE You can still change the Reference as long as the images in your match set have not been matched Once they are the reference image cannot be changed anymore 4 6 6 Moving gels or match sets You can rearrange your gels or match sets in the MatchSets folder to change their position in the list or move them into another match set Just drag your gel or match set to the desired position It is generally inserted after the item you drop it on Whenever there is a possibility to insert it inside or after a match set you will be asked for your choice 4 6 7 Displaying match sets To match gels or match sets they must be open
63. obtained by replacing X by Y ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis 227 Data analysis 228 ny ny 1 Zu 5 In fact the smaller of the two calculated U values corresponds to the number of shifts needed in order that the spot values from the two populations do not overlap For the first example in Figure 10 24 no shifts are necessary since the spot values of classes X and Y are already separated On the contrary for the second example the Mann Whitney test indicates that the first two values from Y or the three last values from X have to be swapped four times in order to separate the samples Figure 10 24 RR ea 0 10 0 2 01 0 05 0 0 bedacghfikj jikghfedcba 598 613 Spot values Ranks Spot values Ranks x Y x Y 0 000 1 5 0 034 1 0 000 1 5 0 045 2 0 044 3 0 056 3 0 059 4 0 058 4 0 068 5 0 064 5 0 144 6 0 069 6 0 237 7 0 075 7 0 240 8 0 078 8 0 249 9 0 104 9 0 255 10 0 106 10 0 308 11 0 126 11 Wy 51 15 Wy 25 41 Ny 6 5 Ny 6 5 Uy 30 Uy G 26 Figure 10 24 Two examples to illustrate the Mann Whitney and Kolmogorov Smirnov tests In a the two classes do not overlap at all whereas in b 4 shifts are needed to completely separate the spot values of the two classes The bold values correspond to the spot values and ranks from class Y the others belong to class X In fact ImageMaster displays the smaller of the two calculated U values in the Statistical Tests repo
64. of pixels 73 3 Spot quantification Once spots are detected ImageMaster automatically computes the amount of protein present in each spot Figure 7 2 illustrates the principles of spot quantification in the Melanie ImageMaster algorithm Measuring the protein quantification values in this way has the advantage of being more robust and reproducible when calculating protein expression variations relative quantification e Intensity The program first calculates the intensity of a spot The intensity is based on the highest calibrated pixel intensities in the spot from which the background has been withdrawn The background is defined as the minimum pixel value in the spot neighborhood e Area The area of a spot is not determined at the spot base because the base is often arbitrary and difficult to determine It is calculated at an intermediary height of the spot More precisely ImageMaster computes the area at 75 of the spot intensity as measured from the peak of the spot The spot outlines displayed in ImageMaster exactly encircle this computed spot area expressed in mm e Vol The volume of a spot is calculated as the volume above the spot outline which is situated at 75 of the spot height as measured from the peak of the spot In Figure 7 2 the measured volume of the spot is hatched Please note that the volume values like the intensities depend on pixel intensity calibration see Section 6 5 1 ImageMaster additionally calc
65. of the Inter Class Intra Class Histograms window not only implies the sorting of the classes according to their central value but also the sorting of the spot values inside each class Figure 10 22 BF ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 223 Data analysis 224 as 15 10 10 0 5 0 5 0 0 abedefghijkl efdabejklhig 4896 a 4896 b Figure 10 22 Inter Class Intra Class Histograms with a unsorted values and b values sorted in ascending order The classes are sorted according to their central value and the spot values within each class are also sorted 10 3 4 Inter Class Report ImageMaster allows you to generate specific reports in which you can numerically display one of the statistical descriptors or overlapping measures for each match and class as well as the maximum value among all the classes To produce an Inter Class Report 1 Select the matches to be studied on the gels or on a report 2 Select the gels to be included in the analysis 3 Choose Analyze gt Inter Class gt Report 4 Inthe pop up list select the value type Intensity Area Vol to be displayed 5 Choose the desired statistics central tendency and dispersion values see Section 10 2 2 in the subsequent dialog box The Inter Class Report Figure 10 23 displays the descriptor selected from the Displayed value list for each class and gives the maximum value among all the classes Max Column It is thu
66. or Wilcoxon test As indicated above the Mann Whitney U test or rank sum test is the nonparametric substitute for the two sample t test when the assumption of normality is not valid It is equivalent to the Wilcoxon rank sum test Once again it should only be used for comparing two unpaired samples The assumptions of the Mann Whitney U test are e The variable of interest is continuous not discrete and the measurement scale is at least ordinal This means that repeated values ties are not acceptable When ties are present in your data there is an approximation provided in the calculations but the exact results no longer hold e The distributions of the two samples are identical although not necessarily normal and differ only in location that is central tendency e The two samples are independent To perform the Mann Whitney test ImageMaster first ranks all the spot values from low to high paying no attention to which of the two classes for example X and Y each value belongs Then each value is given a rank number The smallest number gets a rank of 1 The largest number gets a rank of N where N is the total number of spot values in the two classes If two values are the same then they both get the average of the two ranks for which they tie Finally the ranks in each class are summed thus giving Wy and Wy which are used to calculate the Mann Whitney test statistic U The formula for Uy is as follows The formula for Uy is
67. propagate the information to your gels Note that the updated cells return to their original color If all information is updated the asterisk next to the report title also disappears Please note that you can keep the list of gel description categories for later use To do this click the Create Labels icon in the Gel Description Report In the Gel Descriptions dialog box the following options are available e Click Save and type a file name to store the current gel description categories e Click Load and choose a file name to reload an existing category list ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 121 ED Gel Calibration Report NOTE The gel descriptions defined via the Gels Description Report are only valid for the gels that were selected during the creation of the report To make the list of gel description categories a template for a particular workspace which should be used for any open gels you should define the gel description categories via the Gel Descriptions tab of the Options window go to Tools gt Options The procedure is the same as that described above The only difference is that the defined gel descriptions are automatically displayed in any Gels Description Report displayed Alternatively you can define gel descriptions by selecting gels and choosing Edit gt Gels gt Add Description from the menu You will be asked to choose a gel description category or to type a new one
68. referred or as a reminder when reviewing old work You can add or modify the Comment or Name while viewing the Properties of a match set Note that you cannot modify the name of an open match set To edit the Comment or Name of a match set 1 Right click on the match set 2 Choose Properties from the contextual menu 3 Inthe MatchSet Properties box add or modify the Name or Comment Click OK 4 The changes to Properties are saved ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 53 4 7 Working with classes The Classes 4 folder manages your classes Figure 4 8 You can easily e Create an unlimited number of classes and subclasses e Open classes in a worksheet to carry out statistical analysis Example Workspace 1 projects E Bacteria Gels Al MatchSets i Classes Bacteria 1 ag A a5 ATI FA A_T1_Gel3 mel FA A_TI_Gel2 mel FA A_T1_Gell mel m A_T2 B p B_T2 a B_TI Bacteria 1B a T1 EB A_T1_Gel3 mel EB A_T1_Gel2 mel EB A_T1_Gell mel BE B_T1_Gell mel EB B_T1_Gel2 mel EB B_T1_Gel3 mel tS T2 Bacteria 2 eg Abis Bg A_TIbis F A_T1_Gell mel FA A_TI_Gel2 mel F A_T1_Gel3 mel a A_T2bis Bbis B_Tibis B_T2bis Ca Reports Ga Documents Figure 4 8 The Classes folder 54 Ima
69. selected spots and selects all the unselected ones thus inverting the selection criteria 7 5 3 Reports You can select spots directly from an open Spot Report or DIGE Report You can do this by double clicking on a spot in the report or by selecting one or several spots and clicking the Select on Gels icon in the report toolbar In this way you can also select spots from an Intra Class Report as long as the spots belong to selected matches or from reports on Labels Annotations or Categories if the spots have linked annotations 7 6 Displaying spots 7 6 1 Spot shape Once spots are detected you can display their shapes on the gels in four different ways Figure 7 3 available from the Show gt Spots gt Shape menu e Crossed Draws a cross in the center of gravity of each spot e Outlined Displays the spot border e Filled Shows the area of the spot e Outlined Filled Shows the spot border for inactive spots Outlined mode and the area of the spot for selected ones Filled mode ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA E 0 BIS Oi a b c d Figure 7 3 Possible spot shapes are a Crossed b Outlined c Filled d Outlined Filled Selected spots appear as green crosses green outlines or green surfaces The default spot shapes for newly opened gels can be fixed in the Display tab of the Options window accessible by choosing Tools gt Options in the menu
70. selection of a spot based on its Spot ID Just enter the desired spot identifier in the Select Spots by ID dialog box e By Color When a specific color is assigned to particular spots using Show gt Spots gt Set Color the spots can be selected by choosing the corresponding color from the submenu e In Region This option allows you to select all spots in any regions that were drawn with the Region tool on selected images e From Report File If any Spot Reports were previously saved this function enables the reselection of all spots contained in the specified report file e All Highlights all spots in the selected images ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 135 136 e Refine by Value This feature enables the selection of spots based on their spot value Vol Area Intensity Vol or lntensity coordinates X or Y pl or MW Depending on the detection algorithm used spots can also be selected based on their Saliency Volume Ratio or Slope Make a selection of spots and then choose Select gt Spots gt Refine by Value from the menu Choose the quantitative value you want to use for your refinement Indicate the greater than gt and or lower than lt limits of the interval by checking the appropriate boxes and entering the desired numbers Spots whose values are included in the defined interval remain selected e Inverse Selection If some spots are already selected this option deactivates the
71. set into memory ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Gel Appendix Cut Cuts the selected match set in order to paste it Paste Pastes previously cut or copied items into the selected match set Properties Displays the match set properties MatchSet Name and Comment Open Opens the selected gels with the corresponding master in a new MatchSet worksheet Set Gel as Reference Defines the selected gel as the reference to be used when matching two or more gels The gel icon is tagged red A master gel is automatically created by duplicating the reference gel when opening the match set in a worksheet Delete Applies to non DIGE images only Removes the selected gels from the match set Please note that it does not remove the file from your hard disk The image is only deleted from the active project Add in Classes Adds the selected gels in a class If you would like to add the gels to a new class then enter a new name Otherwise you can choose from the other classes available in the proposed list Copy Copies the selected gels into memory Properties Displays the gel properties Gel Name and Staining A 2 6 Classes folder Create Class Creates a new class in the Classes folder Paste Pastes previously cut or copied items into the Classes folder Class Gel Open Opens the selected class in a new Classes worksheet Create Class Creates a subclass within the selected class Delete
72. set up a 2 DE database on the WWW Electrophoresis 18 2755 2758 Hoogland C Sanchez J C Bairoch A Hochstrasser DF and Appel RD 1999 The SWISS 2DPAGE database what has changed during the last year Nucleic Acids Research 27 289 291 Link AJ ed 1998 Methods in molecular biology Vol 112 2 D Protocols for Proteome Analysis Totowa NJ Humana Press Lopez MF 2000 Better approaches to finding the needle in a haystack optimizing proteome analysis through automation Electrophoresis 21 1082 1093 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Appendix Sanchez J C Wilkins M Appel RD and Hochstrasser DF 1997 Identifying proteins for proteome studies In Creighton ET ed Protein Function a practical approach 24 ed pp 1 27 IRL Press Wilkins MR Williams KL Appel RD and Hochstrasser DF eds 1997 Proteome research new frontiers in functional genomics Berlin Heidelberg Springer Verlag Unlu M Morgan ME and Minden JS 1997 Difference gel electrophoresis a single gel method for detecting changes in protein extracts Electrophoresis 18 2071 2077 Tonge R Shaw J Middleton B Rowlinson R Rayner S Young J Pognan F Hawkins E Currie and Davison M 2001 Validation and development of fluorescence two dimensional differential gel electrophoresis proteomics technology Proteomics 1 377 396 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 277 Appendix 278 ImageMaster 2D Platinum
73. shortcuts annotations gels matches spots show menu SIMOOUN erden ei la ee E ee E es alee edel software features references reqlirements Asus cken E A ata eds oad A datasede aactaloceeese 17 sort by similarity values in inter class histograms values in intra class histograms automatic detection borders color delete delete part detection detection parameters display edit export filter hide ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 285 Index 286 increase size merge pickers preview of detection report save detection parameters spot filtering start ImageMaster statistical tests Kolmogorov ann Whitney report Smirnov wo sample t test Wilcoxon statistics deschiptiVve harresira E cos Guana a elt ie eerie iia alee 198 inter class intra class 196 methodology status bar step tablet control SWISS 2DPAGE gels synthetic gels create SUSTEMTEQUIFEMENES pneis iinn E asair esaat eisi 17 T tabbed groups text link a 3 three dimensional view 102 toolbar tools menu y Twain compatible Scanners aaa a aa ae a S 236 U undo unique identifiers un pinned UUIDs Vv VIEW MEN RES nun RENNER Sic Pett lett a cae 23 WwW WIMGOW TION shoe ee Se EE BE ah DS Aas Oe earn Ake 23 WOFKFOW ee are eu n
74. single step The technology is compatible with almost all protein types Gels with narrow pH ranges permit very ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 7 Two dimensional gel analysis high resolution and large protein loads so that even poorly expressed proteins can be observed However scientists wanting to compare a set of gel images may notice that the quality and reproducibility of 2 D gels are sometimes mediocre Poor resolution high noise levels and large distortions in the protein patterns often hamper the analysis process The reasons for this are extensive unskilled sample preparation leads to unintended biological variability poor sample solubilization produces insufficient resolution vertical and horizontal streaking caused by salt in the IPG strip makes proper detection or quantification difficult dust particles or droplets lead to many spot artifacts etc The crucial algorithms in 2 D gel analysis software have been significantly improved over the years in order to account for such difficulties In addition program interfaces have gained a lot of ground in user friendliness In spite of these advances no image analysis software can solve all experimental problems The desire to draw valuable information from low quality gels remains an illusive goal Before starting a 2 D gel image analysis it is therefore essential to optimize all steps in the gel production process in order to assure the best possible q
75. that there is a gap equivalent to 50 of the current class range to the furthest other class The normalized overlapping is not symmetrical the value from Class A compared to Class B is not the same as the value from Class B compared to Class A ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis ClassA b Class B d Class A Class B Gap 2 0 c b 2 0 Ratio 1 4 c b 1 4 Normalized 1 5 c a b a 2 0 NON OVERLAPPING CLASSES ClassA b Class B o n GQ Class A Class B OVERLAPPING CLASSES Gap 1 0 c b 1 0 Ratio 0 8 c b 0 8 Normalized 0 75 c a b a 0 5 Figure 10 17 Scheme demonstrating how the Gap Ratio and Normalized values are calculated for two non overlapping classes upper part and two overlapping classes lower part Arrows above or below each class range illustrate how the Normalized measure relates to this class range Please note that the above mentioned formulas only apply to Class A for these particular examples Their presence in this manual is only to illustrate the principles of the overlapping measures used in ImageMaster Many different cases and therefore formulas exist Observe that in the Inter Class Histograms and Reports the number 1000000 characterizes the cases where the protein is completely absent from a class in this case ImageMaster cannot compute ranges A value of O for the Ratio or Normalized measures
76. the ImageMaster 2D Elite menu Save the XML file in the same folder as your experiment 3 Start ImageMaster 2D Platinum 6 0 and choose File gt Import gt ImageMaster 2D Elite Experiment in the menu 4 Browse the folder containing your Elite experiment and select the saved XML file 5 You are asked to enter the Source Folder containing the experiment gels indicated by default if you saved your XML file in the experiment folder as well as the Destination Folder for saving the new project in ImageMaster 2D Platinum format Note that you can create a new subfolder by adding the name manually 6 Click OK to confirm your folder settings 7 The import operation takes a few seconds ImageMaster then creates new gels and project files in ImageMaster 2D Platinum 6 0 format and saves them to the given destination folder These files are now ready to be added to the Workspace in ImageMaster Elite experiments All gels in the original experiment are converted into the ImageMaster 2D Platinum format and inserted into a new workspace and project bearing the same name as the experiment see Chapter 4 The generated project can contain several match sets depending on whether or not the initial experiment contained average gels A separate match set is created for each Average Gel User Defined Fields in an experiment are converted to properties of a project and stored in the project file However they are currently not supported by the softwar
77. the Zoom Window can be used to see the visible area in a larger view of the gel Figure 6 5 This is the case when the zoom factor in the Zoom Window is less than that of the gel To see the enlargement of the gel area under the cursor 1 Choose Window gt Zoom in the menu 2 Inthe Zoom Window set the zoom factor by clicking the and buttons The zoom factor must be greater than that of the gel You can also adapt the zoom factor of the gel if needed 3 Position the cursor on the area of the gel where you want to see details 4 The region under the cursor is magnified in the Zoom Window box To see the visible area in a larger view of a gel 1 Choose Window gt Zoom in the menu 2 Inthe Zoom Window set the zoom factor by clicking the and buttons The zoom factor must be less than that of the gel You can also adapt the zoom factor of the gel if needed 3 Position the cursor on the gel you want to view 4 The visible area is localized in a green box in the Zoom Window The Zoom Window can be resized by dragging a corner or edge Spots or annotations are also displayed in the Zoom Window for zoom factors higher than or equal to 1 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 85 ED EN imageMaster 2D Platinum 5 loj x File View Edit Show Select Analyze Reports Tools Window Help s9edsyuon Gels B Spots jo Annotations jo Figure 6 4 Zoom Window used to enlarge the region under the cu
78. the contextual menu 3 Inthe Add Gels box browse the directory where the image files are located select the 1 2 or 3 images belonging to the same DIGE gel and click Open Use the Shift or Ctrl keys to make multiple selections 4 Inthe Create DIGE box specify the DIGE Gel Name the Dye Chemistry DIGE Min or DIGE Sat and a Comment Click OK 5 Inthe DIGE Gel Properties box verify the Gel Image Names and Staining the dyes that were applied to each gel Click OK 6 Inthe DIGE Reference box specify the Standard Gel typically Cy2 if using minimal dyes or Cy3 if using saturation dyes Click OK 7 The DIGE Gel Name appears in the DIGE Gels folder The DIGE Gel Image Names are found below 4 5 6 Moving gels or folders You can rearrange your gels or subfolders in the Gels folder to change their position in the list or move them into another folder Just drag your gel or folder to the desired position It is inserted after the item you drop it on Whenever there is a possibility to insert it inside or after a folder you will be asked for your choice 4 5 7 Displaying gel images To view gel images or work on them they must be opened from the workspace You can select and open individual gels or an entire gel folder Gels that are in ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA subfolders will be opened in separate panes carrying the names of the subfolders Each DIGE gel being the two or three images is also
79. the directory where the backup file is located select its name and click Open In the Restore Backup box choose one of the two options for restoring a workspace By restoring the files using the original file paths you return to your workspace by automatically rewriting the previous file and folder structure By restoring the files in a single folder the workspace is reproduced in a new directory for which you have to enter a file name and location Click Restore The workspace is restored 4 11 2 Backup Restore project With the Backup Project function a single project can be archived including its gels matches master gels reports and other related files This backup is written into a single compressed file with the extension bkp that can be restored when needed Use the Restore Project function to later retrieve the backup To backup a project 1 Right click on the Project Name 2 Choose Backup from the contextual menu 3 Inthe Backup Project box browse to the directory you want enter a file name and click Save 4 The backup file with the extension bkp is archived To restore a project 1 2 Right click on the current Workspace Name at the top of the Navigator Choose Restore Project from the contextual menu In the Restore Project box browse the directory where the backup file is located select its name and click Open ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace 63
80. the exported files E imageMaster 2D Platinum B x File View Edit Show Select Analyze Reports Tools Window Help Close Images Ctrl vWv Close All Save Save As Import P Gel Data m AREN Print gt Image to Clipboard a go tte aut i gt ar Image to File aie m 5 Worksheet to Clipboard AAD DALY vy Et a Worksheet to File fo A To aye W ae Mi a cH SD gt oy Bruker Proteineer SP Ag a pas aS Se Heattcare etary ae Genetix GelPix any Genomic Solutions ProPic Selection Box Master_DIGE Gels i Spots a Annotations fla Figure 11 3 Reference markers IR1 and IR2 IR1 is attached to a pixel cross basis IR2 is attached to a spot Both options can be used in a single gel Spots to be picked are selected highlighted in green ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 243 Data integration 11 3 3 Genetix GelPix spot picker To export a spot coordinate file to the GelPix spot picker 1 Select all spots to be cut with the spot excision robot 2 Choose File gt Export gt Spots to Picker gt Genetix GelPix in the menu 3 Enter a file name and destination folder 4 The spot picker subsequently reads the saved file 11 3 4 Genomic Solutions ProPic spot picker The ProPic spot picker produces a TIFF file of the entire gel holder area with a resolution of 1035 x 1317 pixels each
81. the lines that are selected in the active report 10 3 5 Statistical tests ImageMaster provides three statistical tests two sample t test Mann Whitney U test and the Kolmogorov Smirnov test These tests are used to analyze differences in protein expression between classes of gels The idea is to draw conclusions about the significance of the protein expression changes by extrapolating information from the data you collected For example when you have two samples classes with different means that is different means for the spot values of a particular match you might want to know whether the data were sampled from populations with different means or whether the populations have the same mean with the observed difference being a coincidence of random sampling ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis 225 Data analysis 226 In fact there is no way to definitely conclude which of the two possibilities is true All you can do is calculate the probability of observing a certain difference or larger between sample means in an experiment of this size for populations that in reality have the same mean If the probability is small you can conclude that the difference is not likely to be caused by random sampling and assume instead that the populations have different means NOTE ImageMaster provides qualitative indications about different protein expressions In order to state the results as probab
82. the master gel via an Intra class Report and want to propagate the labels to all matched gels To copy matched labels from one gel to a set of other gels 1 Select the labels to be copied in the source gel ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Annotations 3 ME 2 Select the source gel and the gels into which you want to copy the labels 3 Choose Edit gt Annotations gt Copy Matched Labels from the menu 4 The selected labels are copied to the corresponding spots in the other gels Duplicate labels You can copy selected labels to another category Since the selected labels may belong to different categories this option can be used to merge several categories into a new one However only one label per annotation can be duplicated at a time To duplicate labels 1 Select the labels you would like to duplicate as part of another category 2 Choose Edit gt Annotations gt Duplicate Labels from the menu 3 Inthe Duplicate Labels box pick the recipient category or enter a new category name 4 The selected labels are copied to the chosen category 5 Whena label for the chosen category already exists ImageMaster asks whether you want to keep it or replace it with the new one Rename category ImageMaster allows you to rename any category be it a predefined or user defined category or a Set In the case of a Set the label contents name of the Set are automatically updated on the gel To rename a cat
83. the number of images displayed horizontally and vertically in a pane Choose View gt Pane Layout gt Free in the menu In the Pane Layout box input the following values e Number of Columns Constrains the images positioned one next to the other in the pane e Number of Rows Constrains the images positioned one above the other in the pane ay ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 27 Getting started 28 When the number of open images in a pane exceeds the number of displayed images for example in stacked mode you can make an image visible by clicking on its tab at the bottom of the pane In order to sift through images for example in stacked mode use the Page Up and Page Down keys on your keyboard Alternatively you can choose View gt Pane Layout in the menu and select Previous or Next Switch order You can change the order by which the gel images are displayed by clicking on the gel legend and dragging the image onto another gel It is inserted before this gel Similarly you can re order panes by clicking on their banners and dragging them to their new position You can swap worksheets panes and images To do so use View gt Switch or the Ctrl F shortcut This reverses the previous re ordering operation carried out on a worksheet pane or image Contextual menus When you click the right mouse button on a worksheet banner pane banner or gel legend in the Display Zone a contextual m
84. the predefined Ac category To pack categories 1 Select the gels for which you would like to pack one or more categories 2 Choose Show gt Annotations gt Packed Categories in the menu 3 Inthe Set Packed Categories box the check boxes of the packed categories are selected those of unpacked categories are empty ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Annotations 3 whereas grayed check boxes correspond to categories that have different packing states in the various gels 4 To pack a category check its box Deselect the check box to unpack a category Finally you may need to click once or twice in a grayed check box to unpack or pack the corresponding category 86 Adding modifying annotations and labels You can add and modify annotations and labels in different ways using the Annotation tool Edit menu options Reports or File gt Import function The Annotation tool is helpful if you want to add or modify a single label or just a few labels The Edit menu options are more adapted to the creation of a large number of annotations or labels simultaneously Reports are useful for editing existing annotations 8 6 1 Annotation tool When double clicking on a label while the Annotation tool is activated the Edit Label box is displayed Change the text in this box to modify your label content The Annotation tool should also be used to change the position of an annotation In this case simply select an an
85. the scanning source all TWAIN compatible scanners attached to your PC are automatically recognized by ImageMaster and then launch the scan The scanner software opens giving you the opportunity to change the necessary settings and subsequently initiate the scanning process Once this is done the image is saved in ImageMaster file format and can be added to the Workspace To scan one or more gel images using a TWAIN compatible scanner 1 Choose File gt Import gt Twain gt Select Source in the menu 2 Select the appropriate image capture device from the given list This only needs to be done once unless you want to change to a new image capture device 3 Choose File gt Import gt Twain gt Acquire in the menu 4 The scanner software automatically starts 5 Set the suitable parameters for the scan ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data integration 6 Launch the scan 11 2 Exporting and importing ImageMaster 2D Platinum data 11 2 1 XML format To make the vast amounts of data that can be obtained with ImageMaster available for processing by other applications or to import information coming from external sources ImageMaster uses the common XML format XML is also exploited for saving reports XML stands for extensible Markup Language and was created as a cross platform software and hardware independent tool to structure store and exchange information It allows the creation of customized
86. the spot that will be removed the numbers indicate the successive positions of the mouse cursor The minus sign next to the pen indicates that one is in the deleting mode and the pen must start to draw from a point outside the spot border Split a spot First select the spot to split Only one spot should be selected Then right click outside the spot and while holding down the right mouse button draw a line through the spot at the position where it should be split Figure 7 7 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Figure 7 7 Splitting a spot The grey line shows where the spot will be split the numbers indicate the successive positions of the mouse cursor The cutter indicates that one is in the splitting mode Merge two spots Select the first spot While holding down the Shift key and the left mouse button draw the fusion zone starting from within the second spot going through the first one and finishing within the second spot Figure 7 8 Ls Zs Figure 7 8 Merging a spot The green trajectory defines the region where the two spots will be fused the numbers indicate the successive positions of the mouse cursor The plus sign next to the pen indicates that one is in the adding mode Adding spots When spot editing is enabled the Spot tool can also be used to add a new spot to your gel To add a new spot to an image 1 Choose Edit gt Spots gt Ed
87. those of hidden categories are empty whereas grayed check boxes correspond to categories that take different visibility states in the various gels hidden in some gels shown in others SSS ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 169 8 Annotations 170 4 To hide a category make sure its box is unchecked On the other hand select an empty check box to show the corresponding category You can click once or twice in a grayed check box to hide or show the corresponding label category in the selected gels When you click on an annotation that has both visible and hidden labels all of its labels are displayed on the screen during the time it remains selected Figure 8 8 The hidden labels immediately disappear when the annotation is deselected v 2 e Ldmk gt gt amp a b Figure 8 8 a Unselected annotation with hidden labels b When the annotation is selected the hidden labels become visible 8 5 5 Pack unpack categories By default each spot is displayed with its own label even if several labels hold the same content However to declutter the display you can show a single label for all identical labels In ImageMaster this option is called packing categories Figure 8 9 Basen a b Figure 8 9 a Unpacked and b packed labels for protein P00450 in the Plasma Master Gel SWISS 2DPAGE database The labels belong to
88. to save just those in the file Select only the gels to be normalized make sure the reference gel is deselected Import the normalization by choosing File gt Import gt Normalization from the menu and selecting the previously saved fitting report Two different scenarios can occur If the fitting data of a single gel was saved in the report the normalization will be applied to all or none of the selected gels depending on your choice If the fitting data for two or more gels was saved each normalization in the file is applied to the gel with the corresponding name Alternatively you can select the gels one by one Choose Edit gt Gels gt Edit Normalization in the menu and enter the appropriate Gray Slope and Gray Offset values corresponding to those from the scatter plot for the particular gel Each selected gel is now normalized To check this you can display the new scatter plots All slopes should be close to 1 and all offsets close to 0 Normalized gels will have an asterisk after their name in various reports and displayed spot values correspond to the normalized values ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 113 ED 114 11 You can remove the normalization at any time by choosing Edit gt Gels gt Reset Normalization in the menu Please use caution with respect to this method because if there are many proteins that are expressed differently then you can introduce errors and
89. x File View Edit Show Select Analyze Reports Tools Window Help Create Calibration elals amp m gt amp G D S o Use tablet Kodak step tablet no 2 CC 04 06 08 10 12 14 16 18 20 22 24 0 217 logf t 2 0 863 logf t 0 0308 mean enor 0 0507 Gels i Spots jo Annotations o Figure 6 17 Image of the step tablet with the red calibration step overlay The Create Calibration window shows the calibration curve and the OD values for the different steps on the left NOTE When you select a point in the step tablet list in the Create Calibration window the corresponding step becomes automatically highlighted in green on the step tablet overlay and in the calibration graph This makes it very easy to correlate the three sources of information point in tablet list point in graph and step rectangle The various icons in the toolbar of the Create Calibration window are used to amp Open another tablet definition Save the calibration with the extension cal ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 109 ED 5 Print the calibration graph Copy the calibration graph to the clipboard Display related Reports Two reports are available from the Reports icon in the Create Calibration window These reports can be saved printed and copied to the clipboa
90. you can either type the expression P00 and choose the Regular Expression field in the dialog box or type POO and deselect the Regular Expression field e By Category This feature enables the selection of all labels belonging to one or several categories Use the Shift and Ctrl keys to pick several category names at a time e By Color When a specific color was assigned to particular annotations the annotations can be selected by choosing the corresponding color from the menu ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Annotations 3 e In Region This option allows the selection of all annotations in the active regions that is in all regions that were defined with the Region tool on selected gels e From Report File If any report containing information about labels or annotations and enclosing the columns X Y and GelName was previously saved this function enables the reselection of all labels contained in the selected report file Reports on Categories Labels Annotations and even Spots can be used e All This highlights all annotations in the selected gels e Combine This option can be used to combine the current selection of annotations with a second criterion based on the Intersection Union Difference or Exclusion of a particular category Please see Section 8 4 2 for more information on this feature e Common Labels This option allows the retrieval of all sets of identical labels within a gel or am
91. 1 0034 38 Edition AA E imageMaster 2D Platinum File View Edit Show Select Analyze Reports Tools Window Help waf eE cursor Information zd z 14 Kecam 3 s Detect Spots FileName TestCalibration2 Gels il Spots fo Annotations jo a SpotlD 2147479916 ul Value 0 34963 Detection parameters gt a Pr Area 106 P TE Intensity 1509 Vol 1359 Smooth a C gt 2 Saliency 41 5 Min Area aj CI gt 5 Saliency 0 0000 IV Auto Preview Preview Figure 7 1 Adjusting spot detection parameters in real time To detect spots automatically 1 2 Select the gel images for spot detection If necessary choose Show gt Spots gt Shape gt Outlined to see the spot borders more easily Click on the Region tool Draw a rectangle around an area with representative spots on one or more of the selected gels Note that you can still draw resize or move regions while setting the detection parameters Choose Edit gt Spots gt Detect in the menu The Detect Spots window appears on the screen and the spots in the drawn regions of the selected images are detected with the default parameters If you do not want the program to recalculate the spots in the preview regions for each parameter change turn the Auto Preview ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 129 130 option off To manually refresh the preview regions simply click the Pre
92. 11 0034 38 Edition AA 179 9 Matches 180 NOTE The master images are the nodes for the propagation of matches If a protein is not present in the Reference and so on the Master no matches will be formed even if the protein appears on all other gels see blue spots in Figure 9 1 Consequently the protein will not turn up in the analysis In the example of Figure 9 1 choosing one of the other gels A2 or A3 as the Reference would have been more appropriate To solve this problem spots from matched gels can be copied to the master gel e a2 A3 BI 82 5 8 R Master A Master B o Master AB Figure 9 1 Population matching and the importance of the master image Matches are selected by choosing the corresponding spot which will appear in green One match is selected in match set A In match set B all matches are selected Representative spots in a population that are not in the reference gel blue spots should be manually copied to the Master When several gels are matched to a given Master this Master provides a unique numbering scheme for spots across all gels Each spot in a gel image can in fact be associated to the corresponding Spot ID in the master gel The Spot ID in the master gel is then called the Match ID The fact that you are no longer limited to matching gels but now can match
93. 3 lol x File View Edit Show Select Analyze Reports Tools Window Help mla e el wie Br Bl fvawe Intra Class Report Vol The chosen statistics are Mean 100 and M S D Match ID Mean 100 M S D Separability 4477 1 34843 0 392948 0 516089 4538 0 299013 0 397664 0 556158 4577 1 25715 0 562541 0 945710 4584 0 582289 0 407015 0 479449 4679 1 34106 0 887342 1 05500 4691 1 72898 0 594347 0 735561 4704 2 45890 0 863292 1 06690 1 2 3 4 5 6 7 8 0 182636 0 260009 0 496155 0 641371 0 628942 0 978030 0 502048 0 513872 0 785745 2 03374 0 402549 0 433897 Gels je Spots 6 Annotations jo Figure 5 1 Navigating through the lines of an Intra class Report a Match 4704 is selected in the report When the Select on Gels icon is clicked the corresponding spots are displayed on the gels b Select Next displays the following match with ID 4715 on the gels ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 69 Reports 70 After selecting the line corresponding to Match 4704 and pressing the Select on Gels button all the spots belonging to Match 4704 are selected on their respective gels By clicking the Select Next button the spots belonging to the following match in the report Match 4715 are selected on the screen In this way one can go through all the matches in the report This is just a simple example to
94. 37 138 To show or hide particular spots you can use one of the following options available in the Show gt Spots menu e Show All Makes all the spots on the selected images visible e Hide All Hides all the spots on the selected images e In Region Shows all spots in the region drawn with the Region tool This option is handy when all spots were previously hidden e Only Selected Shows only the selected spots and hides all others This feature is helpful when performing a visual inspection of the spot detection or of the gel analysis results e Hide Selected Hides the selected spots 7 6 4 Show hide spot IDs You can find out the Spot IDs for specific spots via the Cursor Information window see Section 7 9 or a Spot Report see Section 7 9 However sometimes you may want to display the identifiers of selected spots directly on the image To show or hide Spot IDs 1 Select the spots you want to display the Spot ID for 2 Choose Show gt Spots gt Show ID from the menu 3 The Spot IDs for the selected spots are displayed close to each spot center 4 You can hide all Spot IDs again by choosing Show gt Spots gt Hide All ID 7 7 Adding modifying spots Quantitative protein data and in particular the spot volume are highly dependent on an optimal and reproducible definition of the spot borders and a correct splitting of partially overlapped spots To guarantee reproducibility of quantitative work it is therefore
95. 4266 0 239972 0 344238 0 3 345 4594 0 131266 0 0848866 0 646676 8 30224 00686879 0159803 0205525 02 0 177527 0 103055 0 580505 8 02053 0 0988353 0 260546 0 0385055 0 122954 0 0858610 0 698320 7 14862 0 138740 0191090 0 241444 Figure 10 10 Intra Class Report By clicking on the Histograms icon in the toolbar Figure 10 10 you can display the histograms of matches that were selected in the report Please note that the order of the histograms in the resulting Intra Class Histograms window will be the same as that of the selected matches in the Intra Class Report You can take advantage of this characteristic to organize your histograms in a certain way ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 207 Data analysis 208 The Reports icon in the toolbar Figure 10 10 allows you to display reports related to the matched spots The Gel Report item is a reduced version of the original report on gels that can be used for example as a legend for the histograms The Spot Report is a modified report on spots that is useful to compare one spot against the others in the same match see the possible applications in the paragraph below The Report from Selection function creates a new report that only contains the matches that are selected in the active report Spot report In this report the measures available for quantifying the spot values relative to their match are the sam
96. 513872 1 02355 1000000 0 785745 2 15348 0 566332 0 262985 2 04059 0 754605 b Figure 5 2 Refining a selection in your report a The matches in the report were sorted according to their Separability in descending order and the first 10 matches were selected This selection was further refined by only keeping the rows for which the Ratio 100 value is higher than 3 The result is shown in b However when you find a drop down menu next to the icon the ImageMaster tool tip calls it the Reports icon In this particular case the drop down menu gives you a list with several related reports These can include e Report from Selection Generates a new report only containing the selected lines from the active report as describe above e Gel Report Contains summarized information about the gels selected for the creation of the active report and always includes the master gel if one ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 71 ED Reports 72 has been created It can be used as a legend to the gel index a b c on histograms Note that this report is not necessarily the same as the one obtained by choosing Reports gt Gel Report in the menu e Spot Report Isa modified report on spots that is helpful to compare one spot against the others in the same match The usefulness of this report is described in more detail in the Chapter 10 of this manual e Intra Class Report Displays a report
97. 6 0 format and saves them to the given destination folder These files are now ready to be added to the Workspace in ImageMaster 11 1 2 From ImageMaster 2D Elite Any 2D experiments from ImageMaster 2D Elite or Phoretix 2D Elite can continue to be analyzed by the new ImageMaster 2D Platinum project It is important to note that you can convert and use spot shapes and quantification values matching information 1D and 2D calibration results annotations spot filters and much more from the Elite versions However you are not allowed to edit imported spots or compare quantification values from imported gels with new data obtained from the ImageMaster 2D Platinum software This is because spot detection and especially spot quantification are done very differently in the two software versions thus rendering any comparisons inappropriate You can still compare your old gels with a new set of gels analyzed by the latest software although you must redetect the spots and ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 233 Data integration 234 redo the matching All other information such as annotations and 1D or 2D calibrations are preserved To convert ImageMaster 2D Elite experiments 1 In ImageMaster 2D Elite make sure you have synchronized the spot numbers This is necessary to correctly export the match information 2 Export the experiment in XML format by choosing File gt Import Export XML gt Save Experiment from
98. 6 2400 Fax 45 16 2424 Finland amp Baltics Tel 358 0 9 512 39 40 Fax 358 0 9 512 39 439 e France Tel 01 69 35 67 00 Fax 01 69 41 96 77 Germany Tel 0761 4903 490 Fax 0761 4903 405 Italy Tel 02 27322 1 Fax 02 27302 212 e Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7304 Middle East amp Africa Tel 30 210 9600 687 Fax 30 210 9600 693 Netherlands Tel 0165 580 410 Fax 0165 580 401 Norway Tel 815 65 555 Fax 815 65 666 Portugal Tel 21 417 7035 Fax 21 417 3184 e Russia amp other C l S amp N I S Tel 7 095 232 0250 956 1137 Fax 7 095 230 6377 South East Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 Spain Tel 93 594 49 50 Fax 93 594 49 55 e Sweden Tel 018 612 1900 Fax 018 612 1910 Switzerland Tel 0848 8028 12 Fax 0848 8028 13 UK Tel 0800 616928 Fax 0800 616927 e USA Tel 800 526 3593 Fax 877 295 8102 imagination at work User Manual 11 0034 38 AA ProTang Teknikinformation AB 2005
99. 9 270 271 272 273 275 276 276 Contents Contents x ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 1 Introduction 1 1 About ImageMaster 2D Platinum ImageMaster 2D Platinum is a state of the art software application designed to analyze two dimensional electrophoresis 2 DE gels This version of ImageMaster has been developed by a team of top researchers from the Swiss Institute of Bioinformatics SIB in collaboration with Geneva Bioinformatics GeneBio SA and GE Healthcare and integrates the Melanie software It was conceived in close collaboration with biologists and proteomics scientists from Professor Hochstrasser s group in Geneva The application has advanced to its current level primarily due to the input of users worldwide ImageMaster 2D Platinum ensures fast and reliable image comparisons It easily manages multiple image analyses and offers the possibility to automate detection and matching steps However it is the versatility of the software that empowers you the scientist There are numerous interactive tools for optimizing and manipulating data Furthermore attain a higher level of quantitative and qualitative analysis using the robust and sophisticated techniques provided in the application ImageMaster integrates filtering querying reporting statistical and graphing options so that you can easily view compare analyze and present your results From the raw experimental images to the preparation of
100. D Platinum User Manual 11 0034 38 Edition AA 143 spots were detected Information on spots includes the name of the image on which the spots were detected the Spot ID the coordinates of the center of gravity X and Y and the quantification values Intensity Area Vol Intensity and Vol Depending on the spot detection algorithm used the Saliency Vol Ratio and Slope will also be available NOTE In the Cursor Information window the Vol Ratio given corresponds to Volume of the spot under the cursor Volume of the spot on the DIGE reference image To display cursor information 1 Choose Window gt Cursor Information in the menu 2 The Cursor Information window Figure 7 9 appears on the screen 3 Click on the Settings icon to define the data to be displayed in the window 4 Inthe dialog box select the attributes to be shown in the Cursor Information window from the Hidden list using the Shift or Ctrl keys to make multiple selections and transfer them to the Visible list using the right pointing arrow button Similarly you can remove attributes from the Cursor Information window by selecting them in the Visible list and clicking on the left pointing arrow button You can also double click on an attribute to transfer it from one list to the other Click Apply 5 The selected attributes are displayed in the Cursor Information window 6 Position your cursor over the pixel or spot of interest The Cursor Information w
101. EE ENER 4 3 2 Opening an existing workspace 4 3 3 Workspace properties nacecscssccsssssssssssssesssssssssssssssssssssussssssseseseeseseeceeeesssessssssetea AA Working WITH FO SSS zunen nnnakeemeiinuuken 4 4 1 GrFEAating d BF lect n ikea 4 4 2 Inserting an existing project ueceeessssssssssesssesccsssssssessseccessssssneseseseeecsssnuetesseeeeeea 4 4 3 Exporting a project 4 4 4 Project properties 45 Working with gels 4 5 1 Cre ting a subfolder an eaneneensseneneneauen 4 5 2 Importing a hewel srera r e EAEE 4 5 3 Importing a new DIGE gel 4 5 4 Adding an existing gel 4 5 5 Adding an existing DIGE gel 4 5 6 Moving gelsOrfoldersen naee a 4 5 7 Displaying gel images unnnnnnnnsnsssssssssnsnnnnnnnnnnnnnnnennnnnnn 4 5 8 Spot detection 4 5 9 Folder properties 4 5 10 lt G l propertie Ssnin a 4 6 Working with match sets oi cccccccssssssssssssessssscssssssssssssssssssssssssssssssssssssesssssees 4 6 1 Creating a mateh Setiana nonae a ETTER 4 6 2 Creating a match set from a DIGE gel 4 6 3 Adding gels to a match Set cceessssssssssssssssssssssssssssnsussssssssessessesessessessesssssssstss 4 6 4 Copying MATCH SOLS cis csans chiens icles ee acelin ees ae 4 6 5 Setting the match set reference 4 6 6 Moving gels or match Sets nnnnnnnnnnnennssssnennnnnnnnnnnnnnnnnenne 4 6 7 Displ ing matchsetsiiensanaeananenaa ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Contents
102. ES Projection onto Axes 1 2 WVol The chosen statistics are Mean 100 and M S D Match ID Contrib Axe1 a Contrib Axe2 Quality 4 0 0291635 2 97303e 4 0 992559 0 0240521 0 0574727 0 994189 0 0158091 0 0879108 0 989856 0 015460 xl 0 0146218 a al Ba far ale sja Select on Gels Projection onto 4 BE on Gels Reports The chosen stati Match IC Refine Selection Select from Gels kez aua y FE 0 0146218 0 00103070 0 0465930 0 812729 0 0355031 0 994294 b Figure 10 13 Factor Projection Report with a matches ranked based on their contribution to the first axis and b ranked based on their contribution to the second axis Please note that the Factor Projection Report also contains a Quality measure for each match This number gives you an appreciation of whether a match is well represented on the factorial subspace It tells you how close the distance of the projection is to reality Indeed matches with very similar behavior similar expression profiles across gels will be close in space However when projected onto a two dimensional subspace matches that are actually far apart may appear together It is therefore important to look at the Quality measures to judge whether matches are effectively close If the values are high for both matches the chance is great that they are indeed nearby and have a simila
103. F step the strip is placed at the cathodic end of a homogeneous or gradient sodium dodecyl sulfate polyacrylamide gel SDS PAGE possibly along with molecular weight marker proteins flanking one or both ends of the IEF strip In an electric field the negatively charged SDS protein complexes then migrate towards the anode with differing velocities depending on their size Small molecules move faster and further Larger ones move more slowly and cover less distance The proteins are finally visualized by radiolabeling or detected with a variety of staining methods such as silver Coomassie blue or fluorescent stains Adapted image capture devices are used to generate digital images that can be analyzed with a 2 DE image analysis software such as ImageMaster 2 1 2 Applications Two dimensional 2 D gel electrophoresis is a leading research tool in proteomics This technique enables the identification of protein expression changes such as those associated with the exposure to pathogens drugs or other stimuli or alterations linked to different stages of development Thus gel electrophoresis is invaluable in gene expression studies for the discovery of protein targets disease markers and drug candidates or to evaluate drugs for toxicity And this is just to name a few examples 2 1 3 Advantages and limitations 2 DE is currently the most powerful protein separation technique allowing the parallel investigation of thousands of proteins in a
104. File Path and ID The files can be listed in ascending or descending order by clicking on the column header that you want to sort by 4 3 Setting up a workspace In ImageMaster 2D Platinum 6 0 gel images can only be opened from the workspace It is therefore necessary to set up a workspace as soon as you start working with the software ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 37 38 4 3 1 Creating a workspace To create a workspace 1 Click on the New icon in the Workspace toolbar 2 Inthe Create New Workspace box specify the Workspace Name Location and Comment Click OK By default a workspace file with the extension mws is saved in the ImageMaster Workspaces folder found in the user s My Documents directory If you want to save the file in a different folder then browse and open the folder 3 The Workspace Name appears at the top of the Navigator The workspace file is automatically saved 4 3 2 Opening an existing workspace To open an existing workspace 1 Click on the Open icon in the Workspace toolbar 2 Inthe Open Workspace box browse the directory where the workspace file is located select its name and click Open 3 The file is opened in the Workspace window You can also go to File gt Recent gt Workspaces in the menu and choose the Workspace Name from the list This is only possible if a workspace file was saved during the current work session If you open a workspace created with a
105. GE Healthcare ImageMaster 2D Platinum 6 0 User Manual Contents Introduction 1 1 About ImageMaster 2D Platinum al 1 2 What s new in this release al 13 ImageMaster resources nenseenessensseenesssnensennensenensennensenensnnesssnnensnnessanenssnnnn 2 1 3 1 USEEMANUG Bus sec 2 1 3 2 T torials reniee sinne lenkiniensen 2 1 3 3 O lihe help x cua seas cus anne 2 1 3 4 Conventions used 3 1 4 Getting further assistance weeeccccccssssssssssssseessssscssssssssssssessssssesssssssssssseeeess 3 1 4 1 HOW 0 CONTA GUUS zen RRniRe S 1 4 2 Sending eM lla n a e nassen ame 5 1 4 3 System and product information 5 1 4 4 GE Healtheareilistenssl Ace 5 Two dimensional gel analysis 2 1 Two dimensional electrophoresis en 7 2 1 1 TEChNIgUe n anna are 7 2 1 2 Applications 2 1 3 Advantages and limitations 2 2 Differential gel electrophoresis 2 2 1 TOC AIG UG rmen terse tt O dan rset inca ssh Mente h deans 2 2 2 Internal stand rd aadusaliiessnsikeitieiniebiiiiiesiipnihn eiktiigesitien 2 3 Understanding gel images enisinia 2 3 1 Anatomy of Gel IMAGES eceesssssssssssssssssssssssssssssesseseseeseeseessessssstsssssssssunusessses 2 3 2 Spot and gel relationships 2 4 Image analysis workflow anne 2 41 Acgulning datd a 2 4 2 Setting up a workspace euran 2 4 3 Visualizing and calibrating gels e 14 2 4 4 Detecting and quantifying spots 2 4 5 Annotating spots and PIXEIS ee ecc
106. Intra Class Report contains information about selected matches and their comprised spots Match ID central tendency dispersion spot values coefficient ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 177 8 Annotations E of variation range ratio separability etc In addition they can be used to display the labels attached to the spots in the master gel In an Intra Class Report you can add labels or edit existing ones and create new columns corresponding to categories Figure 8 13 Intra Class Report xj la o a e e MES E Value v Intra Class Report Vol The chosen statistics are Mean 100 and M S D Match ID Mean 100 M S D Variation AC ProteinName pI_MW Landmark EB 2967 0 205858 0 0382935 0 186019 P06981 IMP_ECOLI L2 0 108127 0 00384856 0 0355928 0 0422498 0 00589410 0 133506 69036000 0 00628074 0 00503845 0 802206 6 30 76000 3507 0 355613 0 0348053 0 0978742 Pos994 MDH_ECOLI 5 70 1 oo e Figure 8 13 Report on selected matches in selected gels 8 7 5 Inter Class Report An Inter Class Report provides statistical values for sets of gels calculated for all selected matches such as central tendency dispersion and overlapping measures In such a report you can display the labels attached to the spots in the master gel add labels or edit existing ones and create new columns corresponding to categories Figure 8 14 Inter Class Re
107. Label SpotID X and Y If the Spot ID is not known use 1 in this field and ImageMaster will position the label in the corresponding X and Y positions of the gel If X and Y positions are not known use 1 in these fields and ImageMaster will position the label on the spot with the corresponding Spot ID To import labels into open gels 1 Select the gels for which you would like to import labels 2 Choose File gt Import gt Labels in the menu 3 Browse the folder containing the desired Label Report rpt or a text file txt containing the label information Click OK 4 ImageMaster asks for confirmation Answer Yes if you want to proceed 5 The imported labels are added to the selected gels ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 175 8 Annotations 176 Similarly you can import annotations from an Annotation Report or a tab delimited text file containing the required columns SpotID X Y and a column for each category to be imported To import annotations into open gels 1 Select the gels for which you would like to import annotations 2 Choose File gt Import gt Annotations in the menu 3 Browse the folder containing the desired Annotation Report rpt or a text file txt containing annotation information Click OK 4 ImageMaster asks for confirmation Answer Yes if you want to proceed 5 The imported annotations are added to the selected gels 8 7 Annotations and labels in reports Imag
108. Master 2D Platinum User Manual 11 0034 38 Edition AA 155 8 Annotations 156 Is unique When you check the Is Unique box you indicate to ImageMaster that each label on a gel within the new category should be unique That is no identical labels can be created This is the case for the Landmark category for instance In order to prevent confusion as to which spots correspond in different gels there should only be one spot marked with a particular label in each of the gels When the Is Unique box is checked ImageMaster will not accept your label when an identical one already exists You are asked to enter a new label This is always the case for labels of the Landmark category External engine The External Engine field should be used when you want to send HTTP queries to a CGI script on a server A CGI Common Gateway Interface script is a program or script file executed on a Web server in response to a user request It allows for example people from all over the world to query a database that is connected to the World Wide Web The CGI script transmits information such as a database accession number or object identifier from the client to a database engine receives back the results and displays them to the client Virtually all databases on the Web and in particular those containing 2 DE and other protein data see Chapter 11 use CGI scripts to enable data queries ImageMaster takes advantage of this fact by offering the possibili
109. Platinum User Manual 11 0034 38 Edition AA 221 Data analysis 222 Adapt gradations By default the histogram gradations are adjusted according to the spot values in each match However in order to display an identical gradation in all histograms you can deselect the Adaptive Gradations check box at the bottom of the histograms window Figure 10 19 b Figure 10 19 Inter Class Histograms with a adaptive gradations set individually for each histogram and b set according to the minimum and maximum values in all histograms Sort values To simplify the visual search for non overlapping intervals you may want to classify the displayed class values in ascending order by clicking in the Sorted values check box at the bottom of the histograms window Figure 10 20 a b Figure 10 20 Inter Class Histograms with a unsorted values and b values sorted in ascending order Inter Class Intra Class Histograms ImageMaster can draw more detailed Inter Class Histograms for selected matches These histograms not only display the class intervals but also show the individual spot values in each class They can therefore be considered as a kind of mixed representation with properties from both the Intra Class and Inter Class Histograms To display Inter Class Intra Class Histograms 1 Select the matches to be studied on the gels or on a report 2 Select the gels to be included in the analysis 3 Choose Analyze
110. Please note that although the Vol is a rather efficient measure for evaluating protein expression differences between gels the lntensity is not as relevant to use 6 5 3 Intensity normalization using a scatter plot Intensity normalization may in some cases be beneficial or even indispensable to obtaining more precise differential expression values However if your experimental procedure is highly reproducible this image compensation method is not always needed Many differential protein expression studies yield good results without requiring intensity normalization Improper usage of this tool can even be detrimental to your analysis Therefore clearly understand the assumptions that are made in the application of this feature before you decide to use it on your specific set of gels The scatter plot in ImageMaster renders information about the relationship between the spot values from two gels by searching for the linear dependence between the values from one gel and the values from another gel see Section 10 2 1 In numerous cases only a relatively low percentage of the spots in the compared gels are expressed differently Most of the spot values especially Intensity values evaluated in the two gels should be similar and therefore the best fit line of the scatter plot should be close to identity Y x This assumption permits your gels to be normalized based on the best fit line That is to correct the pixel values in one of your gels i
111. Removes the selected classes from the Classes folder Cut Cuts the selected classes in order to paste them Paste Pastes previously cut or copied items into the selected class Properties Displays the class properties Name and Comment Open Opens the selected gels in a new Classes worksheet ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 263 Appendix 264 Delete Removes the selected gels from the class Please note that it does not remove the file from your hard disk The gel is only deleted from the class in the active project Copy Copies the selected gels into memory Cut Cuts the selected gels in order to paste them Properties Displays the gel properties Gel Name and Staining A 2 7 Reports folder Open Opens all the reports contained in the Reports folder in dockable windows Add Reports Inserts one or more reports from the hard disk into the Reports folder Create Folder Creates a subfolder within the Reports folder Paste Pastes previously cut or copied items into the Reports folder Folder Open Opens all the reports in dockable windows Add Reports Inserts one or more reports from the hard disk into the selected folder Create Folder Creates a subfolder within the selected folder Delete Removes the selected folder Copy Copies the selected folders into memory Cut Cuts the selected folders in order to paste them Paste Pastes the previously cut or copied folders or r
112. SSS To import a Selection Box 1 Select the gels to be cropped using a Selection Box 2 Choose File gt Import gt Selection Box and select the previously saved file Click on Open 3 The Selection Box appears on the selected images 4 By clicking inside the box with the Region tool and dragging it you can move the Selection Box to superimpose the anchor on the characteristic spot 5 The gels can now be cropped as described above by using File gt Save As 6 7 Reporting on gels 6 7 1 Gelreport The Gel Report Figure 6 19 displays summarized information about the selected gels such as image height and width in pixels Rows and Columns pixel dimensions of the scanned image PixWidth and PixHeight minimum and maximum gray levels before MinGray and MaxGray and after calibration MinValue and MaxValue as well as the intensity normalization parameters Slope and Offset the calibration function Calibration and calibration units Unit It also shows the Gel ID the full file path of the image the staining method the number of detected or selected spots the number of defined or selected annotations the minimal and maximal values of pl and MW the match set to which it belongs and the class to which the gel was assigned To display a Gel Report 1 Select the gels that should be included in the report 2 Choose Reports gt Gel Report gt Current Template in the menu 3 The Gel Report is displayed 4 Click on the Setti
113. The main difference from Windows Explorer is that the Navigator only gives a symbolic view of your gel images reports and inserted documents This means that you can delete images reports and documents from a workspace without losing the data stored on the hard disk However keep in mind that removing gels from a project may affect your match sets The fundamental organization of the Workspace is fixed At the top of the Navigator is the Workspace Name A workspace contains any number of Projects each of which represents a study of related gels Every project is organized into Gels MatchSets Classes Reports and Documents These default folders cannot be deleted or renamed However you can organize the Workspace according to your needs by adding subfolders to the hierarchical structure Workspace contextual menus Right click on a folder subfolder or file to open a contextual menu from which you choose an action to be carried out Detailed descriptions of all the contextual menus in the Workspace are provided in Appendix A Moving files or folders Move folders subfolders and files in the Workspace by dragging and dropping or using the Copy and Paste options in the contextual menus 4 2 4 File details Detailed information about selected files is given on the right side of the Workspace window The details include the Name MatchSet and Class to which the image belongs dates Created and last Modified Size in bytes File Type
114. This can be done before and during the use of the Adjust Contrast feature In fact the Adjust Contrast window can be left open throughout your work session so that you can adapt parameters at any time in order to highlight faint spots or to help in spot editing decisions This means that you can continue to select gels and other objects while the Adjust Contrast window is open and that you can move the region in your gel at any moment to locally increase the contrast for better viewing As you will see below the Adjust Contrast function may be more interesting in the preview mode than when its modifications are simply applied to the gels NOTE Any changes done by the Adjust Contrast function only influence how the image is displayed on your screen and do not affect the underlying data spot detection and quantitation 6 4 4 Contrast mapping As mentioned earlier modern scanners are usually able to scan 2 DE images with 12 or even 16 bits per pixel 4096 or 65536 gray levels respectively Because common computer screens are only able to display 256 gray levels mapping must be undertaken between the 4096 or 65536 image gray levels and the 256 screen gray levels By default ImageMaster uses a linear mapping function 96 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA a where the lightest point in the image is mapped to 0 white and the darkest point is mapped to 255 black This is illustrated with the gra
115. User Manual 11 0034 38 Edition AA Contents 7 5 5 REDOMES a a e NEA A E Tos Displaulng Spotssese nn nee n EAEN 7 6 1 Spot Shape a zo 7 6 2 SP Ot COMM Pe 0 7 6 3 Showy Wide Spots riia a nines ens de ee et aed ea 7 6 4 Show hide spot IDs s 7 1 Adding Modifying Spots ssersecarcersiicnseussastanndurannata 138 7 7 1 Editing Spots secies ea E RN 139 1 8 MW tid Picalibrati N sisien 142 79 C rsot INFORMATION sssrinin 143 7 10 REPOFLINGON SPOS sissie 145 1 10 17 SpOEREPON a EAN ER AR EA 7 10 2 DIGE histogram ads FAQS DIGETEDON zarten 8 Annotations 8 1 Introduction 2 052288 ccdidicicuntn aS 151 8 2 Predefined label categories un 151 83 _ Creating annotations and labels 152 8 3 1 Creating label categories M 8 3 2 Creating Ssetsn unn en ana EARO al ea 8 3 3 Creating specific link Sye se iie aA 8 4 Selecting annotations and labels 161 8 4 1 ANMOLOLIONAOOL 5 nsen e E E A 161 8 4 2 Select menu er 8 4 3 REDOMS Eee E A AEEA 85 Displaying annotations and labels nu 167 8 5 1 Annotation flag position 8 5 2 Annotation flag color 8 5 3 Annotation flagpole color 8 5 4 Show hide annotations and labels 168 8 5 5 Pack unpack cdtegdtlesun an ansaiennz 170 86 Adding modifying annotations and labels 171 8 6 1 Ahnotation Tool seen 171 8 6 2 Edit menu 4 8 6 3 REPORtS neuen img ERSA o 8 6 4 Importing labels
116. XML and XSL in Chapter 11 You can then use the print option in your browser to get a paper copy Copy the selected actions for subsequent pasting into an open i script Cu Refresh the History All actions are collapsed and the list scrolls fs down until only the most recent operations are visible Display the Gels Report Note that the report only includes the images selected when the History window was first opened This tool is useful for checking which images are included in the current History ES Copy the selected actions to a New Script E 254 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Safety control and automation E Similar to the Undo Redo options there are some actions that are not included in the History The following is a non exhaustive list of exceptions e Modifications applied to the Workspace are not logged e Operations that require file manipulations such as opening saving and printing files are currently not recorded with the History function e Direct actions on reports are not included in the History navigator except when selections or modifications in the report are propagated to the corresponding images 12 4 Script The Script function automates parts of the analysis process ImageMaster routinely carries out a sequence of instructions when you run a script You are empowered to encode desired actions simply by cutting and pasting from the
117. _T1 and B_T2 were copied to be used in populations per treatment T1 T2 rather than growing substrate A B As existing matches are conserved upon copying match sets this can save a lot of work ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace U v v an eN v v v v AT A B_T1 A Er A_T1_Gel3 B_T1_Gel3 A_T2_Gel3 B_T2_Gel3 A_T1_Gel2 B_T1_Gel2 A_T2_Gel2 B_T2_Gel2 A_T1_Gell B_T1_Gell A_T2_Gell B_T2_Gell Figure 4 7 Example of a hierarchical match set structure Case 3 For each match set a reference gel or reference match set must be defined This Reference will be used to create a Master representing the match set In Figure 4 5 Figure 4 6 and Figure 4 7 the reference gels or reference match sets have a red component The choice of the reference gel or reference match set is important as it significantly influences the results of your analysis This is because spots that are not in the Reference will also not be present in the Master unless they are manually copied there The rule of thumb is to choose the gel with the most and the best quality spots as the reference gel To learn more about the Reference the Master and matching please refer to Chapter 9 Please note that the master gel should rather be seen as a spot index It is not possible to display quantification values for a master gel 4 6 1 Creating a match set There are different ways to create a match set To create an empty
118. a History window 1 Select the images for which you would like to display a history 2 Choose Edit gt History gt Show from the menu 3 You can place a marker in the History by choosing Edit gt History gt Insert Marker from the menu or clear the list of actions by choosing Edit gt History gt Clear er ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 253 Safety control and automation CSC Petes S se Actions History 20 06 01 gt Groups Report gt Select All Groups H Groups Report gt Select All Groups H Groups Report G Refine Objects Selection from Report By Value Window SH Selection By Values m Filtered_Column 94 0005 Min_Value 3 00000 EB Max_Value 17 9286 H Select Gel Objects from Report bm Set Spots Color 1 xl Displayed Actions Without Display T Only Gels Action Figure 12 2 History window The History window contains a toolbar and a list of actions Figure 12 2 The functionality of each of the icons is as follows Save the History in an XML type file with extension hst Print the History You can choose to print only selected actions g Note that before printing the History first displays in your default Internet browser The XSL stylesheet located in the Template Script folder of the ImageMaster installation directory is used to transform the XML formatted History into an attractive document find more details about
119. a publication ImageMaster takes you successfully through each stage of an image study 1 2 What s new in this release The most important new features in ImageMaster 2D Platinum version 6 0 are listed below e Population matching e Support of DIGE technology co detection DIGE histograms e Intelligent multi worksheet display e _ Dockable report windows e Workspace with management of gels match sets and classes e Scrollbars for moving and zooming gels e Improved cropping option e Adjust contrast in 3D View e Measure histogram in spot report ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Introduction Introduction 1 3 ImageMaster resources 1 3 1 User manual The ImageMaster 2D Platinum software incorporating Melanie is intended to be intuitive and comprehensive We encourage you to read through the User Manual in order to exploit its full potential This guide explains in detail all the functions and advantages of ImageMaster 2D Platinum If new to the software you will quickly master the fundamental concepts and features If already familiar with the software the manual will inform you of new functionalities and help to optimize your image analyses The chapters in this manual are generally organized according to the logical sequence of a 2 DE gel analysis although expert users will agree that many of the steps can be inverted or repeated at some point The last chapters are more specialized and have n
120. a server ImageMaster allows you to pass on the content of a label as the first parameter to any executable When you double click on a label that has an executable defined in the External Engine field of the label category the executable runs with the label content as a parameter ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Annotations 3 157 8 Annotations 158 To set an executable 1 Inthe Create Category box click on the EXE button located next to the External Engine field 2 Browse the directory where the executable is located and select the correct file name which should carry the extension exe 3 After clicking Open the file with its complete file path automatically inserts into the External Engine field Please note that you can define a different query engine for each label category and therefore you can link one protein spot to different database entries Display properties User defined categories use a gray background color by default You can change the default color by clicking on the color box The new background color is used for all labels of that specific category in the active workspace 8 3 2 Creating sets The predefined category Set was created to help you mark spots with common properties by indicating that they belong to a set The labels in such a category do not contain specific information They only display the name of the set to which they belong Figure 8 1 To create a s
121. aced select Start gt All Programs gt Accessories gt Command Prompt ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Getting started EEE nm 2 When the command prompt appears click on it and enter the command ipconfig all making sure to leave a space between the ipconfig and the all 3 Among the displayed information make a note of the Physical Address but without any dashes or colons If the computer has several Physical Addresses there can be one for each network i e Ethernet card in the computer any of them can be used for identification Please verify that the Physical Address is correctly noted Otherwise the license file will not work 4 Close the command prompt window 3 2 3 Installing the GE Healthcare eLicense server The installation in this section should only be performed when using a floating license If only node locked licenses are used continue from Section 3 2 4 To install the GE Healthcare eLicense server 1 On the computer where the eLicense server will be installed insert the ImageMaster 2D Platinum installation CD 2 Click on the Install GE Healthcare eLicense Server button 3 The installation window is shown Click Next 4 Accept the default installation path and start the installation if the default installation path is changed the paths below need to be updated accordingly 5 A question concerning the Windows Firewall may appear To allow the license server to work properly yo
122. ach class in a match To characterize a class only by the central value for the calculation of the difference or ratio between central values for example set the dispersion percentage slider to 0 Gap Maximum difference between the range of the current class and the range of one of the other classes in the example of Figure 10 17 c b in the case of Class A Negative values indicate overlapping intervals whereas positive values are non overlapping class ranges Ratio Maximum ratio between the lower limit of one of the other classes and the upper limit of the current class in the example of Figure 10 17 c b in the case of Class A Absolute values smaller than 1 indicate overlap whereas absolute values higher than 1 show that there is no overlap In order to easily distinguish matched spots that are over or under expressed in one of the classes the ratio value is preceded by a minus sign when the protein spot is under expressed for the class in question compared to the other class Positive values are attributed to the Ratio value in the over expressed class Normalized Maximum percentage of the current class range not overlapping with the range of one of the other classes in the example of Figure 10 17 c a b a in the case of Class A A value smaller than 1 indicates overlap For example 0 25 implies that 25 of the current class range is not recovered by one of the other classes In the same way a value of 1 5 indicates
123. ader until the column is the width you want While dragging you will notice that the cursor changes its format To simultaneously change the width of all columns hold the Shift key and then drag the boundary of any column header To resize columns so that their whole content is displayed double click on their separator The column to the left of the cursor reverts to its default size You can also alter the row size In this case drag the lower border of the row to reduce or increase its height To enlarge or reduce all rows at once hold down the Shift key and drag the row separator To resize rows so that their whole content is displayed double click on their separator ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Reports 15 Reports 76 Spot Report F x 5 4 4 Sorting data Data in tabular reports can be sorted by the column content If you click once on the header of a specific column a triangle is displayed indicating that the column s numerical data is sorted in descending order and the textual data in ascending order When you click once more on the header the triangle inverts indicating that the data is sorted in the opposite order Note that ascending order means that numbers are sorted from 0 to 9 and text is sorted from A to Z You can sort the items in your table using multiple criteria Since rows containing identical values in a sorted column appear together you can further sort the items in your tabl
124. al of the slider is decreased you can also displace the interval by placing your cursor in the middle of the slider holding down the left mouse button and moving it to the left or right In Figure 6 12 the histogram shows the gray level distribution of the selected image The maximum gray level was set to 9398 by moving the right side of the ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 97 ED slider to the left The red transformation function shows that gray level 9398 is now mapped to the maximum screen gray level 255 or black and that any darker pixels will appear as black E imageMaster 2D Platinum Fie View Edit Show Select Analyze Reports Tools Window Help a an 98 Gels i Spots 0 Annotations o D Image Display Settings FMI Choose the color palette and setthe contrast mapping options Colors Gray T Invert Image A_T1_Gell mel Unit Value z I Only in Region Bending a H gt 0 OK Cancel Figure 6 12 Remapping of the gray levels Adjustments are immediately reflected in the preview region Another way to change the default gray level mapping is the use of a non linear mapping function The Bending parameter expands or compresses the contrast range at the dark or light ends of the range When the bending parameter is positive the image is lighter The image is darker when the bending parameter is negative Figure 6 13 ImageMaster 2D Plat
125. all at the same time need access to the ImageMaster program The number of computers that can simultaneously use ImageMaster depends on the license and is administered via the license server A floating license file must be placed on the computer running the eLicense server This server can either be installed on a computer running the ImageMaster program or on any other network computer It is recommended to install it on a network computer that is always running To be able to start ImageMaster a valid license must be found ImageMaster will check that the license file is available at every subsequent login To obtain and correctly place a license file follow the activation steps below 3 2 1 Access code After ordering ImageMaster a letter including an access code will be send to the order s shipment address The access code is necessary for collecting the eLicense files see Section 3 2 4 Store this access code in a safe place 3 2 2 Finding the physical address of the computer The physical address is necessary when collecting the appropriate eLicense file The address identifies the computer and is used by the licensing system If a node locked license is used it is the physical address of the computer where ImageMaster is installed If a floating license is used it is the physical address of the computer where the license server is installed To find the physical address 1 Onthe computer where the eLicense file should be pl
126. alue Inter Class Histograms x als E Center x Intra Class Histograms Vol The chosen statistics are Mean 100 and M S D Dispersion interval Central tendency Class index Match ID Adaptive gradations F Sorted values Figure 10 18 Inter Class Histograms The Reports drop down menu in the toolbar Figure 10 18 allows you to display information related to the Inter Class Histograms e The Gel Report item is a reduced version of the original report on gels to be used for example as a legend to the classes e The Inter Class Report see Section 10 3 4 lists the numerical values corresponding to the selected histograms e The Intra Class Report displays a report on selected matches for a selected class It is a reduced Intra Class Report where only the spot values of one class are considered The information displayed is that which is usually found in an Intra Class Report see Section 10 2 4 e The Report from Selection function creates a new report that only contains the lines that are selected in the active report NOTE Analogous to the Intra Class Histograms and Reports the Inter Class Histograms and Reports are dual elements they have a reciprocal relationship You can therefore use the Inter Class Report to change the order of your histograms sort your data in the report and then re display the corresponding histograms or to refine your histogram selection ImageMaster 2D
127. alyze Reports Tools Window Help Mouse Selection Intra Class Histograms a x EKAA LEE Histograms on Matches Vol The chosen statistics are Mean 100 and Adaptive gradations Gels je Spots R Annotations jo I Sorted values Figure 5 5 Mouse selection version of the Intra Class Histograms Only the histograms for currently selected objects matches 4767 and 4769 are displayed If the spot from match 4767 is deselected only the histogram of match 4769 will be shown This type of report has the advantage of only displaying information on objects of interest Moreover you do not have to open a new report for each additional item The following report types are available from the Window gt Mouse Selection menu e 3D View e Spot Report e Label Report ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 77 Reports 78 Intra Class Report Intra Class Histograms Inter Class Report Inter Class Histograms ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 6 Gels 6 1 Introduction This chapter presents ImageMaster operations that relate to gels You will learn how to do the following e Change the display of gel images zooming gels adjusting the contrast and looking at intensity profiles or 3D views e Create or verify an intensity calibration e Process your gels rotating flipping scaling cropping e Save print and export gel images To make
128. anned in the wrong direction and you may have to flip them to get their correct mirror image With ImageMaster you can flip gels horizontally or vertically To flip your gels 1 Select the gels to be flipped in the same way in a worksheet 2 Choose Tools gt Gels gt Flip from the menu and select the desired option Horizontally or Vertically 116 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA SSS Sy 3 Enter a new file name in the Flipped Gels box Note that instead of changing each file name individually you can add an extension or replace the extensions of the original image files You can also change the destination folder 4 The flipped images are saved to the disk drive and opened in the ImageMaster window 6 6 5 Scaling gels ImageMaster lets you create smaller or larger copies of selected gels This function is particularly useful for very large images where a reduction in size may significantly decrease the time and memory required for the analysis To reduce or increase the size of your images once they have been opened 1 Select the gels to be scaled in a worksheet 2 Choose Tools gt Gels gt Scale from the menu 3 Enter the horizontal and vertical scale factors in the dialog box 4 Give new file names or modify the file extensions 5 Click OK The scaled gels are saved on the hard disk and opened in the software All spots and annotations are maintained 6 6 6 Inverting gray levels The
129. ard in 2 DE experiments that permits quantitative and statistical analyses First each protein spot in a sample can be compared to its representative spot within the internal standard on the same gel to generate a ratio of relative protein levels Quantitative comparisons of samples between gels are made based on the relative change of sample to its in gel internal standard This process effectively removes the system gel to gel variation enabling accurate quantitation of induced biological change between samples The need to run gel replicates is also eliminated thus reducing the number of gels required per experiment An additional benefit of using an internal standard in DIGE technology is that matching between gels is more apparent The internal standard image is common between all gels in an experiment therefore matching can be performed between internal standard images which have similar spot patterns Conventional 2 DE requires matching between different samples on different gels which introduces differences in spot patterns from sample to sample and gel to gel variation Matching between internal standards allows matching between identical samples so variations in spot patterns are due only to electrophoretic differences 10 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Two dimensional gel analysis 2 3 Understanding gel images 2 3 1 Anatomy of gel images Gels Agel is a 2 DE image that has been digitized and store
130. ase entry as well as the entry s short name in the ProteinName category For example the Human Plasma protein map possesses the Ac label PO2768 and the ProteinName label ALBU_HUMAN for each spot that corresponds to albumin In most 2 DE databases the accession number is the unique entry identifier while the protein name can vary It is therefore strongly recommended to use accession numbers for your queries Nevertheless ImageMaster provides a way to automatically update the protein names in your gels if you use the SWISS 2DPAGE accession numbers To update ImageMaster labels with database protein names 1 Select a gel containing labels with valid SWISS 2DPAGE accession numbers 2 Select all labels you want to update ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data integration ss 3 Choose Edit gt Annotations gt Load Protein Name from SWISS 2DPAGE 4 For each selected label ImageMaster then requests its short name from the SWISS 2DPAGE database using the Ac as identifier and then updates the ProteinName label accordingly eZDPage a DPA POZ649 oso plore lol x File Edit View Favorites Tools Help a Back gt amp A A QSearch GFavorites media A Br 3 w 7 A Address http iwww expasy orgicgi bininice2dpage pl P02649 Go Links ExPASy Home page J SiteMap Contact us SWISS 2DPAGE Search SWISS 2DPAGE F for Go Clear Search b
131. at any moment during your analysis You will learn the details about displaying using saving customizing and editing reports in Chapter 5 Additionally you will find information on specific report types in the various chapters of this document 2 4 10 Controlling and automating gel analyses As with reporting results you can check the operations that were carried out on your gels at any time Do this with the History function that is described in Chapter 12 The latter also instructs you on how to create Scripts for automating parts of your analysis and explains the multiple undo redo feature 2 5 ImageMaster flavors The powerful suite of features that normally apply to 2 DE gels in ImageMaster can now be used in conjunction with DIGE gels There are two modules of ImageMaster 2D Platinum 6 0 available for purchase e ImageMaster 2D Platinum 6 0 The software version to be used with conventional 2 DE gels All menu options related to DIGE are not functional and are grayed out in the graphical user interface e ImageMaster 2D Platinum 6 0 DIGE The fully functional version to be used with conventional 2 DE and DIGE gels You can add and import DIGE gels directly in the workspace You can also co detect DIGE gels using the algorithm created by the GE Healthcare DeCyder software development team match report plot histograms and perform statistical analyses on DIGE gels ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 15 Tw
132. at spot is selected When you double click on a match in an Intra Class Report all gels containing that matched spot are selected Region tool A region is a rectangular area in a gel that is used for different purposes in ImageMaster Certain actions can be limited to this region such as the selection of spots or annotations You can use a region to preview spot detection parameters or gray level adjustments You can also save print or export a region as well as display a 3D view of a selected area To define a region in a gel 1 Click on the Region tool in the toolbar 2 Position the cursor at the top left position of the area you want to select hold down the left mouse button and move the cursor to the bottom right position Release the mouse button at the end point 3 A dashed box outlines the region Figure 6 1 EN imageMaster 2D Platinum loj x Fie View Edit Show Select Analyze Reports Tools Window Help Gels 2 Spots 0 Annotations 0 delta O pI O MW Figure 6 1 Region tool The left gel shows a region during selection no spots displayed the right one after selection ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 81 ED 82 You can move a region by clicking inside the box and dragging it You can also change the size of the box by dragging a corner or edge In order to remove a region select the Region tool if not already done and double click on the gel In order to
133. atinum User Manual 11 0034 38 Edition AA Appendix A Appendix A 1 Contextual menus Contextual menus are available throughout the Workspace and Display Zone In the Workspace right click on a file or folder to open a contextual menu from which you choose an action to be carried out In the Display Zone access contextual menus by right clicking worksheet or pane banners gel legends or inside a gel image These contextual menu options are often duplicates of main menu options but are an alternative and quicker way to perform the action A 2 Workspace contextual menus A 2 1 Workspace name e New Project Creates a project in the workspace e Insert Project Adds an existing project to the workspace e Backup Archives the workspace file with the extension bkp e Restore Workspace Returns an archived workspace to its previous state e Restore Project Returns an archived project to its previous state e Properties Displays the workspace properties Workspace Name Creator File Name Modification Date and Comment A 2 2 Project name e Export Exports a project with copies of its gels matches and documents e Backup Archives the project file with the extension bkp e Delete Removes the project from the active workspace e Properties Displays the project properties Project Name Creator File Name Modification Date and Comment A 2 3 Gels folder Add Gels Adds gels that are already in ImageMaster format with the
134. avigator is a trademark of Netscape Communications Corporation TIFF and Photoshop are 75184 U ppsala trademarks of Adobe Systems Incorporated Sweden All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them GE Healthcare reserves the right subject to any regulatory and contractual approval if required to make changes in specifications and features shown herein or discontinue the product described at any time without notice or obligation Contact your local GE Healthcare representative for the most current information Amersham Biosciences AB a General Electric company going to market as GE Healthcare Amersham Biosciences AB Bjorkgatan 30 751 84 Uppsala Sweden Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 1327 USA Amersham Biosciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Asia Pacific Tel 852 2811 8693 Fax 852 2811 5251 e Australasia Tel 61 2 9899 0999 Fax 61 2 9899 7511 Austria Tel 01 57606 1619 Fax 01 57606 1627 Belgium Tel 0800 73 888 Fax 03 272 1637 Canada Tel 800 463 5800 Fax 800 567 1008 e Central East amp South East Europe Tel 43 1 982 3826 Fax 43 1 985 8327 Denmark Tel 45 1
135. axis Matches 4844 4903 4691 4767 and 4839 are characteristic for population T1 match 4704 is characteristic for population T2 The gel legends are given in the corresponding Gel Report 10 2 6 Heuristic clustering ImageMaster proposes a powerful analysis method to automatically create classes of gels and highlight significant protein spots Heuristic clustering a machine learning algorithm based on artificial intelligence is used to describe the characteristic spots of 2 D gels while using heuristics to speed up the search The algorithm provides the possibility to blindly classify similar gels into two or more classes and to determine the characteristic spots i e matches of each class These characteristic spots often correspond to differently expressed proteins ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis The number of classes that are formed is dependent on the user input ImageMaster first asks for the minimum and maximum number of classes for example 2 and 4 respectively It then splits the gels in 2 3 and 4 classes compares the resulting classifications and according to a similarity function shows the best one The classification step can also be repeated for each of the classes in order to find subclasses In this way a descending hierarchical classification tree is produced The depth of classification or the number of sublevels is established by the Classification Level parameter that mu
136. ber of estimated spots For example for a mammalian lysate run on an 24 cm pH 4 7 Immobiline DryStrip and a large format gel such as the Ettan DALT Gel 20 cm x 26 cm a value of 2500 for the estimated Number of Spots should be satisfactory 7 4 3 Spot quantification Numerical data for individual spots are automatically calculated e g volume area intensity slope and volume ratio and included in the DIGE Reports see Section 7 10 3 Spot Reports see Section 7 10 1 and Cursor Information window see Section 7 9 e Vol Spot volumes sum of pixel intensities within the spot boundary are always expressed with background subtracted Background is subtracted on a spot specific basis by excluding the lowest 10th percentile pixel value on the spot boundary from all other pixel values within the spot boundary The spot volume is the summation of these corrected values e Vol ratio Volume ratios volume of first image spot volume of second image spot indicate the change in spot volume between two images NOTE In the DIGE Histogram and DIGE Report the first and second images are determined by the order in the workspace or the order in the worksheet To obtain the inversed spot ratios change the order of the gels in the workspace or the worksheet In the Spot Report and Cursor Information window the first image is the image mentioned in the FileName the second image is the DIGE reference image ImageMaster 2D Plati
137. blet image obtained in the section above 4 Next load the definition of the control step tablet This Control Tablet File must be specifically adapted to this new step tablet That is it should have been edited with a tool as Windows Notepad so that it contains the 110 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA a SSS appropriate OD values height width and number of steps corresponding to the control step tablet 5 The red calibration step overlay appears on the image of the control step tablet and the Control Calibration window is displayed 6 Select the Spot or Annotation tool in the ImageMaster toolbar and adjust the position of the steps 7 You should now verify that the calibration curve is passing through the data points correctly and with minimum dispersion intervals If this is not the case try to find out why your current calibration does not seem to work properly Applying a calibration To apply a calibration to newly scanned gel images 1 Open and select the gels the calibration should be applied to 2 Choose Edit gt Gels gt Apply Calibration in the menu Select the source of the calibration information This can be an open gel or step tablet image that was already calibrated or you can select a file cal or mel from the hard disk All pixel and spot values subsequently displayed in any reports or in the Cursor Information window correspond to the calibrated values You can remove a calibra
138. ccsssssssccssessccccceccescessesssssssssssssnsnsueeseeesees 14 2 4 6 Matching gelsianaeie rennen 14 2 4 7 Anglyzing datosde aenn anne 14 2 4 8 Integr ting date asesseienaee eu 14 2 4 9 Rep ktingifesults aninion nei aA 15 2 4 10 Controlling and automating gel analyses nnnnnn 15 2 5 ImMageMast r flavo S siinon na n aia 15 Getting started 3 1 Installing ImageMaster 2D Platinum enn 17 3 1 1 System FEGUIFC MEMES sre an a 17 3 1 2 Software installation 17 3 22 elicensing z unseenstsiisrkenstkeiiueengiiuiinertwenaaitign 18 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Contents 3 2 1 ACCESS C00e nein a a a 18 3 2 2 Finding the physical address of the computer 18 3 2 3 Installing the GE Healthcare eLicense server 19 3 2 4 Collecting and placing an eLicense file 20 3 2 5 TOSE UC CL COIS Esine a e a E a S 3 3 Starting and exiting ImageMaster B A ImageMaster window seini eiiie E EERE 3 4 1 Meni bliain AEN REEE 3 4 2 TOODO a E rn Te nero 3 4 3 DISPIQU ZONE eisin a AE 3 4 4 Dockable windows 3 5 ImageMaster options ooaa eseeescssssesscssssesscssseecsessseceessseccssssecsessueccesssecsesseseesse Workspace 41 Introduction e an einen 33 4 2 Workspace window seummmn 35 4 2 1 Workspace toolbaissiisnir ie n 36 4 2 2 Active project 36 4 2 3 NAVIGATO ei a iraa N A EAR died E 37 4 2 4 FHC COUGHS esse an 37 4 3 Setting Up A Workspace u a 4 3 1 CREATING d Works pat soiien EA
139. ct a prior action to be undone 4 Click OK The selected action and all following actions are undone automatically If you are not satisfied with your latest undo or you canceled too many operations you can obviously reapply the actions 252 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Safety control and automation To redo specific actions 1 Select the images for which you would like to redo a certain number of canceled operations 2 Choose Edit gt Redo from the menu 3 Select the action to be redone 4 Click OK The selected action and any preceding actions are redone All operations performed can be reversed with the exception of the following non exhaustive list e Modifications to the Workspace cannot be canceled e Itis not possible to undo operations such as opening saving and printing files e Changes that only apply to all or none of the gels cannot be canceled for example displaying the profile or changing the color palette e Direct actions on reports such as the creation edition or closure cannot be undone However when selections or modifications in a report are propagated to the corresponding images they are included in the undo list and can therefore be canceled 12 3 History Please keep in mind that actions are only displayed for images that were selected when the History window was first opened even if the gel selection subsequently changed and the History was refreshed To open
140. d Data Set Color e Select By Content By Category Combine By Color In Region From Report File Common Labels All Inverse Selection e Report Label Report Annotation Report Category Report Matches e Edit Add Match Delete Match e Show Show ID Hide All ID Show Vectors Hide Vectors e Select By ID For Spots All Refine Selection Inverse Selection Multiple Matches e _ Analyze Intra Class Report Intra Class Histograms e Match Report Generates a Match Report e Zoom Zoom in or out on the selected images or regions You can also choose a factor to reduce or enlarge the selected images or regions by e Show All Makes all spots and annotations visible in the selected gel images e Hide All Hides all spots and annotations in the selected gel images It also hides all match vectors in the selected gel images e Select All Activates all spots and annotations in the selected gel images e Unselect All Deactivates all spots and annotations in the selected gel images m ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 267 Appendix 268 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Appendix 3 B Appendix B 1 Shortcut keys Several menu options can be activated by keyboard shortcuts These are indicated at the right hand side of the corresponding menu option Please note the logic behind the key combinations Ctrl is used for handling gels Shift is used for handling spots Alt
141. d check for possible reasons to explain why your values are systematically biased Stain intensity variations differences in protein loading or image acquisition problems are examples of typical causes To display scatter plots 1 2 Make sure your gels are opened in a Classes worksheet Select a reference gel and the gels for which a scatter plot should be displayed Select matched spots to be included in the scatter plots Generally all matched spots are selected Choose Analyze gt Intra Class gt Scatter Plots Select one of the spot value types Intensity Vol Area lntensity Vol Vol Ratio to be plotted ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 197 Data analysis 198 NOTE To show the correlation between the spot quantities in the gels the Intensity Vol or Vol values are most indicated both for conventional 2 DE gels and DIGE gels The Scatter Plots window shows a scatter plot for each selected gel versus the specified reference gel together with the best fit line correlation coefficient and the number of matches displayed If only one gel is selected other than the reference gel a single scatter plot is drawn This scatter plot is completely resizable when the Scatter Plots window is in floating mode You can drag the corners or borders of the window to make it smaller or larger Selection of two or more gels other than the reference gel displays two or more scatter pl
142. d on a drive It contains the raw input data from which proteins can be detected and quantified The proteins represent the gray values for all the pixels in the image Every pixel is characterized by its X and Y coordinates which represent the horizontal and vertical positions of the pixel on the image The pixel s raw value or gray value is the signal intensity of the pixel Z axis in a three dimensional view The software recognizes gels from several image formats but the gels must be saved in the ImageMaster file format mel in order to store data about spots matches annotations etc If a gel is exported with its objects into a foreign format then the data simply becomes part of the saved image and loses any structure or value Gels can be duplicated deleted cropped filtered flipped or scaled Gels can also be aligned meaning that they are distorted in order to superimpose their image with another gel Various items can be defined on a gel for detailed examination These are spots regions annotations and labels Figure 2 2 Matches match sets and classes are entities that give structure to the relationships between gels Figure 2 3 and will be discussed in the following section Annotations Spots Figure 2 2 Items on a gel region spots annotations and labels Regions A region is a rectangular area of a gel that is defined using the Region tool When a region is set certain actions may be limited to i
143. dapt the zoom factor to decrease the size of the printed image To print gels or selected gel regions 1 To print only regions start by drawing them with the Region tool Define a cropped region in each gel or use the Shift key to select the same region in all open gels 2 Select the gels that you would like to print 3 Choose File gt Print gt Images from the menu 4 Ifa region was selected ImageMaster asks whether you want to print only the selected area Answer Yes or No 5 The selected gels or gel regions are printed To print the active worksheet 1 Choose File gt Print gt Worksheet in the menu 2 The image of the selected worksheet is sent to the printer You can change printing parameters such as printer name paper size paper orientation etc To do so choose File gt Print gt Page Setup from the menu This command calls the standard print window where printer related settings can be modified 126 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 7 Spots 7 1 Introduction The elementary component of a gel is the Spot which delineates a relatively tiny region in the gel where protein is present This shape is automatically differentiated by a spot detection algorithm Each spot in an image is assigned a unique number when the software identifies it called the Spot ID Moreover a spot is quantified its intensity area and volume are computed The ultimate aim of defining spots is
144. define the same region on all gels in the active worksheet hold down the Shift key while drawing the box on one of the gels This region is defined in the other gels based on the corresponding pixels 6 3 Displaying gels 6 3 1 Moving gels There are several ways to change positions in gels You can use the Hand tool Show menu options shortcut keys or scrollbars see Section 6 3 3 You can also double click on a gel to move all gels in the active worksheet to the same position Hand tool The Hand tool is the most straightforward means of moving gels To move a gel 1 Click on the Hand tool in the toolbar 2 Click on the gel and hold down the left mouse button while moving the cursor The image changes position 3 Release the mouse button at the position you want In order to move all gels in the active worksheet by the same displacement hold down the Shift key while changing the position in one of the gels Double click In order to move all gels in the active worksheet to the same position with the same magnification select the Hand tool if not already done and double click on one of the gels The corresponding position in the different gels is estimated by interpolating between the surrounding matches or if such matches do not exist between the two nearest common landmarks i e landmarks with the same name Finally when no such landmarks exist the gels are aligned at the same location using the X and Y coordinates Sho
145. den by some other annotations b Same annotation displayed in front of all other annotations after selection To select all annotations in a region position the cursor at the top left position of the desired region hold down the left mouse button and then drag the cursor to the bottom right position All annotations in the selected region are selected and highlighted in green even those that are hidden To select annotations in more than one region hold down the Shift key while selecting additional regions To deselect all annotations select the gels for which you would like to deactivate all annotations and click on a gel not on an annotation 8 4 2 Select menu In order to select all spots and annotations in the selected gel choose Select gt Select All in the menu or Select gt Unselect All to deselect all spots and annotations You can select specific labels and annotations with the options in the Select gt Annotations menu e By Content This feature enables the selection of labels belonging to one or several categories that must be selected based on their content When the Regular Expression box in the window is not checked the entered string of characters is taken literally and the program selects all labels containing this string By activating the Regular Expression option regular expressions can be used in the search field see Section 8 4 2 for details For example to select all labels containing the string POO
146. deselect the Adaptive Gradations check box at the bottom of the Histograms window Figure 10 6 10 10 a b Figure 10 6 Histograms on matches with a adaptive gradations set individually for each histogram and b set according to the minimum and maximum values in all histograms ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis Sort values Additionally to highlight and classify spot variations you can display the spot values sorted in ascending order by clicking in the Sorted Values check box at the bottom of the Histograms window Figure 10 7 a b Figure 10 7 Histograms on matches with a unsorted values and b with values sorted in ascending order Sort by similarity ImageMaster provides an easy way to look for similar histograms in the Intra Class Histograms window Select one histogram and click on the Sort by Similarity icon in the toolbar Figure 10 5 A new histogram window is displayed in which the selected histogram is depicted in the first position and the other histograms follow according to a similarity criterion This similarity criterion is based on the sum of the squared spot value differences for each gel G The general formula is X 2 Similarity gt vi v G 1 Where v is the spot value for match i vj is the spot value for match j and N is the number of gels displayed in the histogram The result generally is that histograms with similar shapes appear
147. dit Enabled is no longer checked in the menu He tb Figure 7 4 Spot tool when a spot editing is not activated and b spot editing is activated NOTE You are still able to select spots while spot editing is activated Nevertheless we recommend you disable the Edit Enabled option whenever possible in order to prevent accidental modifications to your spots Increase the size of a spot Outline the area that you would like to add by holding down the left mouse button while starting at one point on the selected spot and moving to another point within the spot Figure 7 5 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 139 140 Figure 7 5 Increasing the size of a spot The green line defines the portion of the spot that will be added the numbers indicate the successive positions of the mouse cursor The plus sign next to the pen indicates that one is in the adding mode and the pen must start to draw from a point within the spot border Delete part of a spot In order to erase part of a spot you should also use the left mouse button but now start from outside the initial spot outline and then partially encircle the area of the spot that you would like to suppress Figure 7 6 Please note that the portion of the spot that contains the center of gravity in the initial spot will be conserved Figure 7 6 Deleting part of a spot The red line outlines the portion of
148. e The Comments field in the Experiment Header Information is converted to a Comment for the project Gel images The gel images originally in tiff gel or img format are treated as 16 bit TIFF files and inserted in the newly created project folder In the case of an img file an ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data integration accompanying inf file should be present in the experiment folder When the inf file is missing a warning message is displayed and import is aborted In that case try creating a valid inf file resolution and gel size information must be Known and do the import of the experiment again If the images were initially calibrated the calibration is also applied to the converted gel images Similarly if the Invert Intensities option in the Elite Experiment was turned on the TIFF image is inverted upon conversion of the gel If a master gel was defined in the original experiment this status is also maintained in the new master file tagged in red The Name Title Comments and User Defined Fields in the Gel Header box of the Elite Experiment are transferred to Gel Properties in the ImageMaster 2D Platinum files Average gels Inthe ImageMaster 2D Platinum project an average gel from the Elite Experiment becomes the master of a match set that contains the gels that were composing it Spots Converted spots have spot shapes and quantification values extracted from the experiment f
149. e sample specified by the percentage slider used to suppress outliers from the calculated statistics see Section 10 2 2 The Separability is the highest difference between two consecutively sorted values in the whole sample It measures the greatest gap that you can have if you want to split the spot values in a match into two separate classes To display an Intra Class Report 1 Select a given number of matches possibly all to be studied 2 Select the gels to be included in the histograms The gels can belong to different classes but the class information will be ignored for intra class statistics 3 Choose Analyze gt Intra Class gt Report 4 Inthe pop up list select the value type Intensity Area Vol to be displayed 5 Choose the desired statistics central tendency and dispersion values see Section 10 2 2 and the additional values to be calculated in the subsequent dialog box Figure 10 10 shows a typical Intra Class Report The sliders at the bottom and right side of the window allow you to see the rest of the table The Match ID is followed by the desired statistical values the coefficient of variation optional the 206 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA range ratio optional the separability optional and finally the spot values from each gel These spot values can be normalized as described for Section 10 2 3 in order to simplify the comparison between matches To do this j
150. e MatchSet Creates a new match set that will include the selected DIGE gels as sub match sets The name of the DIGE gel is input as the default Name e Delete Removes the selected gels from the folder Please note that it does not remove the file from your hard disk The DIGE gel is only deleted from the active project e Copy Copies the selected gels into memory You can then paste them into other folders of your workspace e Properties Displays the DIGE properties Name Dye Chemistry and Comment DIGE gel image e Open gt In Worksheet Opens the selected DIGE images in the active Gels worksheet in the Display Zone You can also double click on a file to open it in the active Gels worksheet e Open gt In New Worksheet Opens the selected DIGE images in a new Gels worksheet in the Display Zone e Histogram Shows data associated with detected spots in two selected images from the same DIGE gel Spot data intensity area volume or slope is plotted against log volume ratio The blue curve represents the frequency distribution of the log volume ratios ee ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 261 Appendix 262 Add in Matchset Adds the selected DIGE images in an existing match set or in a new one If you would like to create a new match set enter a new name Otherwise you can choose from the other match sets available in the proposed list Search Searches for the gel on the hard disk in the ind
151. e The frequency i e counts for each bin is displayed on the vertical axis e The Cumulative Histogram is a variation of the histogram in which the vertical axis gives not just the counts for a single bin but rather gives the counts for that bin plus all bins for smaller values of the response variable Yellow guides move with your cursor and indicate at the bottom of the window the frequency Y and the response variable x for each bin The red vertical line represents the mean value Figure 7 11 Measure Histogram SSS ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 147 148 7 10 2 DIGE histogram A DIGE Histogram can be displayed when two co detected images are selected Figure 7 12 It shows data associated with detected spots in the selected images Spot data is plotted against log volume ratio on the X axis using two Y axes e The left Y axis displays the spot frequency The blue curve represents the frequency distribution of the log volume ratios e The right Y axis represents the Measure parameter see below selected from the corresponding drop down menu in the toolbar of the DIGE Histogram window A plotted single data point on the histogram represents an individual protein spot DIGE Histogram E x Measure Max Intensity Gels Gel01 Spike1 Cy3 Gel01 Spike Oy5 fumber of Spots Max Intensity 10 20 18 16 l4 12 10 08 06 04 02 0 02 0 4 06 0 8 10 Log Volume Ratio Figure 7
152. e as above Value Relative Ratio or Normalized You can use these values to select spots that are outliers or on the contrary to select spots that are representative of a match Representative spots In order to select the most representative spots of a match one could for example pick out the spots whose values are close to the mean with less than 20 difference To do this choose the Mean 100 as central tendency and then select all spots that have a Ratio Displayed value list between 0 8 and 1 2 Another possibility is to choose spots with a Median Statistics Ratio Displayed value list of 1 which will select the spots that are in the middle of the match Outliers Outliers can be selected by choosing values that are outside the range given by the mean two times the standard deviation In fact with a normal distribution at most 5 of the values should be outside this interval In practice you can find such outliers by using the Mean 100 and Mean Squared Deviation 100 and then choosing all spots with a Normalized value Displayed value list higher than 2 When you calculate the median and choose Ratio values Displayed value list larger than 2 you select spots that are at least 2 times higher or lower than half of the spots from the match The Midrange 100 and Half range 100 statistics combined with a Normalized value Displayed value list of 1 allow you to select spots that are the highest or th
153. e based on a simple interpolation between the two nearest common landmarks landmarks with the same name Otherwise when the gels are not aligned and have no common landmarks the operations are based on the same image location 6 4 Viewing signal intensity 6 4 1 Loading unloading gels The digitized gel is composed of individual pixels each of which is characterized by its horizontal and vertical positions X and Y coordinates and its signal intensity raw pixel value The depth of an image see Section 6 1 3 determines the number of possible pixel gray values However no matter what the original image depth ImageMaster remaps an image to 256 gray levels for display In the normal view of an image raw pixel values do not need to be stored in memory This corresponds to the default unloaded mode in ImageMaster For some operations such as spot detection adjustment of the contrast mapping and displaying the Profile ImageMaster relies on the raw image data The program automatically loads the pixel gray values before performing such an operation When the operation is done ImageMaster generally unloads the raw data in order to free up memory ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA NOTE The raw image data must be manually loaded in order to display pixel values in the Cursor Information window The Loaded mode displays the original pixel gray values for the image When your computer has limited memory i
154. e by specifying additional columns for ranking Note that if you would like to sort your data by two or more criteria you should sort by the least important columns first and finish with your principal criterion In Figure 5 4 the principal criterion is the Spot ID of the master gel Match ID and the secondary criterion is the spot Volume gla Bo a s e all 2 B wl Information on selected Spots FileName Spot D M SpotD Vol Intensity Area vol intensity 92 A_T1_Gel2 A_T2_Gel3 3736 5174 4914 4132 62 1134 00 4914 85 2900 627 000 10 3100 0 0809608 0 130000 0 00178785 0 0770059 0 0370558 0 145559 0 177300 Figure 5 4 Rows in a Spot Report sorted by two criteria Spots were first sorted according to their Volume in descending order then according to their Match ID 5 5 Mouse selection reports Mouse selection reports Figure 5 5 are similar to any standard report in terms of functionality icons etc The main difference is that they are interactive The reports contain only the items you select with the mouse while using the Spot or Annotation tools You can select several items at a time by holding down the Shift or Ctrl key but when deselecting them they immediately disappear from the report ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Reports 5 EN imageMaster 2D Platinum loj x File View Edit Show Select An
155. e contextual menu 3 Inthe Create Folder box specify a Name and Comment for the new folder and click OK 4 The Folder Name appears in the Reports folder Right click on the Reports folder a subfolder or report file to open a contextual menu from which you choose an action to be carried out Detailed descriptions of all the Reports folder menus are provided in Appendix A 4 8 2 Adding reports When saving a report it is automatically inserted into the Reports folder of the Project it has been created from However you can also add report files from disk To add reports 1 Right click on the Reports folder or a subfolder 2 Choose Add Reports from the contextual menu A ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 59 3 Inthe Add Report Files box browse the directory where the files are located select the names and click Open Use the Shift or Ctrl keys to make multiple selections 4 The files are added to the Reports folder or subfolder 4 8 3 Opening reports You can open a report from the Reports folder by double clicking on it or by selecting Open from the contextual menu 4 8 4 Moving reports or folders Move folders subfolders and files within the Reports folder by dragging and dropping or copying and pasting 4 9 Working with documents The Documents folder manages all project related files other than your gel images and tabular reports These can include text files spreadsheet files Microsoft PowerPoi
156. e created on the hard disk and inserted into the current project The new gel images contain only the parts of the gels that lie within the specified regions Any spots and annotations that were present in the saved region on the original gels are maintained You can also export and import a Selection Box to ensure that the final size of your cropped gels is identical even between work sessions A Selection box is a region with an anchor attached to it You position the anchor on an easily recognizable protein spot and as the region moves with the anchor you will always crop a similar part of the gel To export a Selection Box 1 With the Region tool define a region on a gel that you would typically like to crop While holding the Alt key click on a characteristic spot that can easily be found in all gel images An anchor appears on the clicked position Note that this anchor may be located inside or outside the gel region If you are not satisfied with the position of the anchor you can redefine it by pressing Alt again while clicking on a new location You can also remove the entire Selection Box by double clicking in the gel image Select the gel in which you defined the Selection Box Choose File gt Export gt Selection Box to save the Selection Box to a file with the extension cpt Crop Tool Enter a destination folder and file name Click on Save ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA a
157. e flagpoles of the selected annotations are displayed with the chosen color after deselecting the spots 4 The colors remain displayed this way until you choose Show gt Annotations gt Set Color gt Reset All to revert all selected annotations to the default cyan or until you exit ImageMaster 85 4 Show hide annotations and labels You can quickly cover entire gel images with a considerable number of annotations and labels which are not always necessary at any given moment in the analysis Therefore you can hide all or part of them ImageMaster not only gives you the possibility to hide certain label categories the program also allows you to hide certain selected annotations Figure 8 7 Note that the visibility of an annotation or label plays a role in its selection As a rule annotations and labels can only be selected and manipulated with the Annotation tool when they are visible on the gels To select hidden annotations or labels the menu options should be used To show or hide all annotations and spots in the selected gels choose Show gt Show All or Show gt Hide All in the menu Hide All also hides the match vectors To show or hide specific annotations you can use one of the following options available in the Show gt Annotations menu e Show All Makes all the annotations on the selected gels visible e Hide All Hides all the annotations on the selected gels ImageMaster 2D Platinum User Manual 11 0034 38 Edi
158. e lowest in their match ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA e The Midrange 50 and Half range 50 combined with a Normalized value Displayed value list superior to 2 will select spots that are at least 2 times higher or lower than 75 of the spots from the match NOTE In order to restrict the number of selected matches you can remove the matches that contain too many outliers You can also use this functionality to search for unusual gels If a gel contains more outliers than the others it may be atypical or abnormal You should determine the reasons for this abnormality before continuing the analysis 10 2 5 Factor analysis The visual task of comparing gels is rather difficult when dealing with a large number of gels that consist of thousands of spots In particular it can be hard to assess whether different sample populations exist and to characterize their different protein expression profiles Factor analysis helps here by finding a way to condense the information contained in such huge data sets into a smaller number of factors or dimensions that explain most of the variance observed The factor analysis tool is used to examine the interrelationships between large numbers of variables that is spot values for a series of gels and to explain these relationships for example gel populations in terms of common underlying factors or associations with specific spot patterns Factor analysi
159. e menu 2 Enter your personal details in the Customer Information box Please note that this data is used to update our customer database In this way we can contact you with certainty when it is time to send new patches or installation files 3 Inthe Online Support box specify whether you want to ask a question make a suggestion or report a bug Depending on your choice the software asks you subsequent questions that help us to supply you with an appropriate answer 4 Click on the E mail button A new message is created with your default e mail software automatically directed to imagemaster support ge com Highlight the lt Paste clipboard gt text with your mouse and then Edit gt Paste the contents of your clipboard into the mail message The e mail now contains your questions and contact information as well as useful data about your ImageMaster version work session and computer system If the problem is file specific then attach any relevant files Send us this message Alternatively you can print the clipboard contents and send it to us by fax 1 4 3 System and product information To quickly obtain product and system information for your own benefit or during a technical support call choose Help gt System amp Product Information from the menu A window shows the useful data about your ImageMaster version and computer system You can also get information about your ImageMaster version by looking at the release number g
160. e note that the 1 1 or 100 zoom is recommended when capturing an image with a scanner Shrinking the image often leads to a loss of quality or the ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 79 ED 80 inability to resolve data Increasing the size of the image is useless because it does not add any information Camera based systems do not capture at a set scale When using such systems you generally end up with different resolutions in the final images Although this is not a problem for ImageMaster working with identical resolutions certainly facilitates image manipulation for example zooming or alignment 6 1 3 Image depth The range of potential gray levels in an image varies according to the image depth In the case of an 8 bit image one pixel has 256 possible gray values 0 to 255 Images scanned with a higher image depth contain more information A 16 bit image 65536 gray levels will reveal more subtleties We strongly recommend an image depth of at least 12 bits for gel analysis When carrying out an intensity calibration the raw pixel values are converted into real world units generally optical density or OD This means that the range of gray values is the same no matter what the original image depth Some scanning devices produce images that are already calibrated ImageMaster takes into account the conversion tables or calibration formulas stored in the files exported by such devices If necessa
161. e other match sets available in the proposed list e Delete Removes the selected gels from the folder Please note that it does not remove the file from your hard disk The image is only deleted from the active project e Search Searches for the gel on the hard disk in the indicated directory when a gel with the appropriate ID is not found at the usual location This is the case when a red mark covers the gel icon generally indicating that you moved the original gel file or replaced it with another one e Cut Cuts the selected gels in order to paste them e Properties Displays the gel properties Gel Name and Staining 260 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Appendix SSS A 2 4 DIGE Gels folder e Add DIGE Gel Adds DIGE images that are already in ImageMaster format with the extension mel to define a DIGE gel e Import DIGE Gel Adds DIGE images that are not already in ImageMaster format to define a DIGE gel e Paste Pastes previously cut or copied items into the DIGE Gels folder DIGE gel e Open gt In Worksheet Opens the selected DIGE gels in the active Gels worksheet in the Display Zone You can also double click on a file to open it in the active worksheet e Open gt In New Worksheet Opens the selected gels in a new worksheet in the Display Zone e Detect Performs spot detection on the selected DIGE gels You are asked to estimate the Number of Spots to be found in the gels e Creat
162. e that the classical reports in ImageMaster contain particular data types and to export the comprehensive information of an analysis set the creation of several reports would often be necessary This is why ImageMaster offers an additional feature for exporting gel data 11 2 3 Export gel data ImageMaster allows the export of all gel related data except for the gel image itself into a single XML file This XML file can include all available information on a set of gels together with spots shape quantification aligned coordinates annotations and match information Figure 11 1 To export gel data to an XML file 1 Select all objects gels spots annotations that should be exported to an XML file No preliminary selection is necessary if all gels in the active worksheet should be exported potentially with all their spot annotation and match data 2 Choose File gt Export gt Gel Data to XML in the menu ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data integration ss 3 Select the data to be exported from the Export Gel Data to XML File dialog See below for more details about the items that can be exported 4 If objects are selected ImageMaster asks whether you want to save only the selected objects or not If you answer Yes just the selected objects are exported No means that you want to export all information on all open gels 5 Enter a name and destination folder for the file to be g
163. e ugeigeh 13 33 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA worksheets close rename select workspace archive backup classes create documents gel icons gels folder hierarchy matchsets navigator open properties reports restore save structure toolbar window X SAMLE O A1 ALOI AALTEN AE TEE NENIET IET EAEE AAE At Rha tet MN a ea 237 Aslistylesheet ann eltern EEE EEEE ET 237 Z ZOOM WINGOWE ars ih ea en ea S a Be Ne A ee 85 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Index 287 Index 288 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA This version of ImageMaster has been developed by the Swiss Institute of Bioinformatics in collaboration with GeneBio and GE Healthcare All intellectual property rights on this User Manual as well as on the ImageMaster 2D Platinum software belong to the Swiss Institute of Bioinformatics No part of this User Manual may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopy recording or any information storage or retrieval system without permission in writing from the Swiss Institute of Bioinformatics 1992 2005 Swiss Institute of Bioinformatics All rights reserved Swiss Institute of Bioinformatics CMU Rue Michel Servet 1 CH 1211 Geneva Switzerland ImageMaster 2D Platinum provides access to several databases on the Inte
164. e used and what precautions should be taken into account However keep in mind that the results are generally only indicative and should always be checked carefully ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 195 Data analysis 196 NOTE For all data analysis functionalities described in this chapter you must choose the spot quantification value on which the calculations should be based You can choose between Intensity Area Volume lntensity and Volume In addition the Saliency is available for gels detected with the ImageMaster algorithm and the Volume Ratio and Slope for spots detected with the DeCyder co detection algorithm For conventional 2 DE gels the Volume or Volume are generally indicated For DIGE gels the Volume Ratio should be selected in order to use the Internal Standard 10 2 Intra class statistics 10 2 1 Scatter plots In order to analyze gel similarities or experimental variations such as disparities in stain intensity or sample loading one can produce Scatter Plots for matched spots Figure 10 1 Scatter plots give an idea of the relationship between the spot values for example Intensity Vol and Vol from two gels by searching for the linear dependence between the spot values of one gel variable X and the corresponding values in a reference gel variable Y Select on Gels Reports Scatter Plot Scatter Plots i x alal 3 m Scatter Plots Intensity
165. eCyder software development team 7 2 SpotID Each spot in a gel has a unique identifier called the Spot ID Spot IDs of deleted spots are not reused ImageMaster attributes a new ID to each new spot When a spot is split the child spot for which the coordinates are closest to the parent spot keeps the existing spot ID the other child spot gets a new ID When two spots are merged the resulting spot is attributed the ID of the initial spot that was closest to the new center of gravity ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 127 128 Through the combination of Spot ID and Gel ID every spot in any of your experiments is therefore uniquely identified thus preventing any confusion Be aware that when you edit a spot making it smaller or bigger after having generated a report the spot values in the existing report are not updated and are therefore incorrect 7 3 Detecting spots in non DIGE gels The Melanie ImageMaster spot detection algorithm is optimized to give relevant biological results with minimum user interaction Only a few easy to use parameters need to be defined in order to automatically locate spots in your image NOTE When you look at a detected spot in the 3D View see Section 6 4 6 you will notice that the borders are not localized at the base of the spot especially for intense spots This is normal because the spot contours displayed effectively reflect the quantified parts of the spot and
166. eMaster provides various reports including specific information about annotations labels and categories Intra Class and Inter Class Reports can display the content of annotations and labels attached to spots in the master gel As pointed out in Chapter 5 these reports are editable although to different extents In Section 8 6 3 you will find that the editing feature is particularly useful for adding or modifying annotations and labels 8 7 1 Label report The Label Report displays information about selected labels such as the gel name category label content Spot ID and X and Y coordinates Figure 8 10 The Label column in this report is editable thus giving you the possibility to modify the existing label contents Label Report xj uls Blo a l le el Information on selected labels FileName Category Label 94 0002 AC P13009 94 0002 ProteinName RPOB_ECOLI 94 0002 Ac P00575 ECOLI Landmark Pi 1 00 Mw 240000 ECOLI pI_MW 1 00 240000 Figure 8 10 Report on selected labels in selected gels ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Annotations 3 8 7 2 Annotation report The Annotation Report displays information on selected annotations in selected gels Thus for each annotation you will find the gel name Spot ID X and Y coordinates and the label contents for all chosen categories Figure 8 11 You can modify the label contents for each category or even add n
167. ecedence from high to low for regular expressions Combining sets of annotations You can select annotations based on multiple criteria You can do this by combining sets of annotations in particular ways You can select a first set of ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 165 8 Annotations 166 annotations on your gels and then combine this set with a second set according to one the following schemes see also Figure 8 5 e Intersection Selects spots that are in the intersection of two criteria e Union Selects spots that belong to either one criterion or the other e Difference Selects spots that belong to either of the two criteria but excludes those that belong to both of them e Exclusion Selects spots that are found only in the first criterion and are not found in the second criterion The possibility of combining sets of annotations is particularly useful when you have different sets defined Imagine for example that you marked interesting spots found from Factor Analysis with labels of the category Set Factor Analysis Similarly you marked significant spots emerging from a Student t test with labels of the category Set Student t test You could now select all spots from the first set by choosing Select gt Annotations gt By Category from the menu and picking Set Factor Analysis from the Select Labels by Category box When you subsequently choose Select gt Annotations gt Combine gt Intersect
168. ed in a MatchSet worksheet ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace When opening a match set with gels in it all gels are displayed except for the Reference The Master of the match set recognizable through its red legend is shown instead This is because the master and reference images are the same and matches are automatically created between the two The Reference therefore no longer needs to be matched When necessary the reference gel can be displayed by choosing Show gt Show Reference from the menu When opening a match set with submatch sets in it all master gels of the submatch sets will be displayed except for the one that was chosen as a reference for the opened match set Instead the master image of the opened match set is shown To open a match set 1 Select the match set to be opened Right click on its name 2 Choose Open from the contextual menu to open the match set 3 The match set is opened in a new MatchSet worksheet in the Display Zone 4 6 8 Matching Once a match set is opened in the Display Zone you are ready to match the gels against the Master See Chapter 9 to learn more about matching Note that the gel or match set icons change when matching is completed 4 6 9 Match set properties The Name and Comment are properties of match sets The Comment describes and or explains the content of the match set It serves as a useful source of information to which a coworker is
169. egion is lost e Synchronize all 3D Views is used to propagate the orientation for example the Up Rot and Zoom parameters of the current view to all other 3D views You can perform the same action by holding down the Shift key while adjusting the center point of the axes in one of the gels by right clicking e Spot Shape sets the way the spots are displayed e Display axes can remove the axes from the 3D View The last icon Adjust Contrast enables you to quickly change the gray level setting of the peaks NOTE An interactive version of 3D View is available from the Window gt Mouse Selection menu Its functionality is much the same as the standard 3D View but it interactively displays the third dimension for the current mouse selection region or spots When you select a new spot on your gel the 3D view as well as that of matched spots in other selected gels automatically displays When you deselect the spot its 3D view immediately disappears from the report 6 5 Calibrating and normalizing gels ImageMaster offers several possibilities to compensate for image differences caused by variations in experimental conditions for example protein loading or staining and scanning properties such as image depth This section describes the available features The terms normalization and calibration as used within ImageMaster are also clarified Please note that you need to master the notions of spots and matches to full
170. egory 1 Select the gels for which you would like to rename a category 2 Choose Edit gt Annotations gt Rename Category in the menu 3 Inthe displayed category list select the one you would like to rename 4 Enter the new category name in the next dialog box 5 ImageMaster immediately applies the modifications to all labels on the selected gels ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 173 8 Annotations 174 Deleting annotations and labels Two possibilities are available from the Edit gt Annotations menu for deleting selected labels or annotations e Delete by Category With this feature only the selected labels belonging to the categories specified from the category list are deleted e Delete This option deletes all selected labels Please note that if you select annotations all their labels are selected and therefore deleted 8 6 3 Reports This section describes the functionalities in ImageMaster that are helpful for editing annotation and label content in reports that contain information on these objects see Section 8 7 Please see Chapter 5 for details about other standard tools available in the various report toolbars including icons to save print or copy reports or report lines to navigate between reports and gels to refine report selections and so on Creating annotation categories Annotation Reports Intra class and Inter class Reports contain the Annotate icon in their toolbar Clicking
171. el properties The Gel Name and Staining are Properties of any gel image file You can modify the Staining while viewing the Gel Properties ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace 45 46 To edit the Staining 1 Right click on the image name 2 Choose Properties from the contextual menu 3 Inthe Gel Properties box modify the Staining Click OK 4 The changes to Properties are saved The Name Dye Chemistry and Comment are Properties of DIGE gels They can similarly be edited by right clicking on a DIGE gel not the individual images and choosing Properties from the contextual menu 4 6 Working with match sets The MatchSets Gal folder manages your match structures Figure 4 4 You can easily e Create an unlimited number of match sets and submatch sets enabling you to match individual gels or gel populations e Define a reference gel or reference match set that serves as the Master for the matching e Opena match set in a new worksheet to carry out matching e See which gels or match sets have already been matched e Use your match sets to create classes A match set includes gels or populations of gels in submatch sets that should be matched together This is a major innovation over previous versions of the software You are no longer limited to matching gels You can now match populations of gels and what more is different levels of populations This is called population matching
172. elative volume higher than 0 02 In other words you would like to analyze spots according to two criteria First the spots should have a relative volume higher than a certain threshold in order to select spots that are meaningful in terms of volume Second these spots should be present in at least half of the selected gels You may select this special set of spots by performing the following steps To filter spots in matched gels 1 Select your six matched gels Please note that the master gel will not be included in the refinement The reference image must be made visible to be included in the selection choose Show gt Show Reference 2 Select all spots in the gels by choosing Select gt Spots gt All from the menu 3 Subsequently refine the current spot selection based on the relative spot volume by choosing Select gt Spots gt Refine by Value from the menu picking the Vol and keeping only the spots whose Vol is gt 0 02 4 Spots corresponding to this first criterion remain selected 5 Choose Select gt Matches gt Refine Selection from the menu and Keep the Matches whose Number of Selected Spots is gt 3 by clicking the first option and moving the slider to the value 3 6 The remaining matches meet both specified criteria As you may notice there are endless possibilities for combining selections of spots and matches Your own experiments and needs will drive the usage of this function Multiple matches
173. en detected see Chapter 7 the visible spots of the specified reference gel are displayed in blue on the image Figure 6 8 Thus you can easily compare the position and size of the red spots in the current gels with the blue spots from the reference gel a a A lv Figure 6 8 Spots overlapped with a outlined spot shapes and b filled spot shapes selected spots in the front gel and pair vectors are displayed ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 89 ED To activate the Transparency mode in the active worksheet 1 Choose Show gt Gels gt Transparency gt Show in the menu 2 Inthe Transparency Settings window specify the reference gel check uncheck the desired options and click OK 3 ImageMaster displays your gels with the chosen options NOTE You can first activate the Transparency mode and then align your gels with respect to the reference gel You can even add landmarks in the Transparency mode Nevertheless it is recommended to perform any operations related to gel alignment especially the definition of landmarks before entering the Transparency mode to avoid a slow down due to the recalculation of the overlaid images You can change the default color to be used for displaying the spots from the specified reference gel Overlapped spots You can do this by going to the Display tab in Tools gt Options 6 3 5 Grid lines Display Grid Lines over selected gels to
174. enerated 6 ImageMaster creates a file on the hard disk that can be read by other applications You can also view the contents of the file with your browser Choose data to be exported r Gels Descriptions Pixel Annotations M Spots Quantification i Annotations 7 pl MW V xY align I Shape M Matches Figure 11 1 Export Gel Data to XML File dialog box The following items can be exported to the XML file Gels Minimal exported items for gels are gel IDs gel names and summarized information about gels as present in the Gel Report This includes image height and width in pixels pixel dimensions of the scanned image minimum and maximum gray levels raw and calibrated intensity calibration parameters number of spots number of annotations gel class and match set assignments In addition the following data can be added to the exported file e Descriptions User defined gel descriptions as entered in the Gel Description Report e Pixel Annotations Exports all annotations placed on individual gel pixels Sasa ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 239 Data integration Spots Minimal exported properties for spots are X and Y coordinates on the gel and Spot ID In addition the following data can be added to the exported file Quantification All quantification values as appearing in the Spots Report Annotations Annotations linked to spots pl MW Calculated pl and MW values if available X Y a
175. ent gels when they bear identical labels of the same category e pl_MW green This category contains the known isoelectric point pl and molecular weight MW information which is subsequently used to compute approximate pl and MW values for any point in a gel You should enter the pl value first and MW value second separated by a space By replacing one of the values by 1 you indicate to the program that no value is set probably because you do not know it e Comment pale pink This category is an example of a general category where users may store their comments User comments can also be entered in any other user defined category see Section 8 3 1 e Set yellow This is a generic category used to mark spots having common characteristics All labels from a Set category share the same text or in other words the same set name Figure 8 1 exemplifies a set called Pick containing spots that need to be exported to a spot excision robot See Section 8 3 2 for more details Please note that the default colors for the different label categories can be changed You can do this by going to the Categories tab in the ImageMaster Options accessible by choosing Tools gt Options in the menu 83 Creating annotations and labels You can create two types of annotations The first one visualized with a cross basis is simply connected to a pixel and has the same coordinates as that pixel The second type of annotation concerns tho
176. ent labels from the same category You can do this by linking additional annotations to the spot To add a label to an existing annotation 1 2 Click on the Annotation tool in the toolbar Double click at the basis of the annotation to which your label should be added In the Create Annotation by Click box enter the name of a new category or choose one of the existing categories by clicking on its name When a new category is created the Create Category window will appear Please see Section 8 3 1 to find out more about the parameters requested Type the content of the label in the next dialog box and click OK The label is added to the existing annotation ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Annotations 3 153 8 Annotations 154 NOTE Alternatively you can add labels by selecting annotations or spots and choosing Edit gt Annotations gt Add Label in the menu This option is particularly useful when you want to add identical labels to a series of spots or annotations because you can create many labels at a time instead of having to define each one individually At any time ImageMaster allows you to link an annotation to a spot or do the opposite This is helpful because sometimes you may miss a spot to which you wanted to link a newly created annotation or you may have decided that an annotation should not be linked to a given spot To link an annotation with a spot clic
177. enu appears containing the main options used to work with images panes or worksheets If you right click directly on a visible gel image then another contextual menu offers the possibility to edit show or select objects like gels spots annotations and matches These short menus duplicate some of the commands found in the menu bar but are ordered differently and are quicker to access at any time A list of all contextual menus is given in Appendix A Status bar The status bar is an important resource because it indicates the total number of gels spots and annotations that are currently selected in the active worksheet 3 4 4 Dockable windows The workspace and reports are displayed in dockable windows that can be set according to your preferred style of working with numerous windows at a time Dockable windows can be docked i e fixed in place against the left right top or bottom edges of the ImageMaster window and always lie on top when visible A visible window can be in Dockable pinned Auto Hide un pinned or Floating mode Dockable pinned The pinned mode i amp d enables dockable windows to be locked into position around the edges of the ImageMaster window Once a window is pinned you can move it to a different location by dragging the title bar Figure 3 5 ImageMaster displays blue arrow guides to indicate where the window may be docked By ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Getting started
178. eports in the selected folder Properties View or edit the Name or Comment for the folder Report Open Opens the selected reports in dockable windows You can also double click on a report file to open it Delete Removes the selected reports from the folder Please note that it does not remove the file from your hard disk The report is only deleted from the active project ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Appendix SSS Sy e Search Searches for the report on the hard disk in the indicated directory when a report with the appropriate ID is not found at the usual location This is the case when a red mark covers the report icon generally indicating that you moved the original report file or replaced it with another one e Copy Copies the selected reports into memory e Paste Pastes the previously cut or copied items below the selected report e Properties Displays the report properties Path Type Message and Comment The Type item tells you whether your report is a Spot Report Gel Report Match Report etc The Message gives information on the spot value or statistics that were used to generate the report or other data depending on the report type The Comment field displays the report comment that was defined by the user A 2 8 Documents folder e Open Opens all the files contained in the Documents folder with the appropriate software depending on the extensions As in Windows Explorer f
179. er gels ccssssssssssssssssssssssssssssessesssssesesesssssssssssssnssssses 12 Safety control and automation VAT lhtrod ction tern seele 12 1 1 Action descriptors i 124 2 Displ ayed actionsasauasasehaaene na 12 1 3 History and script Navigators eeeeeseesssssssssscsccscsscscseccccessssssssssssssssssssanenseee 12 1 4 Gel selecti Aruaia aari 12 2 Undod teda cessano user asccanctettcacel anna 12 3 History 12 4 Script A Appendix AT SC nt xtu l MENUS sospes ke A 2 Workspace contextual menus oecceccccccsssssssssssesesssccscssssssssssnssesseesessesssses A 2 1 W rkspace NOME sisin aee R A ty A 2 2 Project name A 2 3 Gels folder A 2 4 DIGE Gels folder R A 2 5 M tehSets folder unse nt A 2 6 Cl ssestolden aunenn E E RE A 2 7 Reports folder A A 2 8 DOCUMENTS Tolder au unanenisainansiennneimaannanae A 3 Display zone contextual MENUS oieseecccsssssssssseesssssccccsssssssssseeesssseesesssees A 3 1 Worksheet pane or legend A 3 2 IMAGE antreten keins AEEA NE B Appendix BL Shorte t Kelsas mn O aa BE T Gel Sshorleulsmetee ee een BS SPOLShOHIENS I EOAR OE AEA B4 Annotation shofteutszu eeeesnnne ine Bide M teh sh rteuts 2a een ann C Appendix C 1 Software references ananeeeuseeennnneennnnnesnununnennnunnenununnenennn C 2 Statistical methods asasscassssasseeeeneeneeen C3 PURE SP SACHIN nannten ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 26
180. es that have been made to the images and image data An additional filter can be combined with any of the above three filters When the Only Gels Action box is checked only actions that operate on selected images are displayed Thus you can exclude operations that act at the level of the program window for example layout settings or that result in the generation of reports By default this box is not checked and therefore all operations in the filter types above are listed 12 1 3 History and script navigators The Action Descriptor lists in the History and Script windows have the form of a navigator in which some Action Descriptors are preceded by a or node allowing the item to be expanded or collapsed and an icon indicates whether it can be scripted or not The corresponding action does not require any user dependent input such as the selection of a region and can therefore be scripted gt Actions identified by this icon cannot be scripted because they require user intervention Even if selected in the History navigator they are not copied to any newly generated script 250 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Safety control and automation Once an action is expanded you see one or both of the following icons Identifies a Parameter This represents a major property of the action For instance the Select Rows from Report action is characterized by a Window parameter d
181. escribing the title and type of the report the Sort parameter indicating the column in which the report data was sorted and the Selection parameter containing information about which items in the report were selected Identifies an editable Value that characterizes an action or an action parameter 12 1 4 Gel selection The listed actions only include the operations relevant to the gel images selected when first opening the History window or activating the Undo or Redo functions So even if the gel selection changes and the History is refreshed the Action Descriptors only apply to the originally selected gels Therefore if you want a history for one or more images that were not selected during the creation of the current history you must select the image s in question and open a new History window Similarly if you want to undo a general action for example spot detection on specific images you should first select the desired gels and then choose the Undo function from the menu 12 2 Undo redo ImageMaster allows you to cancel any unwanted modifications You can return to an earlier state of your analysis by using the Undo option Figure 12 1 You no longer lose whole parts of your analysis simply because you made an error and could only recover a previously saved state Moreover ImageMaster not only offers a multiple Undo function it also enables you to restrict the canceled actions to specific images Please
182. et 1 Select one or more spots 2 Choose Edit gt Annotations gt Add Label from the menu 3 Inthe Add Label box click on the category called Set and add a keyword which will become the name of the set For instance to mark spots that should be exported to a spot excision robot the final category name can be Set Pick 4 Alabel containing the name of the set for example Pick is attached to the selected spots note that the string Set is not displayed on the labels Normally when a new category is created it is immediately shown on the screen Newly created sets are an exception to this rule A new set is visible or hidden depending on the current visibility state of the generic category Set 8 3 3 Creating specific links As seen earlier it is possible to link labels to remote database entries by defining an external search engine for a particular category see Section 8 3 1 In addition ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Annotations 3 to this functionality ImageMaster offers additional possibilities to link your gel objects to Web pages mass spectrometry data text files or any other files located locally or on the Internet By using specific keywords in the labels of any category you can create links to particular objects including Web pages files and text To create a specific link for example to a Web page file or text you should add an annotation to a gel and include the following i
183. ew categories Annotation Report 1 ala e 3 s e B Information on selected annotations FileName pl MW Landmark plMw ProteinName 94 0002 6 46142 240000 1 00 240000 94 0002 5 60000 94070 0 5 60 1 94 0005 6 30000 76000 0 6 30 76000 94 0002 6 34569 70111 0 P06981 IMP_ECOLI 94 0005 5 60000 67677 0 94 0005 6 16926 56617 0 P06981 IMP_ECOLI 94 0002 5 32432 44044 0 P02917 LIVJ_ECOLI 94 0002 6 90000 36000 0 6 90 36000 1 2 3 4 5 6 T 8 Figure 8 11 Report on selected annotations in selected gels 8 7 3 Category report You can display information about all labels belonging to selected categories and selected gels The information displayed in this report Figure 8 12 is the same as for a report on labels Figure 8 10 and the label contents can be edited as well Category Report a alal e gt m Information on selected categories Category FileName Ac 94 0005 P36996 1052 6 75263 12176 0 Landmark 94 0002 772 5 36216 18485 0 307 Landmark 94 0002 L2 2889 632 382 6 34569 70111 0 308 Landmark 94 0005 L1 4057 251 781 5 05287 22286 0 309 Landmark 94 0005 L2 3082 609 363 6 16926 56617 0 310 p _Mw 94 0002 1 00240000 2335 670 22 6 46142 240000 Figure 8 12 Report on selected categories in selected gels 8 7 4 Intra Class Report An
184. ews or histograms the printing window with the normal printing options is displayed When you print tabular reports things are a bit different The report table to be printed is first displayed in your default Internet browser This is because the XSL stylesheet located in the Template Reports folder of the ImageMaster installation directory is used to transform the XML report into an attractive table find more details about XML and XSL in Chapter 11 You can subsequently use the print option in your browser to get a paper printout Copy to Clipboard Export your data directly into another program You can for example copy tables to spreadsheet software like Excel or graphics to programs such as Word or Adobe Photoshop To do so first select the desired lines in a table or the desired graphics in a window using the Shift or Ctrl keys Then copy the selection to the clipboard by choosing the Copy to Clipboard button in the toolbar or the Copy to Clipboard option in the contextual menu available by clicking the right mouse button Paste directly into the preferred software ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Reports 67 Reports 5 2 2 Adding comments a Add a Comment to the report You can add comments to any report to note relevant information Since the report comment is available at any time it is very convenient to keep information about selecti
185. expressions available to search patterns in labels Please not that the term element used in the description indicates a character or a subexpression The characters have a special meaning in certain contexts You must exclude them from the expression with a backslash if you want them to be taken literally This means that if your labels contain any of these special characters you must precede them with a backslash if you want to include them as normal characters in your search expression Note that you must also release the backslash character itself from the expression For example the search pattern R returns the result R 3 24 but not R 2 87 Nevertheless bracketed expressions are an exception to the rule Inside bracketed expressions all special characters including the backslash lose their special meaning for instance matches exactly any of the characters inside the brackets The order of precedence for the regular expressions described above is as shown in Table 8 2 For example the regular expression abc2 3de matches either the string abc2 or the string 3de rather than the string abc2de or abc3de because concatenation has a higher order of precedence than alternation Escaped characters lt Special character gt Bracket expression u Grouping 0 Single character duplication m n Concatenation Anchoring Alternation Table 8 2 Order of pr
186. extension mel Import Gels Adds new gels that are not already in ImageMaster format Create Folder Creates a subfolder into the Gels folder ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 259 Appendix e Paste Pastes previously cut or copied items into the Gels folder Folder e Open gt In Worksheet Opens all gels contained in the folder in the active Gels worksheet in the Display Zone e Open gt In New Worksheet Opens all gels contained in the folder in a new Gels worksheet in the Display Zone e Add Gels Adds gels that are already in ImageMaster format with the extension mel e Import Gels adds new gels that are not already in ImageMaster format e Create Folder Creates a subfolder into the selected folder e Delete Removes the selected folder e Cut Cuts the selected folder in order to paste it e Paste Pastes previously cut or copied items into the selected folder e Properties Displays the folder properties Name and Comment Gel e Open gt In Worksheet Opens the selected gels in the active Gels worksheet in the Display Zone You can also double click on a file to open it in the active Gels worksheet e Open gt In New Worksheet Opens the selected gels in a new Gels worksheet in the Display Zone e Add in Matchset Adds the selected gels in an existing match set or ina new one If you would like to create a new match set enter a new name Otherwise you can choose from th
187. fferent gels Figure 6 16 When using this feature the various regions are displayed in a single window using identical scales and orientations allowing direct comparison of spot positions and heights 3D View x TEE Up 45 Rot 40 Zoom 1 4 M Lighting I Spots overlay Figure 6 16 Multiple 3D views for the comparison of corresponding spots or regions in different gels ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA To display multiple 3D views of several corresponding gel regions 1 Use the Shift key to select the same region in all selected gels or define a particular region for each gel Alternatively you can select one or more spots in each gel 2 Select the gels for which you would like to display and compare the 3D views 3 Choose Reports gt 3D View from the menu 4 When spots are selected and a region is defined ImageMaster asks whether your 3D View should be based on the selected region or highlighted spots 5 ImageMaster displays a single window with side by side 3D views for each active region or area around selected spots Zoom operations and rotations are applied to all gels in a multiple 3D View Right clicking on the desired position can change the point on which the axes are centered When holding the Shift key at the same time this change is applied to all the 3D views in the window The 3D View is considered to be a normal report and as such provides most of t
188. for overlapped spots and match vectors in an image Grid settings e Type Specify whether the grid graduations should just be regular subdivisions of the visible space in Fixed mode or a subdivision in terms of real coordinates at whole multiples in Adaptive mode see Section 6 3 5 e Units Choose the default grid units to be used for displaying a grid over a gel Note that you can still set specific grid properties for each gel see Section 6 3 5 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Getting started e Divisions Number Specify the default number of grid subdivisions to be displayed in the horizontal and vertical directions see Section 6 3 5 Categories e Set the constraints for the label categories see Section 8 3 1 and define the color for the labels of each category Gel descriptions e Add or delete gel descriptions and change their constraints see Section 6 7 2 Quantification e Volume quantification Indicate if you wish to compute the spot areas in mm2 based on gel resolution default for new workspaces or pixels default for workspaces containing imported ImageMaster 2D Elite experiments Database e Annotations folder Select the folder where all linked files should be located These files are linked to spots or pixels in a gel via file link labels see Section 8 3 3 e Database ImageMaster offers full support for the Bruker Proteineer suite In this context the Server Na
189. fying your script ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Safety control and automation e Move the actions in a script by dragging and dropping them into another position They are placed below the point of your cursor e Copy actions from any history or script using the Copy tool and paste them into your script using the Paste tool Pasted actions are inserted at the end of your script or below the currently selected action but can be moved simply by dragging and dropping them into another position e Suppress one or several actions in your script by selecting the actions and clicking the Cut icon e Modify the values that characterize the actions or action parameters To do so right click on the Value icon and choose Modify from the contextual menu Enter a new value e Oblige ImageMaster to ask for user input while the script is running It is practical for example if you want to postpone the choice of statistics until the moment you actually run the script To do this right click on the Action Descriptor and choose Set User Action from the contextual menu The script will stop at the designated point and display a standard dialog box relating to the operation thus enabling you to adjust your input Once all the user definitions are made click the OK button and the script will continue to run The Tools menu in the ImageMaster program menu bar offers additional functionality for creating or handling scrip
190. geMaster 2D Platinum User Manual 11 0034 38 Edition AA A class is a set of gels or gel populations that you can compare with other such entities In the Classes folder you state your biological questions Your goal by comparing classes and therefore the gels within those classes is to find the protein expression variations between different biological states NOTE To compare gels in a class or within different classes the gels should have a common node in the match set that is used to construct the classes Without a common node the gels cannot be matched together and statistical comparison is impossible This means that all gels to be compared should belong to the same higher level match set NOTE The same series of gels may be found in several match sets Therefore several possibilities will exist when adding gels to a class The decision of which match set to use can have significant impact on the statistical analysis The use of classes can again be illustrated with the example of the bacteria gels described in the previous section see Section 4 6 Figure 4 8 illustrates three possible class setups based on the match set configurations Bacteria 1 Figure 4 5 and Bacteria 2 Figure 4 6 The hierarchical structure of the class Bacteria 1 in Figure 4 8 is based on the match set Bacteria 1 and uses the same configuration The class Bacteria 1 contains two sub classes A and B which in turn have two sub classes each U
191. gt Gels gt Zoom in the menu and select one of the following options e In Zoom in on the selected gels e Out Zoom out on the selected gels e 1 16 Reduce the original image by a factor of 1 16 e 1 8 16 Use one of the intermediary zoom factors e 32 Enlarge the original image by a factor of 32 e Fitin Screen Show the full image in the cell ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 83 E imageMaster 2D Platinum lol x File View Edit Show Select Analyze Reports Tools Window Help aIedSHOMA Gels B Spots fo Annotations fo Figure 6 2 Magnify tool The upper left gel is displayed without zoom factor The upper right gel was zoomed 2 fold whereas the lower gel was zoomed out by a factor of 0 25 EN imageMaster 2D Platinum lol x File View Edit Show Select Analyze Reports Tools Window Help Gels B Spots 0 Annotations fo Figure 6 3 Magnifying glass 84 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Magnifying glass To temporarily enlarge an area of a gel 1 Click on the Magnify tool in the toolbar 2 Hold down the Ctrl key and click on the region you want to enlarge 3 The area under the cursor is magnified Figure 6 3 Zoom window The Zoom Window has two functions First it can be used to enlarge the area of the gel under the cursor Figure 6 4 This is the case when the zoom factor in the Zoom Window is greater than that of the gel Second
192. he contextual menu Right click on the destination class and choose Paste from the contextual menu 3 Inthe Create Class box specify a Name and Comment Click OK Repeat for every copied match set 4 New classes appear in the Classes folder or destination class They contain the gels from the copied match set Right click on a class subclass or gel to open a contextual menu from which you choose an action to be carried out Detailed descriptions of all the Classes folder menus are provided in Appendix A 4 7 2 Adding gels toa class There are different ways to add matched gels to a class To use contextual menus to add matched gels to a class 1 Select one or more gel images in a match set Use the Shift or Ctrl keys to make multiple selections Right click on one of the images 2 Choose Add in Class from the contextual menu 3 Inthe Add Gels in Class box type in a name if you want to place the gels in a new class Or choose an existing class from the list Click OK 4 The gels are added to the class To copy and paste gels to a class 1 Select one or more gels in a match set Use the Shift or Ctrl keys to make multiple selections Right click on one of the gels 2 Choose Copy from the contextual menu 3 Right click on the destination class 4 Choose Paste from the contextual menu 5 The images are added to the class To drag and drop gels to a class 1 Select one or more gels in a match set Use the Shift or Ctrl keys to make multiple selections
193. he applied filter you can continue to use the current selection or on the contrary delete the selected spots or exclude them from your analysis by hiding them for instance A second filter can be applied You can select matches that is the spots in the matches that are representative of your population Spots that are only present in one of five gels of a match set are not representative of the match set and should be excluded from the analysis You can select matches by the number of spots they contain For example select all matches that have at least 4 or 5 spots In other words you can select spots that are present in at least 4 or 5 gels To apply a match filter 1 Make sure the gels have been matched 2 Select all gels for which a match selection should be refined ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Matches 9 3 Select spots or matches can be the result of a previous spot filter 4 Choose Select gt Matches gt Refine Selection from the menu 5 Enter the threshold for the refinement of matches see Section 9 3 1 For instance you might Keep the Matches whose number of selected spots is gt 4 or 5 6 The remaining matches satisfy the specified criterion You can obtain all spots that do not satisfy this criterion by choosing Select gt Spots gt Inverse Selection NOTE You should check your results carefully Detection or matching problems may cause the exclusion of spots that a
194. he standard icons in its toolbar More information about the icon functionalities is found in Chapter 5 In short the 3D image can be saved in PNG TIFF or BMP formats and printed or copied to the clipboard for insertion into another program The Select on Gel Image icon makes it possible to highlight spots on the related gel image once they have been selected in your 3D View To select spots on the 3D View just left click on their center or on the tip of their peak when viewed from the side With the Select on Gels Reports option in the Select on Gels drop down menu the spots are not only selected in the corresponding gels but also in any open reports Finally the Select from Gels feature does the exact opposite any spots selected on the corresponding gel region are highlighted in the 3D View The Show icon in the 3D View toolbar groups a series of special features e Show Region on Gel causes the gel to be moved so that the region corresponding to the 3D View and more particularly the center of the axes in the 3D View becomes visible This option is useful for finding a specific point on the gel when no spots were detected e Set Region on Gel not only moves the gel so that the region corresponding to the 3D View becomes visible but also reselects the region on the gel One ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 105 ED 106 should be careful when using this option because any previously selected r
195. hen matching is completed ImageMaster provides the total number of matches found NOTE Matches are saved on disk when saving the gel files However the information in the workspace project is necessary to reconstruct the match sets and retrieve the matches This is the reason why it is important to regularly save the workspace which in turn saves the projects and why the workspace is saved by default when closing the software 9 2 2 Specifying starting matches When the gels are very distorted or different automatic matching may fail In this case it can be necessary to define starting matches or landmarks that is corresponding spots that are present in each of the gels to be matched However the number of landmarks or starting matches should be kept to a minimum Often a single landmark may suffice to successfully initialize the matching procedure NOTE The wise choice of landmarks or matches is imperative for obtaining adequate matching results Landmarks essentially correct global deformations of gels Therefore it is recommended not to put landmarks on spots in locally distorted regions because this can worsen the matching results around such regions To correctly match gels you should perform a first matching round using some carefully selected landmarks If some locally deformed regions are not properly matched it is best to delete any wrong matches that were generated manually add a few new matches and then to
196. hin a class of gels are studied with the intra class statistics tools These tools include e Scatter plots to analyze gel similarities or experimental variations e Descriptive statistics of central tendency and dispersion which can be calculated and displayed in Intra Class Reports and Histograms e Factor analysis enabling the identification of similar gels and of spots that are characteristic for a particular population of gels e Heuristic clustering an artificial intelligence technique to automatically classify sets of gels and highlight significant protein spots ImageMaster can also help find significant protein expression between classes of gels For this purpose two complementary statistical methods for inter class analysis are available e Overlapping measures which summarize each class of gels as an interval and measure the overlap between these intervals The overlapping values can be displayed in Inter Class Histograms and Reports e Statistical tests which assess the difference between populations and give an estimate for the significance of the difference that is how high is the probability that this difference is only due to chance NOTE The statistical tools in ImageMaster include all the options that the expert user might expect Nevertheless those who are less familiar with the statistical methods can simply use the default settings Whenever possible this manual indicates how the statistical data can b
197. ible without being concerned about noise spots that can be filtered with the Saliency and Min Area parameters e Saliency The Saliency parameter is a measure based on the spot curvature It indicates how far a spot stands out with respect to its environment Real spots generally have high saliency values whereas artifacts and background noise have small saliencies Although the Saliency is an efficient quantity for filtering spots it is also highly dependent on the images for example image resolution and depth Some gels need a saliency value of 10 for correct filtering Others may necessitate a value of 5000 To estimate the saliency range to use with your images you can display a Cursor Information window or Spot Report and look at the saliency value given for a spot that you would like to suppress Enter this value in the Saliency field of the Detect Spots window The spot detection algorithm then discards all spots with saliencies smaller than the specified threshold ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA e Min Area After setting an appropriate Saliency to filter out all noise spots ee there may still be noise in your gel that cannot be eliminated without suppressing real spots This often happens with dust particles that consist of a few very dark pixels Get rid of these artifacts by using the Min Area parameter It eliminates spots that have an area smaller than the specified threshold expressed in number
198. ic clustering ocscsssssssssssssssssssssssssssssssssssssssssssecsesseseeceesessessesssssssnisnanseee 214 IitersClass statistics anea E REEE E om 216 Specifying classes 216 Overlapping measures Inter Class Histograms Br Inter Class Repoft unsenennseee namen aus 224 St tistic ltests auususlassenen ai E A a 225 Data integration 11 1 21 2 4 11 1 2 11 1 3 11 2 11 2 1 11 2 2 11 2 3 11 2 4 11 3 11 3 1 11 3 2 11 3 3 11 3 4 Converting into ImageMaster 2D Platinum format 233 From earlier versions of ImageMaster and Melanie 233 From ImageMaster 2D Elite nennen 233 From Twain compatible scanners uuu 236 Exporting and importing ImageMaster 2D Platinum data 237 KMETA E er e a aa Aea astra 237 ImageMasterTeports sereen on a eE E E ERRA 238 Export gel dAlar seiiuindeiikunkunitesetsund ktituknaikisih 238 Imp rtigelidatd iausa eeiser anna 240 Exporting to spot EXCISION robots eenneneeeenn 241 Bruker Proteineer SP spot picker ER GE Healthcare Ettan spot picker nnnnennennensssenneennnnnnnennnnnnnnne Genetix GelPix spot picker eennnnnnnnnennsenssssnnennnnnnnnennnnnennnnnnn Genomic Solutions ProPic spot picker ennenesesessenenennnnnn 244 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 11 4 Connecting to external protein databases oun 11 41 Setting the databse nosei in a AR 114 2 Querying the datebasenauuneneeenenseensannennnn 11 4 3 SWISS 2DPAGE mast
199. ic gel normally looks similar to the reference image ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 191 9 Matches 192 NOTE The spots from the reference gel image should be selected as well if the synthetic gel to be created should take into account the quantification values of this gel Therefore remember to display the reference gel in the match set by choosing Show gt Show Reference and to select the spots to be included in the synthetic image The Master of the match set does not contain quantification information and will only be used for determining the spot positions in the new synthetic gel 9 7 1 Spot filtering Synthetic gels are created based on selected spots in selected gels In order to obtain the cleanest possible synthetic gels without artifacts or insignificant spots you can refine your spot selection using the various filtering options in ImageMaster You can perform a type of filtering that consists of selecting and possibly eliminating spots that do not satisfy certain criteria of size volume and so on To apply a spot filter 1 Select all gels to be filtered 2 Select all spots on the selected gels 3 Choose Select gt Spots gt Refine by Value from the menu 4 You are asked for the value type to be utilized for filtering and the thresholds to be employed Click OK when you finish entering the latter 5 Only spots meeting the specified conditions are kept 6 Depending on t
200. icated directory when a gel with the appropriate ID is not found at the usual location This is the case when a red mark covers the gel image icon generally indicating that you moved the original image file or replaced it with another one Copy Copies the selected images into memory Properties Displays the DIGE gel properties Gel Name and Staining A 2 5 MatchSets folder Create MatchSet Creates a new match set into the MatchSets folder Paste Pastes previously cut or copied items into the MatchSets folder MatchSet Open Opens the selected match set in a new MatchSet worksheet Create MatchSet Creates a sub match set within the selected match set Delete Removes the selected match set from the folder Copy Copies the selected match set into memory Cut Cuts the selected match set in order to paste it Paste Pastes previously cut or copied items into the selected match set Properties Displays the match set properties MatchSet Name and Comment SubMatchSet Open Opens the selected match set in a new MatchSet worksheet Set MatchSet as Reference Defines the selected match set as the reference to be used when matching two or more match sets The match set icon is tagged red Create MatchSet This option is available only if the sub match set is empty It creates a sub sub match set within the selected sub match set Delete Removes the selected match set from the folder Copy Copies the selected match
201. icates must be incorporated within each experimental 8 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Two dimensional gel analysis design This requires the separation and analysis of a large number of samples and slows the process if each sample must be separated on a different gel Differential gel electrophoresis DIGE is a method for pre labeling protein samples prior to two dimensional electrophoresis By allowing different samples to be run on a single gel one of which can be an internal standard it can significantly simplify the above mentioned problems The system is based upon the use of up to three cyanine dyes possessing unique fluorescent properties The dyes Cy2 Cy3 and Cy5 are mass and charge matched N hydroxy succinimidyl ester derivatives that differentially attach to lysine residues in a protein Therefore the labeled proteins migrate simultaneously on the 2 DE gel but still produce distinct excitation and emission spectra The design of a DIGE experiment is straightforward Figure 2 1 Protein samples are labeled with the different DIGE dyes and subsequently mixed on the same 2 DE gel Electrophoresis is applied to separate the proteins according to molecular weight and isoelectric point The gel is then scanned using instrumentation such as the Typhoon Variable Mode Imager GE Healthcare to generate overlaid multi channel images for each gel A Pool A B B Cy3 Cy2 Cy5 Y gt 4
202. ich will correspond to the master gel for the Intra Class and Inter Class Reports Note that the cells of updated labels go back to their original color When all labels in your report have been updated the asterisk behind the report title also disappears NOTE A word of warning is expressed here Any changes made to a gel addition or modification of labels by using the Update Gel icon can be canceled with the undo function This is not the case for reports Editing operations on reports cannot be reversed Notice that categories of the type Set or categories using the Boolean Data Type are displayed in the form of check boxes in some reports Editing these labels corresponds to checking the box in order to indicate that the item belongs to a Set box checked or not empty box or takes the value 0 empty box or 1 box checked for a Boolean Data Type To check several boxes i e lines simultaneously 1 Select the rows you want to change the checked state 2 Hold down the Shift key while clicking in the box of one of the selected rows of course in the relevant column All selected lines are now in the same activated state Click in the check box a second time if the state should be reversed ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Reports 73 ED Reports 74 5 4 Customizing reports 5 4 1 Settings The presence of the Settings icon indicates that the current report is completely
203. icking Load and choosing a file name you can load an existing template file There is no limit to the number of report templates that you can save or use If you save a report template in the ImageMaster Menus folder of the User Documents directory it is available at any time from the menu Reports gt Report the templates are automatically loaded when ImageMaster starts If the template is saved in or loaded from any other folder it is added to the menu Reports gt Report gt Recent Templates and is only accessible during the current work session ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA The last report template used is available by choosing Reports gt Report gt Current Template from the menu Spot Report Settings Set the spots attributes to be displayed in the report Landmark Visible attributes FileName ProteinName Master gel a Load Save Landmark Ac gt ce Figure 5 3 Spot Report Settings window You can add items to or suppress items from the Visible attributes list using the corresponding blue arrow buttons 5 42 Column order You can reorganize the columns of a table directly in the report window To do so drag the column heading to its new position that is to another column to which it will be inserted to the left 5 43 Column and row size You can enlarge or reduce the column size Drag the boundary on the right side of the column he
204. icking on its banner By default ImageMaster displays panes in tiled mode To stack panes 1 Select the worksheet 2 Choose View gt Worksheet Layout gt Stacked in the menu 3 The panes are stacked 26 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Getting started SSS You can also fix the number of panes displayed horizontally and vertically in a worksheet Choose View gt Worksheet Layout gt Free in the menu In the Worksheet Layout box input the following values e Number of Columns Constrains the panes positioned one next to the other in the worksheet e Number of Rows Constrains the panes positioned one above the other in the worksheet Images A pane can contain one or more gel images Each image has a legend with its name in the upper left corner The color of the legend indicates whether the image is selected green or red for a master image or not gray or pink for a master image In order to select an image click on its legend To close a gel image 1 Select the gel image Use the Shift or Ctrl keys to make multiple selections 2 Choose File gt Close Images in the menu 3 The image is removed from the pane Images can be laid out so that they do not overlap tiled images or so they do overlap stacked images By default ImageMaster displays images in tiled mode To stack images 1 Select the pane 2 Choose View gt Pane Layout gt Stacked in the menu 3 The images are stacked You can also fix
205. ighlights significant matches that may allow identification of different populations and their characterization by specific spot patterns Most of the ImageMaster reports share a certain number of features One of these is the standard report toolbar that offers common functionalities such as saving 66 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA printing selecting objects or navigating through the gel data A second feature is that several reports are editable more specifically those that display annotation content and properties In addition most of the reports are customizable This means that you can suppress or move columns resize columns or rows and sort your columns in descending or ascending order based on their values as can be done with spreadsheet software such as Microsoft Excel Finally the lines in tabular reports are numbered indicating the number of selected objects used to create the report 5 2 5 2 1 Report toolbars Standard tools a Save a report Enter the File name in the displayed window and choose the desired format in the Save as type field Tables can be saved in the ImageMaster report format XML file carrying the extension rpt in text format with tabulations separating the columns txt or as a Microsoft Excel Workbook xIs Graphics can be saved in PNG TIFF or BMP formats 8 Print a report When you print graphical reports such as scatter plots 3D vi
206. ight click on the Workspace Name at the top of the Navigator 2 Choose Insert Project from the contextual menu 3 Inthe Insert Project box browse the directory where the project file is located select its name and click Open Use the Shift or Ctrl Keys to make multiple selections 4 The Project Name and the folders gels reports and other documents included in the project appear in the Navigator 4 4 3 Exporting a project Exporting a project allows you to save a project together with copies of its gels matches master gels and other documents to which new IDs are attributed so that you can import it into a new workspace or send it to a colleague Please note that the exported gel file structure is based on your project organization Exported projects can be included in any workspaces as explained above To export a project 1 Right click on the project to export 2 Choose Export from the contextual menu 3 Choose a destination folder for the exported project and click OK 4 4 4 Project properties The Project Name Creator File Name Modification Date and Comment are Project Properties The Project Comment is helpful because it describes and or explains the project It serves as a useful source of information to which a coworker is referred or as a reminder when reviewing old work You can add or modify the Comment or Project Name while viewing the Project Properties To edit the Project Name or Comment 1 Right click on the
207. ile As spot areas and the associated volumes in ImageMaster 2D Elite are calculated in terms of pixels and not mm the Compute area in mm2 box in the Quantification tab of the ImageMaster Options accessible by choosing Tools gt Options in the menu is not checked to reflect this fact Please note that it is prohibited to edit spots from imported files If you need to compare imported gels with new ones you must do an automatic spot detection in ImageMaster 2D Platinum Comments on spots previously visible in the Edit Spot Fields box are converted to spot annotations of the Comment category Any User Defined Fields for spots entered through the Edit Spot Fields box or Measurements window are converted to annotations of a category with the name of the original User Defined Field Note that User Fields of type Document are converted to annotations that contain a file link the label content is file file path Filtered spots not displayed in the Elite version are imported into ImageMaster 2D Platinum but are flagged with annotations of type Set Filtered_Spot You can easily select these spots Select gt Annotations gt By Category and decide if you want to keep delete or hide them Similarly all spots that were selected for picking are flagged with annotations of type Set Picked_Spot Matches If gels were matched in the original experiment the spot matches are converted into ImageMaster 2D Platinum matches ImageMaster 2D Platin
208. iles with unrecognizable extensions are not opened e Add Files Inserts one or more files from the hard disk within the Documents folder e Create Folder Creates a subfolder within the Documents folder e Paste Pastes the previously cut or copied items in the Documents folder Folder e Open Opens all the files with the appropriate software depending on the extensions As in Windows Explorer files with unrecognizable extensions are not opened e Add Files Inserts one or more files from the hard disk within the selected folder e Create Folder Creates a subfolder within the selected folder e Delete Removes the selected folders with their files e Copy Copies the selected folders into memory e Cut Cuts the selected folders in order to paste them e Paste Pastes the previously cut or copied items in the selected folder e Properties Displays the folder properties Name and Comment er ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 265 Appendix 266 File e Open Opens the selected files with the appropriate software depending on the extensions You can also double click on a single file to open it e Delete Removes the selected files from the folder Please note that it does not remove the file from your hard disk The file is only deleted from the active project e Copy Copies the selected files into memory e Paste Pastes the previously cut or copied items below the selected file e Properties
209. ilities the data points protein values in the case of ImageMaster must respect the restrictive assumptions made by the various statistical tests see below If an assumption is not met even approximately the significance levels and the power of the test are invalidated Matters are made even worse when several assumptions are violated Since this is often the case in studies of protein expression ImageMaster only displays the numerical values for each test and not the probabilities and lets the user search in the appropriate tables for the corresponding significance levels If the appropriate test was chosen and the assumptions were met the probabilities found can be used and the results accepted If this is not the case the numerical values given by ImageMaster still allow you to classify the differences between the sample means according to their relative significance Two sample t test The two sample t test is generally used to determine whether the mean or median of a variable differs between two populations It is based on the following assumptions e The data are continuous not discrete e The data follow the normal Gaussian bell shaped probability distribution e The variances of the two populations are similar e The two samples are independent There is no relationship between the individuals in one sample as compared to the other as there is in a paired t test The t test statistic is based on the difference
210. indicates that the particular class is entirely covered by another one ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 219 Data analysis NOTE For non expert users it is recommended to compare only two classes at a time in the Inter Class Report see Section 10 3 4 This is because only in this case one gets the direct relationships difference ratio or percentage overlap between the two classes As indicated in their definition the Gap Ratio and Normalized values always calculate the MAXIMUM difference ratio or percentage respectively with respect to ANY of the other classes This means that when the Ratio values for three classes e g A B C are compared for instance the software calculates the ratio of A with respect to B and of A with respect to C but only displays the highest value in the column for class A The number shown does not indicate with respect to which class the value was obtained The idea is to quickly enable you to find a match i e protein marker that distinguishes the current class from any of the other classes Once such protein markers are found the complementary tools in the software such as the Inter Class Histograms see Section 10 3 3 can be used to study the match in more detail 10 3 3 Inter Class Histograms As with intra class statistics you can visually investigate the statistical and overlapping descriptors of classes by displaying Inter Class Histograms To display
211. indow displays the desired information If the Value field shows Unloaded the raw pixel values are unavailable In this case choose Edit gt Gels gt Raw Image gt Load to load the image data for the gels The order of the items in the Visible attributes list determines the order of the items in the Cursor Information window You can therefore adjust the display by adding your attributes in sequential order to the Visible attributes list 144 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Cursor Information F Bl FileName A_TI_Gell SpotlD 4541 z VAE 15298 cursor information 7 E Setthe attributes for the cursor information window Area 6 53 Intensity 11792 Hidden Visible ise ae FileName Saliency 3655 coc Figure 7 9 a Cursor Information window and b Cursor Information dialog box for specifying the Settings Please note that these settings can be made permanent by going to the Cursor Information tab in the Options accessible by choosing Tools gt Options in the menu Your adapted settings can also be saved and used as templates For this purpose the following options are available from the Cursor Information dialog box e Save Allows you to save the new Cursor Information template e Load Opens an existing template file 7 10 Reporting on spots 7 10 1 Spot Report The Spot Report summarizes all relevant information about the selected spots their identification number
212. indows see Section 3 4 4 Since every report displayed on the screen is listed in the Window menu it can easily be located by clicking on its name In order to quickly hide all pinned report windows you can go to Windows gt Minimize All In order to close all report windows choose Windows gt Close All in the menu 5 1 1 Reports menu From this menu you can display reports by 3D View A three dimensional view of selected gel regions or areas around selected spots Gel Summarized information about the selected gels such as Gel ID intensity calibration parameters number of detected spots number of annotations gel resolution and size etc Gel Description User defined properties of gels such as sample type date of the experiment operator name treatment conditions pH range SDS gel percentage or staining Gel Calibration Summarized information about the selected gels including file name ID file path calibration coefficients type of file creator and creation date ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Reports 65 ED Reports Spot Specific information about selected spots such as Spot ID and coordinates quantification values attached labels etc Label Information on selected labels such as content category Spot ID if the label is linked to a spot and annotation coordinates Annotation Information for selected annotations about chose
213. ints Since ImageMaster does not have any information about the experimental possibly non linear pl scale the calculated values are only approximate In the case of the MW of the spots the procedure is similar except that ImageMaster searches for the closest spots above and below the spot for which the MW will be determined and it makes a logarithmic interpolation Extrapolating pl and MW values is more complicated For instance if the pl of a spot on the extreme right side of your gel is to be determined the program looks for the two closest spots to the left of the spot in question If these two spots are sufficiently distant from each other in order to decrease the error the value for the spot in question can then be extrapolated Normally the pl and MW values in the Spot Report or Cursor Information window should be the same as in the defined pl_MW annotations However this is only the case if the annotations are attached to actual spots and not just to pixel positions in the image If an annotation is attached to a pixel the pl and MW values for the spot that lies closest to it will be slightly different from that of the pixel to which the annotation is attached You can solve this ambiguity by linking the annotation to the spot 7 9 Cursor information The Cursor Information window can be used at any time to display information on pixels x and Y coordinates pl and MW estimates or raw pixel values and spots if ImageMaster 2
214. inum User Manual 11 0034 38 Edition AA c Figure 6 13 Region of a gel showing a contrast adjustment a Original image with bending parameter 0 b same area with bending parameter set to 2 c same area with bending parameter set to 2 To adjust the contrast in an image 1 Select one or more gels Draw a region in these gels to get a preview of the contrast mapping modifications 2 Choose Show gt Gels gt Adjust Contrast in the menu 3 Inthe Image Display Settings box select an image from the list The grey level histogram of the chosen image is shown and can now be used for adjusting the contrast 4 Specify the Unit you want to use see below and indicate whether you want to modify the contrast mapping as a function of the complete image range or only of the selected region see below 5 Change the minimum and maximum gray levels by displacing the slider borders or by typing valid numbers in the boxes at the lower left and right corners of the histogram You can also use the bending scroll bar 6 These settings are applied to the regions in the selected gels 7 When you are satisfied with the changes click OK to apply the settings to all the selected gels All contrast mapping changes are applied to selected gels only and are saved with the image file Thus the next time you open these gels the modifications are still active In some cases you may want to save a specific mapping fu
215. ion AA Two dimensional gel analysis Class A Class B Al o A2 B1 B2 Match set AB Figure 2 3 Definition of matches match sets and classes Spots S1 S2 S3 and S4 together form a match Note that the spot in Master AB has the same number as the corresponding spot in A1 This is because A1 is the reference image for Match set AB on which the Master AB is based The Match set AB contains all the gels to be matched A1 A2 B1 and B2 and is represented by a master image Master AB A class regroups gels from a particular biological state that has to be compared with another such state The spots in the selected match green are under expressed in Class B with respect to Class A 2 4 Image analysis workflow A typical image analysis both for non DIGE and DIGE gels would consist of the following steps Please note that the specificities of DIGE gel analysis are explained in the corresponding chapters 2 4 1 Acquiring data Your gel images must first be digitized using an image capture device This will generally be done with a separate software You can open gels from TWAIN compatible scanners from within ImageMaster Please consult Chapter 11 for more details about this In Section 6 1 you will also find recommendations on image scanning format resolution depth 2 4 2 Setting up a workspace You must set up a workspace to open and work on gel images The workspace allows you t
216. ion from the menu and pick the category Set Student t test only spots that belong to both sets remain selected SetA Set B SetA Set B Intersection Union SetA Set B SetA Set B Difference Exclusion Figure 8 5 Combining sets of spots in ImageMaster To combine sets of annotations 1 Select annotations according to a first criterion by using the options in the menu or by picking the desired annotations with the Annotation tool ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Annotations 3 2 Choose Select gt Annotations gt Combine in the menu and select one of the four possible operations Intersection Union Difference or Exclusion 3 Choose the label category corresponding to your second criterion in the dialog box and click OK 4 The result of the logical operation is selected according to Figure 8 5 8 4 3 Reports You can select labels or annotations directly from a displayed report on Annotations Labels Categories or even Spots at least for spots that are linked to annotations Do this by double clicking on an item in the report or by selecting one or several items and clicking the Select on Gels icon in the report toolbar 8 5 Displaying annotations and labels You can change the way annotations and labels are displayed but these modifications will not be saved in your ImageMaster file 8 5 1 Annotation flag position Sometimes you may want to move an annotation flag because you are preparing a
217. is used for handling annotations Ctrl Shift is used for handling matches Some exceptions do exist The most important ones are the following two shortcuts used for undoing and redoing actions carried out on a gel Shortcut Menu Command Ctrl Z Edit gt Undo Shift Z Edit gt Redo ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 269 Bd Appendix B 2 Gel shortcuts Shortcut Menu Command Ctri A Select gt Gels gt All Ctrl F View gt Switch Ctrl I Show gt Gels gt Grid Lines gt Show Ctrl J Show gt Gels gt Grid Lines gt Hide Ctrl N Select gt Gels gt Inverse Selection Ctrl S File gt Save gt Worksheet Ctrl W File gt Close Images Ctrl lt Up gt Show gt Gels gt Move gt Up Ctrl lt Down gt Show gt Gels gt Move gt Down Ctrl lt Left gt Show gt Gels gt Move gt Left Ctrl lt Right gt Show gt Gels gt Move gt Right Ctrl 1 Tools gt Toolbar gt Hand Ctrl 2 Tools gt Toolbar gt Magnify Ctrl 3 Tools gt Toolbar gt Region Ctrl 4 Tools gt Toolbar gt Spot Ctrl 5 Tools gt Toolbar gt Annotation lt Page Up gt View gt Pane Layout gt Next lt Page Down gt View gt Pane Layout gt Previous Shift lt Up gt Show gt Gels gt Zoom gt In Shift lt Down gt Show gt Gels gt Zoom gt Out F1 Show gt Show Reference Master F2 Show
218. ist of more than two gels In any event your objective should always be to work with the largest possible sample sizes ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 231 Data analysis 232 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data integration 11 Data integration 11 1 Converting into ImageMaster 2D Platinum format 11 1 1 From earlier versions of ImageMaster and Melanie ImageMaster recognizes gel files that were saved with ImageMaster 2D Platinum 4 9 or 5 and Melanie 2 3 4 or 5 If you add files from these versions in the Workspace Gels folder you will recover spots and annotations but no match data However you can convert entire analysis sets such as a single folder containing all matched gels into the current ImageMaster file format This way all match information is recovered without having to redo the gel matching To convert ImageMaster 2D Platinum or Melanie data 1 Choose File gt Import gt ImageMaster 2D Platinum or Melanie Data in the menu 2 Inthe Batch Files Conversion box indicate the Source Folder containing your gels and associated match files in one of the old format by browsing to the desired directory 3 Provide a Destination Folder name where your analysis set should be saved in ImageMaster 2D Platinum format You can manually add the name of a new subfolder if needed 4 Click OK ImageMaster then creates new gels and match files in ImageMaster 2D Platinum
219. it Enabled from the menu 2 Click on the Spot tool ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 141 142 3 Double click on the desired location in your image 4 Use the slider to set the disc radius in the Adding Spot dialog box The disc radius expressed in number of pixels determines the radius of the circular spot to be drawn 5 Anew spot circle with the specified radius is drawn at the desired location In addition you can add spots to an image by copying them from other gels provided the images were previously matched see Chapter 9 for more details on matching To do this perform the steps described below Note that ImageMaster automatically creates matches between the selected spots and the newly created ones To copy spots from one matched image to another 1 Select spots on a matched image 2 Select one or more images you want to copy the spots to 3 Choose Edit gt Spots gt Copy From Image 4 The new spots are added to the selected images Once new spots are added to an image using either of these methods you can make modifications to the newly created spot using the procedures described above Deleting spots To remove spots from an image just select the spots you want to delete and choose Edit gt Spots gt Delete from the menu 7 8 MW and pl calibration If you have a gel with pl MW standards ImageMaster can compute approximated pl and MW values for all the spots pixels in this image
220. ity distributions are continuous e The measurement scale is at least ordinal e The two samples are mutually independent In the Kolmogorov Smirnov test the data points in each sample spot values for a particular match in a class are sorted in ascending order and converted into an empirical distribution function EDF This function gives the fraction of data points to the left of a given value z In the second example from Figure 10 24 the ordered data points from class X are 0 034 0 045 0 056 0 064 0 069 and 0 078 The fraction of data points to the left of each of these z values can easily be calculated and plotted full line in an Empirical Distribution Plot Figure 10 25 It is clear that no data lie strictly below 0 034 17 0 17 1 6 of the data is strictly smaller than 0 045 33 0 33 2 6 of the data is strictly smaller than 0 056 50 0 50 3 6 of the data is strictly smaller than 0 064 and so on The same procedure can be followed for the second sample class Y in our example the dashed line in Figure 10 25 The Kol mogorov Smirnov test statistic D is then defined as the maximum distance between the empirical distribution functions EDF of the two samples In the example cited here D is 0 63 0 83 0 20 If D is greater than a particular decision limit critical value found in a Kolmogorov Smirnov table there is a statistically significant difference between the two samples However the test provides no insight
221. iven under About ImageMaster in the Help menu Choosing Help gt About ImageMaster displays the program splash page 1 4 4 GE Healthcare listens The GE Healthcare staff is attentive to your suggestions Many of the new features and enhancements in this software are a direct result of conversations with our customers We truly appreciate any comments criticisms or ideas that would help us to improve the software Please do not hesitate to contact us by e mail at imagemaster support ge com ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Introduction Introduction 6 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Two dimensional gel analysis 2 Two dimensional gel analysis 2 1 Two dimensional electrophoresis 2 1 1 Technique Two dimensional electrophoresis 2 DE can effectively resolve thousands of proteins in a complex biological mixture by separating the proteins based on two independent properties isoelectric point pl and molecular weight MW Separation in the first dimension is achieved by re hydrating an immobilized pH gradient IPG strip with the protein sample and subsequently applying an electric field The charged proteins migrate within the pH gradient until they reach their isoelectric point where they lose their net charge and therefore their mobility Non equilibrium pH gradient electrophoresis NEPHGE can also be used to accomplish this separation After the isoelectric focusing IE
222. k on the annotation basis and drag it to a spot If for some reason an annotation already exists within a spot but is not yet linked you can also select the annotation and choose Edit gt Annotations gt Link with Spot To unlink an annotation from a spot select the annotation and drag it outside the spot or choose Edit gt Annotations gt Unlink from Spot 8 3 1 Creating label categories As mentioned above ImageMaster offers a series of predefined categories However you can also create your own categories which are always displayed with gray backgrounds You can easily do this by adding an annotation using the procedure described above and typing the name of a new category You must enter the constraints for the new category in the Create Category box Figure 8 2 Please note that the category constraints can also be modified at a later stage by going to the Categories tab in the ImageMaster Options accessible by choosing Tools gt Options in the menu However it is better to set the constraints while creating the category otherwise you may have to adapt the existing labels to make them fit the new restraints Note that user defined categories are only available from the category list as long as there is at least one label of this category in the open gels ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Annotations 3 EEE er Set category constraints for category Notes Categories
223. kflow Figure 4 1 as major operations such as spot detection matching and statistical analysis are to be prepared in the workspace To understand how this works you should note that every project in the workspace is organized into five default folders Gels MatchSets Classes Reports and Documents e First gels are imported in the Gels Ga folder where possible relationships between gel images are specified e g DIGE The gels are then opened in the Display Zone and spot detection is carried out e Subsequently MatchSets GA are created to specify how the gels will be matched together Each match set is characterized by a reference gel and can belong to a higher level match set Once match sets are defined the corresponding gels can be opened and effectively matched e Sets of gels that will be compared are grouped in Classes Ga This is where the biological question is stated Classes must be defined in order to ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 33 proceed with statistical analysis and the discovery of protein variations differences and similarities in a series of gels e Saved ImageMaster reports are inserted in the Reports a folder The objects of interest in these reports can be sorted selected visualized on the gels and exported in different formats e Documents related to the project can be inserted in the Documents ia folder Graphical files containing exported gel images histograms 3D view
224. ksheet to File from the menu 2 Enter the desired folder file name and file type in the Export Image As dialog box To the clipboard Alternatively you can export images to the clipboard for direct pasting into another software The procedure is very similar to exporting images to files However you can only export one gel image at a time to the clipboard To export a gel or a selected gel region 1 If you want to export a gel region first outline it with the Region tool 2 Select the gel to export 3 Choose File gt Export gt Image to Clipboard from the menu 4 If no region was selected the entire gel image is sent to the clipboard Otherwise only the region is exported 5 You can then paste the image into the preferred software To export a view of the worksheet to the clipboard 1 Choose File gt Export gt Worksheet to Clipboard from the menu 2 You can then paste the image of the worksheet into the preferred software 6 83 Printing gels ImageMaster provides various printing options Print selected gels or regions of gels one image per page or even the active worksheet Whatever your choice the image is printed as it is displayed on the screen retaining objects and properties such as spots annotations contrast mapping and pseudo colors alignment zoom grid etc A ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 125 ED NOTE With a zoom factor of 1 the printed gel image takes the full paper width You can a
225. l Bacteria 1 B B_T1 EB A_TI_Gelt mel EB B_T1_Gei2me Bacteria 1BNB_TI M B_T1_Gel3 mel Bacteria 1 8 B_T1 EB A_T2_Gel3 mel Bacteria 1A 4_T2 EB A_T2_Gel2 mel Bacteria 1 A A_T2 M A_T2_Gelt mel Bacteria 1A A_T2 M 8_T2_Geit mei Bacteria 1 B B_T2 EB B_T2_Gel2 mel Bacteria 1 B B_T2 EB B_T2_Gel3 mel Bacteria 1 B B_T2 EB A_T1_Gelt mel Bacteria 2 Abis EB A_T1_Gel2 mel Bacteria Navis EB A_T1_Gel3 mel Bacteria 2 Abis EB A_T2_Gel2 mel Bacteria 2 Abis EB A_T2_Gel3 mel Bacteria 2vAbis EB A_T2_Gelt mel Bacteria 2 Abis B B_T1_Gel3 mel Bacteria 2 Bbis M B_T1_Gel2 mel Bacteria 2 Bbis EB B_T1_Gelt met Bacteria 2 Bbis EB B_T2_Gel3 mel Bacteria 2 Bbis CT aa Figure 4 9 Selected gels in the Classes folder 4 7 1 Creating a class There are different ways to create a class To create an empty class 1 Right click on the Classes folder 2 Choose Create Class from the contextual menu 3 Inthe Create Class box specify a Name and Comment Click OK 4 The Class Name appears in the Classes folder To create a class from a match set 1 Select one or more match sets from the MatchSets folder Use the Shift or Ctrl keys to make multiple selections SSS 56 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace 2 Left click on one of the match sets a Hand cursor appears Drag and drop the folders into the Classes folder or into a destination class Or right click on one of the match sets and choose Copy from t
226. l the landmarks e None Unaligns and restores the images Note that the addition of suspect landmarks or a bad distribution of the landmarks can worsen the alignment results Therefore these mistakes should be kept to a minimum and the following rules considered when defining landmarks e For alignment using the Global option landmarks should be well distributed over the entire gel This means that they have to cover both the X and Y directions On the other hand when using the Exact option landmarks should especially be defined around the distorted regions of the gels e Landmarks should only be defined on clearly corresponding spots Moreover one should differentiate between spots that effectively represent the same protein form and spots that could be linked based on biological arguments variants of the same protein such as different phosphorylation states Protein variants definitely should not be used as landmarks e Landmarks should be placed on small sharp spots of similar area rather than on large diffuse ones which may differ considerably in size because in the latter case the error in the position will be much more substantial e Landmarks should be added gradually so that you can monitor their individual influence on the alignment which is automatically updated after each landmark addition Incorrectly placed landmarks may seriously decrease the alignment quality If you add all your landmarks at once you
227. laced in the designated DIGE Gels folder that is activated in ImageMaster 2D Platinum DIGE Each DIGE gel holds one two or three gel images and can be processed with the specific DIGE functionalities The DIGE Gels folder can not contain subfolders To create a subfolder 1 Right click on the Gels folder or a subfolder 2 Choose Create Folder from the contextual menu 3 Inthe Create Folder box specify a Name and Comment for the new folder and click OK 4 The Folder Name appears in the Gels folder Right click on the Gels folder a subfolder or gel to open a contextual menu from which you choose an action to be carried out Detailed descriptions of all the Gels folder menus are provided in Appendix A 4 5 2 Importing a new gel You can Import and automatically convert new gel images with tif png gel img or scan extensions into ImageMaster format with the extension mel At this level you can choose a reduction factor to be applied when importing the images Thus you automatically decrease the image resolution and therefore the file size of large gel files To import a new image 1 Right click on the Gels folder or a subfolder 2 Choose Import Gels from the contextual menu 3 Inthe Import Image box input the Reduction Factor and File Format and click OK 4 Inthe Import Gels box browse the directory where the image file is located select its name and click Open Use the Shift or Ctrl keys to make multiple selections
228. last option in the Tools gt Gels menu lets you change the gray levels of selected gels by inverting them This means that if your image shows white spots on a black background the inversion displays black spots on a white background the required mode for analysis in ImageMaster To invert the gray levels of your gels 1 Select the gels to be inverted in a worksheet 2 Choose Tools gt Gels gt Invert Gray Levels from the menu 3 Enter new file names in the Invert Gels box Note that instead of changing each file name individually you can add an extension or replace the extensions of the original image files You can also change the destination folder 4 The inverted images are saved on the hard disk and opened in the ImageMaster window er ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 117 ED 118 6 6 7 Cropping gels With ImageMaster you can crop your gels That is you can create new gels that only contain the defined regions of selected gels together with the spots and annotations that were located in those regions To crop gels 1 Define regions in selected gels using the Region tool Delimit a particular area in each gel or use the Shift key to select the same region in all gels Choose File gt Save As from the menu For each selected gel enter the destination folder and file name Click on Save ImageMaster asks you if you want to save only the selected area Answer Yes New gels ar
229. latinum User Manual 11 0034 38 Edition AA Annotations 3 To check several boxes rows simultaneously within a given category column 1 Select the rows for which you would like to change the checked state 2 Hold down the Shift key while clicking in the check box of one of the selected rows obviously in the relevant column All selected lines are now in the same checked state Click in the check box a second time if the state should be inversed Updating gels Subsequently the Update Gel icon should be used to make the modifications definitive and to propagate the information to your gels Note that the cells of updated labels return to their original color When all labels in your report are updated the asterisk behind the report title also disappears NOTE You should be aware of the fact that the annotations displayed in the Intra class and Inter class Reports are those from the master gel This means that any modifications including the creation of new columns corresponding to categories and the addition of new labels can only be applied to the master gel Note that this is not really a problem since you can easily propagate selected labels to matched spots see Section 8 6 2 8 6 4 Importing labels and annotations You can import annotations and labels from a file into open gels You can import labels from a Label Report for example or from any other tab delimited text file containing the columns Category
230. lease note that the string text must first be inserted followed by the text you would like to associate with the spot To connect general information about the gel other tools are better adapted Comments can be attached to match sets and classes folders and projects in the workspace as well as to any report that is saved on the hard disk Additionally you can specify gel descriptions see Section 6 7 2 8 4 Selecting annotations and labels 8 4 1 Annotation tool Annotations and labels can be selected with the Annotation tool as long as they are visible on the gel The selected labels or annotations are highlighted in green and displayed in front of the other annotations Figure 8 4 If the annotation is attached to a spot the spot is also selected Remember that the opposite is also true if you select a spot with the Spot tool the attached annotation is selected as well To select a label make sure the Annotation tool is activated and then click on the label You can select more than one label by using the Shift key To select an annotation activate the Annotation tool and click on the annotation basis To select more than one annotation select the first one then hold down the Shift key and select additional annotations ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Annotations 3 161 8 Annotations 162 a Figure 8 4 a Annotation hid
231. lign Aligned spot coordinates if the gels were aligned Shape Spot shape descriptions using directional chain codes Matches Exports all match information between all or selected spots in the exported files 11 2 4 Import gel data Similar to the export function gel data can be imported from an XML file previously generated by ImageMaster or produced by another application the file should contain the appropriate tags recognized by ImageMaster To import gel data 1 Select a single gel you want to input data from an XML file 2 Choose File gt Import gt Gel Data from XML from the menu 3 Browse the appropriate folder and pick the desired file name 4 If data from several gels is present in the file ImageMaster asks you to specify the gel from which data should be imported 5 Select the data to be imported from the Import Gel Data from XML File dialog box See below for more details about the items that can be imported Some options may be inactivated if the necessary information is not present in the opened file 6 ImageMaster imports the desired information The following items can be imported from an XML file Figure 11 2 Gels You can import the following data from existing gels 240 Calibration Intensity calibration parameters Slope and Offset Descriptions User defined gel descriptions entered in the Gel Description Report ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data integratio
232. lities between matched spots 10 2 2 Descriptive statistics Statistical analyses of the spot values within a class of gels allow you to analyze variations in protein expression among gels ImageMaster provides the most commonly used descriptive statistics of central tendency and dispersion which can be calculated and displayed in the Intra Class Reports and Histograms ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis These descriptive statistics are in fact numbers that summarize the spot values from a match The central tendency allows you to localize the central value representing the data whereas the dispersion indicates how closely the data points fall around the center Based on these measures you can isolate matches composed of spots whose quantification values are unusual The detected variations can be due to an inadequate detection or matching operation or can result from protein expression changes among gels Therefore these statistics are not only useful for analyzing extracted data but also for verifying process The descriptive statistics can be chosen with the following interface Figure 10 2 Central tendency Mean 4 bj 100 C Midrange C Reference Dispersion Mean squared deviation Ki 100 C Mean absolute deviation C Half range size Figure 10 2 Statistics common to the Intra Class Report Intra Class Histograms Inter Class Report and Inter Class Histograms
233. lues supplied with your step tablet are generally expressed in diffused density DD you have to convert them to OD values For this purpose the manufacturer of the step tablet should provide the appropriate relationship For the Kodak Step Tablets no 2 and 3 for example this is OD 1 4 DD ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 107 ED 108 To create a calibration once a correct Calibration Tablet File is generated 1 Scan the step tablet and import the image file into the ImageMaster software If necessary rotate the image such that the light steps are displayed at the top Choose Tools gt Calibration Tablet gt Create inthe menu The Load Step Tablet Definition box opens Browse the folder where you saved the Calibration Tablet File specifically tailored to your step tablet see above and Open the file A red calibration step overlay appears on the image and the Create Calibration window is displayed Figure 6 17 Select the Spot or Annotation tool in the ImageMaster toolbar and adjust the position of the steps by dragging the overlay while holding the left mouse button To help you differentiate the darkest steps you can adjust the contrast Please note that the size of a step on the red overlay not the red box but the distance between two short horizontal lines should correspond exactly to the size of a step on the image If this is not the case you must adjust the height of the tablet in the
234. lysis of two dimensional electrophoresis images Il Algorithms Electrophoresis 18 2735 2748 Appel RD and Hochstrasser DF 1998 Computer analysis of 2 D images In Link AJ ed Methods in Molecular Biology Vol 112 2 D Protocols for Proteome Analysis pp 363 381 Totowa NJ Humana Press ExPASy Molecular Biology Server 2003 The Melanie 2 DE analysis software Online http www expasy org melanie Miller MJ Olson AD and Thorgeirsson SS 1984 Computer analysis of two dimensional gels automatic matching Electrophoresis 5 297 303 Pun T Hochstrasser DF Appel RD Funk M Villars Augsburger V and Pellegrini C 1988 Computerized classification of two dimensional gel electrophoretograms by correspondence analysis and ascendant hierarchical clustering Applied and Theoretical Electrophoresis 1 3 9 Vargas RJ 1996 Two dimensional gel electrophoresis computer analysis systems from image acquisition to protein identification Ph D thesis Faculty of science Geneva University Wilkins MC Hochstrasser DF Sanchez J C Bairoch A and Appel RD 1996 Integrating two dimensional gel databases using the Melanie II software Trends in Biochemical Sciences 21 496 497 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Appendix 275 Appendix 276 C 2 Statistical methods Armitage P and Berry G 1987 Statistical methods and medical research Oxford London Blackwell Scientific Publications Kim JO and Mueller
235. m of all the sample values divided by the sample size e by just selecting Midrange the midpoint of the sample value is calculated that is the middle location between the two sample extremes e to obtain a Trimmed Mean or Midrange you should select Mean or Midrange and discard the desired percentage of outliers with the percentage slider e to obtain the Median you should select Mean and discard 50 of the values at each of the extremities that is select 0 in the percentage slider e byselecting Reference the value from the spot in a specified reference gel is taken as the central tendency Dispersion The dispersion measures the variability of the sample data as indicated by how clustered or scattered the data points are around their center value There are numerous measures of variability standard deviation range interquartile range and many others Like the statistics for central tendency these measures make use of all the available sample data and can be heavily influenced by outliers Therefore you can also restrict the sample to the central values by trimming out the extreme values with the percentage slider e by selecting Mean Squared Deviation M S D the square root of the average squared difference of each sample value to the center location is calculated e by selecting Mean Absolute Deviation M A D the mean of the absolute difference between each value and the central value is calculated It is n
236. match gels with minimum user intervention Nevertheless sometimes you may need to help the matching process by specifying a few starting matches or landmark annotations see below Before adding any landmarks or starting matches we recommend trying to match your gels with only a single landmark or possibly none at all In some cases no landmarks will be required More often a single landmark is sufficient for quick and efficient matching If matching takes a long time and no matches seem to be found the counter shown during matching always shows 0 or increases slowly you may want to cancel the matching procedure and define a few landmarks Subsequently you can repeat the automatic matching procedure below To match the gels in a match set 1 Open the match set in a new MatchSet worksheet 2 Select the gels to be matched in the worksheet 3 Choose Edit gt Matches gt Match Gels from the menu 4 If matches already exist you are asked whether you want to keep them or not If you answer Yes ImageMaster keeps the existing matches and ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 181 9 Matches 182 just looks for additional ones If you click No the matching procedure is started from scratch only annotations of the landmark category if any are considered 5 All selected gels are matched with the master image Please note that the matching step might take some time depending on your computer capacity 6 W
237. match set 1 2 Right click on the MatchSets folder or a match set Choose Create MatchSet from the contextual menu In the Create MatchSet box specify a Name and Comment Click OK The MatchSet Name appears in the MatchSets folder or match set chosen in step 1 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 49 50 To create a match set from a gel folder 1 In the Gels folder select one or more folders containing gels with saved spots Use the Shift or Ctrl keys to make multiple selections Left click on one of the folders a Hand cursor appears Drag and drop the folders into the MatchSets folder or into a destination match set Or right click on one of the gel folders and choose Copy from the contextual menu Right click on the destination match set and choose Paste from the contextual menu In the Create MatchSet box specify a Name and Comment Click OK Repeat for every copied folder New match sets appear in the MatchSets folder or destination match set They contain the gels from the copied gel folders Right click on the MatchSets folder a match set or gel to open a contextual menu from which you choose an action to be carried out Detailed descriptions of all the MatchSets folder menus are provided in Appendix A 4 6 2 Creating a match set from a DIGE gel A DIGE gel is an inherent match set As the different images in a DIGE gel have identical co detected spots ImageMaster automatically create
238. me User Name and Password field are used for exporting gel data to the Bruker ProteinScape database If the Bruker tools are not installed these parameters are inactive grayed out A ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 31 Getting started 32 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace 4 Workspace 4 1 Introduction The Workspace is the command center of ImageMaster It can be seen as the place where all gel matching and analysis data is centralized and from where all operations carried out in the software are controlled It is where you perform the following actions e Manage gels match sets classes reports and documents e Import new gels and directly convert them into ImageMaster format e Open gels for viewing and processing e Define match sets and reference gels match sets for matching e Create classes for statistical analysis e Add comments to describe and explain your experiment e Save all this information in the workspace file with the extension mws The workspace is a systematic and efficient way to clarify your research and avoid unnecessary work The workspace ideally should reflect the structure and design of your research This encourages you to think right from the beginning about the way the study should be constructed and about the information you would like to obtain from your analysis The workspace also guides you through the gel analysis wor
239. ms Section 10 2 3 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis 213 Data analysis Comments on factor analysis Factor analysis is used to examine the protein expression pattern within each match The quality of the factor analysis output depends on the quality of spot matching Hence it may be useful to exclude spots that are not well matched across all gels using the Select gt Matches gt Refine Selection function from the menu as described in Section 9 3 1 Nevertheless in cases where a majority of spots were properly matched the inclusion of all matches in the factor analysis can yield good results with no preliminary match filtering necessary This statistical method based on data variation and their standard deviations highlights the natural formation of classes among the gels and allows identification of matches i e matched spots that are characteristic of these classes However one should be very critical when analyzing factor analysis plots as the results can be greatly influenced by outliers bad matches and so on Factor analysis can provide valuable indications in some cases but not in others Intra Class Histograms xj als m e Value gt Histograms on Matches Yol The chosen statistics are Mean 100 and M S D Adaptive gradations F Sorted values 214 Figure 10 14 Intra Class Histograms ranked based on their contribution to the second
240. n EEE Spots Imports spots Note that spot shape descriptions using directional chain codes need to be present in the file Annotations Imports both pixel and spot annotations NOTE When several types of objects are imported at the same time all import operations should take place correctly If this is not the case because of ambiguous or erroneous file formatting none of the objects are imported Choose data to be imported Gel F Descriptions Calibrations Annotations Spots Figure 11 2 Import Gel Data from XML File dialog box 11 3 Exporting to spot excision robots Please note that in addition to exporting spots to an excision robot it may be useful to annotate the exported spots This allows you to easily select them later on for adding experimental data such as mass spectrometry information 11 3 1 Bruker Proteineer SP spot picker ImageMaster can export spot coordinates directly to the Bruker Proteineer SP spot picker For more details about this functionality please see the documentation provided by Bruker Daltonics To export a spot coordinate file to the Bruker Proteineer SP 1 Select all spots to be cut with the spot excision robot 2 Choose File gt Export gt Spots to Picker gt Bruker Proteineer SP in the menu 11 3 2 GE Healthcare Ettan spot picker To use the Ettan Spot Picker two adhesive markers should be placed on the gel before scanning These markers are used for the calibration of the coordi
241. n If some matches are already selected this option deselects the selected matches and selects all the unselected ones thus inverting the selection criteria Multiple Matches Selects only the multiple matches in the selected gels see below Refining a match selection ImageMaster allows you to refine an initial match selection based on the presence or absence of matched spots in the selected gels By using the sliders in the Refine Match Selection window Figure 9 2 you can choose the threshold x for the refinement of the matches corresponding to selected spots Selected spots is gt X Keeps matches that have selected spots in at least X of the chosen gels Selected spots is lt X Keeps matches that have selected spots in less than X of the chosen gels Unselected spots is gt X Keeps matches that have unselected spots in at least X of the chosen gels Unselected spots is lt X Keeps matches that have unselected spots in less than X of the chosen gels Refine Match Selection Keep the Matches whose number of selected spots is gt Ki 6 selected spots is lt unselected spots is gt unselected spots is lt Figure 9 2 Refine a match selection based on the number of selected spots in each match ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA For example suppose you have a set of 6 matched gels for which you would like to study the matches that contain at least 3 spots with a r
242. n categories and their labels together with annotation coordinates and Spot ID if the annotation is linked to a spot Category Information on all labels belonging to selected categories The displayed information is the same as for the report on labels Match A list of selected matches among a number of selected gels in terms of corresponding Spot IDs Match Statistics A list of the matching results number of matches and percentage of matches between the master and the selected gels 5 1 2 Analyze menu From this menu you can display reports and histograms by Intra Class Information about each selected match such as its Match ID value for each spot in the match and chosen statistical measures calculated on all spots in the match Scatter Plots also provide information such as slope offset correlation coefficient and fitting error that compare the spot values for two gels Inter Class Central tendency dispersion and overlapping measures for classes of gels computed for all selected matches Differences between the spot values in two classes can also be quantified with Statistical Tests such as the Student t test Mann Whitney U test and Kolmogorov Smirnov test Also look at DIGE Reports and histograms on DIGE gels display information such as Volume Ratios and frequency distribution of these ratios Heuristic Clustering Classifies sets of gels and h
243. n earlier version of ImageMaster you will be asked if you want to convert it If you click Yes ImageMaster migrates the workspace so that you can use the new features in ImageMaster 2D Platinum 6 0 NOTE Once you open and save a workspace in ImageMaster 2D Platinum 6 0 you are no longer able to work with the file in an older version of ImageMaster Be careful not to open version 6 0 workspace files in older versions because this can corrupt your workspace file 4 3 3 Workspace properties The Workspace Name Creator long File Name Modification Date and Comment are Workspace Properties The Comment is helpful because it describes and or explains your workspace It serves as a useful source of information to which a coworker is referred or as a reminder when reviewing old work You can add or modify the Comment or Workspace Name while viewing the Workspace Properties ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 4 4 Workspace To edit the Workspace Name or Comment 1 Right click on the Workspace Name at the top of the Navigator 2 Choose Properties from the contextual menu 3 Inthe Edit Workspace Properties box add or modify the Workspace Name or Comment Click OK 4 The changes to Properties are saved Working with projects A workspace can contain different projects A project normally includes all gels along with related data produced and analyzed during the course of a specific gel study In p
244. n illustration or just want to see what lies underneath To interactively change an annotation s flag position click on one of the labels and drag the flag to the desired position Figure 8 6 s T PO7016 in TD ECOLT a b Figure 8 6 a Default and b modified annotation flag position 8 5 2 Annotation flag color As mentioned inSection 8 2 each predefined label category is automatically created using a different background color pink for the Ac category blue for landmarks etc to help you visually identify the various label categories You can change the default colors by going to the Categories tab in the ImageMaster Options accessible by choosing Tools gt Options in the menu selecting an annotation category and modifying its Color in the Display properties The new ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 167 8 Annotations 168 background color is used for all labels of that specific category in the current workspace 8 5 3 Annotation flagpole color Annotation flagpoles can be displayed on the screen using three different colors other than the default cyan To change the color of the annotation flagpoles 1 Select the annotations for which you would like to change the flagpole color 2 Choose Show gt Annotations gt Set Color in the menu and select one of the proposed colors Please note that the Default option corresponds to cyan 3 Th
245. n such a way that the best fit line approaches identity This normalization therefore minimizes the number of spots that are expressed differently and can be used to compensate for sample loading variations ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA To normalize intensities based on a scatter plot 1 10 Select the gels that might need normalization including the reference gel to be used for the scatter plots The gels and the reference gel should be matched belong to the same match set Select all matches Display the scatter plots by choosing Analyze gt Intra Class gt Scatter Plots Make sure you choose Intensity as value type This is important because normalization does not work with other value types If the slope in a scatter plot is close to 1 and the offset close to 0 then normalizing the data is not appropriate However if these values are not close to 1 and 0 then try normalizing your gel using the following procedure Select the scatter plots of the gels that need to be normalized Choose the Fitting Report option from the Reports drop down menu in the toolbar of the Scatter Plots window and save this Fitting Report The Fitting Report gives the gray slope gray offset and correlation values for the scatter plot best fit line for each gel as well as the number of matches that were selected to display the scatter plot If you only select some of the lines in the report you will get the chance
246. nal to the spot intensity It can be rotated in any direction to view the interesting spot s from all sides thus facilitating spot editing or matching decisions ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Show Adjust Contrast BE imageMaster 2D Platinum File View Edit Show Select Analyze Reports Tools Window Help zie Gels i Spots fl Annotations i M Lighting I Spots overlay Figure 6 15 3D View for the active region of gel A_T1_Gell To display the 3D View of a gel region 1 2 Select a region in one or more gels Or select one or more spots Choose Reports gt 3D View in the menu When both spots are selected and a region is defined ImageMaster will ask whether your 3D View should be based on the selected region or selected spots ImageMaster displays a 3D View for the region or the area containing the selected spots Figure 6 15 shows the region in gel A_T1_Gell and the corresponding 3D View window At the top of this window you see the typical toolbar You can turn the gel by clicking on the arrows at the right side of the gel image By default the three axes are displayed the X axis in brown the Y axis in blue and the Z axis in purple You can change the point on which the axes are centered by right clicking on the desired position This position then becomes the new center of the image Note that this is a way to move the view up or down depending on the position yo
247. nates that is for determining the correspondence between the X and Y positions of the il ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 241 Data integration 242 analyzed gel image and the coordinates of the actual gel located on the spot picker Figure 11 3 Once the gel has been digitized and analyzed with ImageMaster 2D Platinum the software can generate a pick list This list contains the location in pixels of the center of each spot you wish to pick as well as the pixel coordinates of the centers of the two reference markers To export a file with spot coordinates for use by the spot picker you first have to open the image files and perform image analysis spot detection is mandatory You should then annotate the reference markers If a reference marker is well detected during the automatic spot detection process nice round spot perfectly centered on the marker you can just add an annotation on the marker spot The basis of such an annotation is displayed as a small square and its coordinates correspond to the center of the spot If a reference marker is not well detected irregular shape or consisting of several spots it is better to delete the existing spot s and create an annotation on the pixel that is in the center of the marker This kind of pixel annotation has a crossed basis and its coordinates correspond to the pixel it is attached to Note that the two options can be used in a single gel one marker
248. nction so that it can be applied at a later time to other gels with similar gray level properties To do this click on the Save icon in the Image Display Settings window and enter a file name as well as the desired destination folder ImageMaster saves the mapping ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 99 ED 100 function with the extension glt Gray Levels Transformation The mapping functions stored in the default folder ImageMaster Menus folder in the user s My Documents directory are always available from the Open icon in the Image Display Settings toolbar It is possible to load the gray mapping function of any open gel by selecting the gel s name from the Current list in the dropdown menu attached to the Open icon in the Image Display Settings window Unit Two different units are used for displaying the gray level minimum and maximum e Value Uses the raw pixel values as displayed in all the reports When a calibration is done these correspond to the calibrated pixel values e Percentage Chooses the scale as a percentage of the total gray level range in the histogram Only in region By checking the Only in Region box in the Image Display Settings window ImageMaster only considers the gray levels that are present in the selected region Only in Region is very useful in combination with the Unit and the choice of a relatively small region In this case you enter an adaptive mode that allows you
249. nd choose Reports gt Gel Calibration gt Plot to view the calibration curve See below for details about the calibration curve Creating a calibration If you intend to calibrate the image capture device yourself you need to scan a calibration step tablet or calibration strip along with your gels These step tablets have known intensity values expressed in optical density OD or diffuse density DD published by the manufacturer of the step tablet Please note that for the purpose of 2D gel analysis it is only useful to calibrate the image capture device when working in transparent mode Normally no calibration needs to be done when you do reflective scanning With some equipment both a transparent and a reflective calibration strip are provided When calibrating be sure to use the appropriate calibration step tablet NOTE The OD values for the step tablet have to be specified in a Calibration Tablet File together with other information such as the height and width of the tablet and the number of steps An example of such a Calibration Tablet File Kodak2 tab can be found in the Template Tablet folder of the ImageMaster installation directory This Calibration Tablet File is made for use with the Kodak Step Tablet no 2 If you do not use this specific step tablet you can copy the file and edit the data to make your own Calibration Tablet File You can edit the file with tools such as Windows Notepad As the intensity va
250. nd 3951 in another gel In further data analysis and quantification e g Vol in the Intra Class Report the two spots are considered as being one the volumes have been summed 9 3 2 Reports You can select matches directly from a displayed Match Report see Section 9 6 1 You can do this by double clicking on a row in the report or by selecting one or more rows and clicking the Select on Gels icon in the report toolbar 9 4 Displaying matches A match describes the relationship between individual spots As mentioned above selecting matches corresponds to selecting their constituent spots However this is not always a straightforward way to visualize the correlation 186 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Matches ex EEE between spots ImageMaster therefore provides the following features for easier visualization of matches 9 4 1 Show hide matches Matches can be displayed as blue vectors that link the locations of the spots in the displayed gel and the matched spots in the master image Figure 9 4 These match vectors are always minimized with respect to the center of the visible gel area and enable you to evaluate the matching results more easily You can change the default color for the match vectors by choosing Tools gt Options from the menu and going to the Display tab To display match vectors 1 Open a MatchSet or Classes worksheet 2 Choose Show gt Matches gt Show Vectors f
251. nd MW estimates When the original image data of the gels is loaded the raw pixel values are shown Note that if spots are detected then the Cursor Information window also displays spot information ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 95 ED To display cursor information 1 Choose Window gt Cursor Information in the menu 2 Inthe Cursor Information window click on the Settings icon to specify the information and display order see Section 5 4 1 for more details on how to change save and load settings Click OK 3 Position the cursor over the pixel you want The Cursor Information window displays the related information If the Value field is unloaded the raw pixel values are not available In order to see the raw image data choose Edit gt Gels gt Raw Image gt Load in the menu 6 4 3 Displaying gray levels A 2 DE gel image is traditionally displayed as a gray level image where gray levels represent the signal intensity Sometimes the displayed gray levels are so low that small spots are hardly visible In order to emphasize these very faint spots you can adapt the brightness and contrast of the image You can also display images using pseudo colors These types of operations are carried out with the Adjust Contrast function Generally it is useful to preview the modifications that will be done to your image To get such a preview it is sufficient to select an interesting region in your gel
252. ndow Tabbed groups Dockable windows can be organized into tabbed groups This feature extends your ability to maximize the use of limited screen space by combining multiple dockable windows into one window In order to form a tabbed group drag the title bar of a dockable window into the center of another You will see the nested tabs at the bottom of the docked window In order to separate a tabbed group drag a tab away from the docked window or double click on the tab The workspace cannot be added to a tabbed group In addition tabular reports and graphical reports cannot be grouped together 3 5 ImageMaster options You can set various parameters that influence your work in ImageMaster These settings are accessed by choosing Tools gt Options in the menu More detailed information about the options are provided in the related chapters of this manual However an overview of the settings per tab is given below General e Raw Image Indicates whether the raw image data of newly opened gels should be kept in memory If the available RAM on your computer is low then it is better not to use this option See Section 6 4 1 for details on loading raw image data Cursor information e Set the attributes to be shown in the Cursor Information window see Section 7 9 Display e Indicate the default spot colors for normal and selected spots and spot shapes to be used Crossed Outlined Filled Outlined Filled Choose the color
253. nds to the area at 75 of the spot height when measured from the peak 132 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA When detecting spots ImageMaster also computes the Saliency for each spot see Section 7 3 2 It is displayed together with the above mentioned quantification values in the Cursor Information window or a Spot Report 7 4 Co detecting spots in DIGE gels The co detection algorithm is designed to simultaneously process 1 2 or 3 images derived from a single gel e Single detection one image e Double detection two images e Triple detection three images Single detection is performed on images of fluorescently post stained gels used for picking a case where there is a single image associated with the gel Double and triple detection takes advantage ofthe inherent comigration benefits of the CyDye DIGE Fluor dyes A set of co run images 2 images in double detection and 3 images in triple detection are merged together thereby incorporating all spot features in a single image Spot detection and spot boundary definition is then performed using pixel data from all the individual raw images and the merged image The resultant spot map is overlaid back onto the original image files Since the spot boundaries and the detection areas are identical for all images the spots are effectively already matched This process results in highly accurate volume ratio calculations 7 4 1 Procedure To perform spot de
254. ng report contentu ssu seeunssuuusteusssunneemseasn 54 Customizing reportsu aueansensueneuneusnananmaunkunn 5 4 1 SOLU GS a alerts E EAO bir 5 4 2 ColUMmn Order e n 8 asian S S 5 4 3 Column and row size 5 4 4 Sorting data sed 5 5 Mouse selection reports oiceeeccssssssssssseeesssssccscsssssssssusssesssscsessssssssssseessssesessen 6 Gels 6 1 ZHtLOdUELON Dieses ren hn 6 1 1 Image f rmat an a na Eileiheikinusen 6 1 2 Image resolution 6 1 3 Imagesdepthun ena2u Rei 6 2 Selecting gels and gel regions u 80 6 3 Displa ing Gels isasi AARNA 82 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA v Contents vi 6 3 1 MOVING GEIE nlnsenssessnn sense nenne 82 6 3 2 ZOOM GEIS sis sce E cee eee erie lca ae ern eee A 83 6 3 3 Scrollbai Sns E E RRRA 87 6 3 4 TFQAS POLE CY En RER RER 88 6 3 5 GFIG NINOS a Rena Ne IE 6 3 6 Aligning gels 6 4 Viewing signal intensity on ececccccccsssssssssssesssessscccccssssssssuussesssssecceessssssseeees 94 6 4 1 Loading unloading GEIS eessssssssssssssssssssssssssecsessesseseseessessssssssssssssissssnuueaeees 94 6 4 2 CUFSOF INTORMEHION aaassccitsssisessesecosdtesianctecavnzesitsctsseststcbeugetessccesessceabienaatas 95 6 4 3 Displaying gray levels ceeccsessssssssssssssssssssssssssssssessessseseeseseeeseseesssessssssssssssesss 96 6 4 4 Contrast mapping 6 4 5 ROVERE EEA E teeter NED ee 6 4 6 SID VICW i e einama a a E EREN 6 5 Calibrating and normali
255. ngs icon in the report toolbar to customize the report see Chapter 5 for more details on report customization ee ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 119 ED Gel Report x S e Ie o Bl Information on selected Gels FileName MatchSet Gray Slope Gray Offset MinGray MaxGray 94 0002 Ecoli New matching 1 28091 25 6309 63 0000 2211 00 94 0005 New matching 1 00000 2355 00 ECOLI Ecoli New matching 1 00000 2361 00 Master_New matching 1 00000 2361 00 Figure 6 19 Gel Report The gel 94 0002 was normalized as revealed by the asterisk next to its name and the values for Gray Slope and Gray Offset 6 7 2 Gel description report Besides the above information on selected gels you can enter experimental data about your gel images to be used for later reference by yourself or any colleagues This information can include sample type date of the experiment operator name pH range SDS gel percentage or staining All this data can be entered in the dedicated Gel Description Report and stored in your gel files Figure 6 20 The Gel Description Report is available from the Reports menu Initially no descriptions are defined in the report and only a column with the gel names is available To define descriptions for your gels 1 Select the gels for which you want to add descriptions 2 Choose Reports gt Gel Description Report in the menu 3 The Gel Description Report is di
256. notation and drag its basis to the new location When you drag an annotation linked to a spot outside of that spot ImageMaster asks if you want to remove the link with the spot Similarly when you move an annotation onto a spot ImageMaster asks if you want to link it with that spot 86 2 Edit menu Various options for modifying copying pasting duplicating and deleting labels or annotations are available from the Edit gt Annotations menu With the exception of the Modify function all of these features can be used to edit add delete modify several annotations at a time Modify Once an annotation is selected you can change the content of its labels and add new categories by choosing the Modify option and entering the desired modifications in the Edit Label box Please note that only one annotation can be selected at a time When picking a single label only the content of the selected label can be modified Copy paste annotations You can select annotations in a given gel image and copy them to the corresponding locations in other gels This is a simple means of creating a set of similar annotations in a series of gels and can be useful for quickly adding landmarks for instance Subsequently you just need to adjust the annotation positions ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 171 8 Annotations 172 To copy annotations from one gel to a set of other gels 1 Select the annotations you would like to
257. nse file should be placed in the ImageMaster installation folder which is by default C Program Files GE Healthcare lmageMaster 2D Platinum For a floating license the file should be in the folder C Program Files GE Healthcare eLicense server Licenses on the computer running the license server see Section 3 2 3 To collect and place a license file 1 Goto the web site http www elicensing amershambiosciences com gtlweb login 2 _ Enter the access code and press Continue 3 Click on Collect Licenses 4 Step 1 Select the product to collect mark the check box 20 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Getting started 5 Step 2 Register your product 6 Step 3 Enter the Physical Address Host ID of the computer where you will install the license file The expected type of Host ID shall by default be ETHERNET Ensure that the Physical Address is correct Click on Continue 7 Ifthe data shown in the next screen is correct select to Collect License Otherwise use Back to edit 8 Save the license to a file or e mail the license It is strongly recommended to save the license file Close the Download Complete window after saving the file If the license is e mailed the content is delivered as raw text in the e mail body The text must then be copied to an ASCII text file given the extension lic 9 Place the license file in the appropriate folder if this was not already done when saving the licen
258. nstall ImageMaster from a CD ROM or by downloading the installation package over the Internet When you insert the CD ROM into the appropriate drive on your computer the Setup Wizard starts automatically and guides you through a series of screens Alternatively you can double click on the icon of the installer file for example SetupIMPv600 msi to launch the install program The ImageMaster installer creates a default directory on your hard disk called Program Files GE Healthcare lmageMaster 2D Platinum in which the program files are placed If you want to save the default directory in a different folder then browse and open the folder before continuing the installation ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 17 Getting started 18 Once installation is complete it is recommended to restart your computer 3 2 eLicensing An electronic license eLicense file is required to enable ImageMaster to run once installation is completed There are two types of eLicenses e Node locked license Machine license This type of license is for a single computer It is practical when only one or a few computers are used for working with ImageMaster A node locked license file must be placed on the computer running ImageMaster e Floating license Concurrent license This type of license can be used by all computers connected via network to a computer with a license server installed It is useful when many users but not
259. nt The Spot tool lets you select or edit individual spots The tool k changes when in the Edit Enabled mode The Annotation tool is used to add edit move or select ae annotations and labels 3 4 3 Display zone Gel images are opened from the Workspace see Chapter 4 for details and subsequently displayed in the Display Zone Figure 3 4 The Display Zone is laid out in three levels Worksheets Panes and Images Their layouts can be customized according to your requirements A Status Bar is located at the bottom of the Display Zone 24 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Getting started EN imageMaster 2D Platinum lol x File View Edit Show Select Analyze Reports Tools Window Help MA N a9edsy1o Gek B Spots psi Annotations jo d Figure 3 4 The Display Zone a Worksheet b Pane c Image and d Status Bar Worksheets Images are always displayed in worksheets Each worksheet has a banner which is the horizontal bar located at the top containing the worksheet name and data set type enclosed in brackets The color of the worksheet banner indicates whether it is selected green or not gray The selected worksheet is always at the front of the Display Zone In order to select an inactive worksheet click on its banner The worksheet is brought to the front of the Display Zone To rename a worksheet 1 Select the worksheet
260. nt presentations or mass spectrometry data The Documents folder can also contain pick lists and graphical files such as histograms 3 D views scatter plots factor analysis projections exported gel regions or gel windows with bmp tif or png extensions When saving a graphical file in ImageMaster it is automatically inserted into the Documents folder of the source Project 4 9 1 Creating a subfolder You can create folders and subfolders in the Documents folder in order to better organize your project related files To create a subfolder 1 Right click on the Documents folder or a subfolder 2 Choose Create Folder from the contextual menu 3 Inthe Create Folder box specify a Name and Comment for the new folder and click OK 4 The Folder Name appears in the Documents folder Right click on the Documents folder a subfolder or file to open a contextual menu from which you choose an action to be carried out Detailed descriptions of all the Documents folder menus are provided in Appendix A ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 4 9 2 Adding files As mentioned above any project related file generated by any software can be inserted in the Documents folder of the project To add files 1 Right click on the Documents folder or a subfolder 2 Choose Add Files from the contextual menu 3 Inthe Add Document Files box browse the directory where the files are located select the names and click Open Use the
261. num User Manual 11 0034 38 Edition AA NOTE When using single detection the volume ratio value is 1 0 for all spots since there is no second image 7 5 Selecting spots 7 5 1 Spot tool Spots can be selected with the Spot tool as long as they are visible on the image Once selected they are highlighted in green Figure 7 3 unless the default spot colors were modified see Section 7 6 2 If an annotation is attached to a spot the annotation is also selected Similarly if you select an annotation or label with the Annotation tool the linked spot is also selected To select a spot make sure the Spot tool is activated and then click on the spot To select more than one spot select the first one and then hold down the Shift key while clicking on additional spots To select all spots in a region position the cursor at the top left position of the desired region hold down the mouse button and then drag the cursor to the bottom right position All spots in the designated region will be selected and highlighted in green To deselect all spots select the images in which you would like to deactivate all spots and click in one gel not on a spot 7 5 2 Select menu Additionally you can select spots by using the items in the Select gt Spots menu Some of the following are particularly useful for filtering out artifacts especially Refine by Value possibly in combination with Inverse Selection e By ID Allows the
262. o defined place in the general procedure of an image analysis study 1 3 2 Tutorials You can familiarize yourself with ImageMaster 2D Platinum by working through the online tutorials These tutorials offer a step by step guide on how to analyze your gel images and report results They are supplied with gel images and related files enabling you to carry out an entire image analysis using the various tools available in the software You can access the different tutorials from the Help menu in the main program window For each of the tutorials the option Help gt Tutorials gt Tutorial X gt Open will prepare your environment restore the necessary files and display the tutorial text The option Help gt Tutorials gt Tutorial X gt Result shows the expected outcome after running the corresponding tutorial 1 3 3 Online help The user manual is also available online from within the ImageMaster software To consult the online manual 1 Choose Help gt User Manual from the menu 2 ImageMaster opens the online document 3 Find the desired section from the Contents Index or through a Search ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 1 3 4 Conventions used Before getting started with ImageMaster you should have a working knowledge of your computer s operating system and its conventions including how to use a mouse and standard menus and commands You need to be able to adeptly open save and close files If you wi
263. o dimensional gel analysis 16 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Getting started 3 Getting started 3 1 Installing ImageMaster 2D Platinum 3 1 1 System requirements In order to install and run ImageMaster your computer must satisfy the following requirements e Microsoft Windows 2000 or XP operating systems e Administrative permission to install ImageMaster e Atleast 256 MB RAM for ImageMaster 512 MB is recommended and 768 MB RAM for ImageMaster DIGE 1024 MB is recommended The amount of memory required is mainly determined by the number and size of image files to be processed simultaneously Increased memory therefore enhances the performance when many and or large images are analyzed e A high quality display To take full advantage of the software including the 3D View feature the color resolution should be set to 24 bit 16 7 million colors However a color resolution of 8 bit 256 colors is generally sufficient It is recommended to use a screen resolution of at least 1024 x 768 pixels e At least 60 MB of available disk space for program files and approximately 400 MB for the full installation of tutorials e Internet Explorer 6 Microsoft Corporation Netscape Navigator 6 Netscape Communications Corporation Mozilla 1 4 Mozilla Foundation or higher versions A browser enables you to print reports and to access scientific databases on the Web 3 1 2 Software installation You can i
264. o organize your gels into projects to define match sets and classes and to keep accompanying data such as reports and image documents in project related folders Your preferred ImageMaster settings are also saved in the workspace file Detailed information about the workspace is found in Chapter 4 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 13 Two dimensional gel analysis 14 2 4 3 Visualizing and calibrating gels The next step is to handle the gel files open save print close manipulate the gels images select move zoom stack align possibly transform the gels rotate crop scale and view the signal intensity adjust contrast profile 3 D view At this stage it may also be necessary to calibrate your gels Information about all these features is given in Chapter 6 2 4 4 Detecting and quantifying spots Once you are familiar with your gel images you can perform automatic spot detection In Chapter 7 you will learn everything you need to know about spots including how to select display and edit spots as well as how to view their properties and quantification values 2 4 5 Annotating spots and pixels Individual pixels and spots in a gel image may be labeled with annotations These annotations can be used in functionalities such as calibration alignment and matching or be utilized to mark spots with their particular characteristics Chapter 8 explains how to create use select and display labels categ
265. o the spot picker Annotate these spots with a category of type Set so that you can easily re select them at a later stage 2 Place your gel on the ProPic robot bed produce the ProPic image and open it in ImageMaster Use the gel image as is meaning do not resize crop flip or rotate the image 3 Align your analytical gel image to the ProPic image using a sufficient number of landmarks see Section 6 3 6 for details on how to align gel images 244 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data integration EEE 4 Select all spots to be cut with the spot excision robot from the analytical image 5 Make sure only the analytical gel image is selected 6 Choose File gt Export gt Spots to Picker gt Genomic Solutions ProPic 7 Enter a file name and destination folder The file automatically is given a tds extension TwoDSpotlist 8 ImageMaster exports the aligned X and Y coordinates for each selected spot That is the coordinate system of the ProPic image will be used 9 The spot picker subsequently reads the saved file 11 4 Connecting to external protein databases A key advantage of the ImageMaster program is its ability to link spots on gel images to protein data in 2 DE or other databases 2 DE databases contain information on proteins identified on 2 DE images such as pl and MW values bibliographical references to protein related literature information on protein functions etc ImageMaster can
266. on atta Litas control calibration cursor information D dataan lysis 35 differential gel electrophoresis DIGE cases add gelsto workspace histogram u import gels in workspace internal standard report dispersion display zone displayed actions dockable windows documents add files to workspace create folder in workspace open edit ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA menu edit spots eLicensing_ exit ImageMaster export Gelidatato XML une EHI 238 F factor analysis interpret bh IQ 10 ns oe ELAES aa ea Rvs ee file menu fitting report floating floating license folder DFOPETLES nn EAEE A E AOT 45 TaiK oA AEEA E EE E EE E EE E E 276 G GE Healthcare Ettan spot picker GE nn A A add to workspace align anatomy calibrate calibration report create folder in workspace crop delete description report erase export gray levels grid lines IDs import in workspace invert gray levels load image data move open print properties rename rotate synthetic unload image data ZOOM gels folder Genetix GelPix spot picker Genomic Solutions ProPic spot picker graphical user interface ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Index 281 Index 282 grid lines
267. on criteria and other protocols for later use and in case other people work with the report Comments are automatically saved in the report file when the latter is saved NOTE Notice that any comment can but need not be saved separately using the Save button in the Comment window Previously saved comments as with any text file can subsequently be opened and reused by clicking the Open icon Comments can also be printed copied or pasted as a whole or in part using the corresponding icons 5 2 3 Selecting and navigating Reports have the property of being navigable meaning that you may reselect on the gels one or more items gels spots matches etc that were picked in a report or histogram You can also scroll through the items listed in a report systematically viewing each of them on the gels by order of appearance Double click on a report line or histogram to select and display the corresponding object on the gells If you want to select several items or go through a list of items systematically use the following icons in the report toolbar Select on Gels When clicking on this button any items corresponding to the selected report lines or histograms use Shift and Ctrl keys for multiple selections are selected on the open gels The open gels are displaced in order to show at least one of the selected objects Select Next You can use this icon to select the following object listed in the rep
268. on selected matches for a selected class It is a kind of reduced report on matches where only the spot values of one class are considered e Inter Class Report Lists the numerical values corresponding to selected histograms e Fitting Report Scatter Report These reports are only available when displaying scatter plots and are detailed in Chapter 10 of this manual e DIGE Report Is a modified report on spots for DIGE gels which allows to display Volume Ratios between two selected images of the same DIGE gel in addition to the other quantification values for spots 5 3 Editing report content Some reports in ImageMaster on matches classes annotations labels and categories are editable although to different extents Within the Label and Category Reports you can edit existing annotations on any gel used for the creation of the report In the Intra Class Inter Class and Annotation Reports you can additionally create new columns corresponding to label categories and add new labels However as the annotations displayed in some of these reports Intra Class Inter Class are those from the master gel any modifications are only applied to the latter This is not a problem since you can easily propagate selected labels to matched spots see Section 8 6 2 The ability to edit reports gives you the possibility to easily mark any interesting data from within the report and to propagate this data to your master gel resulting in the
269. on the Annotate icon allows you to insert a category as an extra column by selecting its name from the category list containing all the categories present in the open gels You can create a new category by typing its name in the upper field of the displayed window In this case you are asked for the category constraints Adding or modifying labels Once you created or displayed the desired categories you can directly add new labels to the appropriate cells or edit existing ones Just double click in a cell to start typing your label When finished a single click in any cell quits the editing mode The cell corresponding to the modified label appears in dark green when the related line is selected or in gray otherwise You can quickly see that modifications were made to your report when an asterisk follows its window title Please note that when you want to maintain a current selection while editing labels you should hold down the Shift key while double clicking in a cell You can release the Shift key as soon as you are in the editing mode Notice that categories of the type Set or categories using the Boolean Data Type are displayed in the form of check boxes in some reports Editing their labels corresponds to checking the box in order to indicate whether the corresponding item belongs to a Set checked box or not empty check box or takes the value 0 empty check box or 1 checked box for a Boolean Data Type ImageMaster 2D P
270. ong a series of gels When two or more gels are selected this returns the common labels for all the categories chosen in the pop up dialog box When only one gel is selected this operation corresponds to selecting packed labels that is labels from the specified categories containing the same content see Section 8 5 5 e Inverse Selection If some labels are already selected this option deactivates the selected labels and selects all previously unselected ones thus inverting the selection criteria Regular expressions Regular expressions provide a mechanism to select specific strings from a set of character strings Regular expressions use symbols and syntax elements to describe a generalized pattern ImageMaster invokes the standard Extended Regular Expressions to search patterns in labels the essentials of which are summarized in Table 8 1 A ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 163 8 Annotations Syntax Description Example Matches any one character e oli matches eaoli eboli ecoli Matches any character listed between the brackets a z indicates the range of characters between A and Z and 0 9 is any numeral from 0 to 9 Pla d matches Pa Pb Pc Pd Matches any character except those listed between the brackets P bd matches Pa Pc Pe but not Pb or Pd atches the preceding element zero or one times P0 1 matches P1 and P01 Matches the p
271. option ImageMaster relies on the intensity of every pixel in the gel to display the Profile For this reason the raw image data are automatically loaded when using this utility If the memory resources on your computer are low please do not forget to unload the raw image data when you finish using the Profile function see Section 6 4 1 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 101 Gels 1 Spots D Annotations 0 Figure 6 14 Profile feature Red curves represent the intensity variations of the gel in the vertical left and horizontal top directions at the position of the mouse cursor Green lines indicate the exact position of the cursor whereas the numbers indicate the minimum and maximum gray levels in a specific Profile view To view profiles on your gels 1 Choose Show gt Gels gt Profile in the menu 2 Position your mouse cursor over a gel The horizontal and vertical profiles at the cursor position are displayed 3 Once the Profile feature is no longer needed choose Show gt Gels gt Profile from the menu 6 4 6 3D view Another way to examine the intensity variations in a gel is by looking at the three dimensional 3D view of a gel region Figure 6 15 In this type of view the X and Y axes represent the pl and MW values whereas the pixel intensity is plotted along the third dimension Z axis The resulting image shows a peak for each protein spot with a peak height that is proportio
272. or the rotated gel If you choose one of the predefined angles the rotated image is directly saved to the disk drive and opened in the ImageMaster window If you select the Free rotation option the image appears with a grid on it The bold horizontal grid line plays the role of landmark to help you visualize the rotation It becomes the new horizontal in your rotated image Make sure the Region Spot or Annotation tool is activated Then click on any position on the gel and rotate the grid while holding the left mouse button Release the button when the bold line is parallel with what should be the new horizontal in your image Figure 6 18 You can also manually enter a rotation angle in the Rotation Too dialog box Press OK to confirm your rotation spots and annotations are preserved or Cancel to return to the initial orientation of the gel ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA a 115 ED EN imageMaster 2D Platinum p lol x File View Edit Show Select Analyze Reports Tools Window Help alien Rotation Tool Turn the image A_T1_Gell_SavedRotmel by rotating the grid so that the bold line defines the new horizontal reference Angle 0 Figure 6 18 Rotation Tool The grid is rotated until its bold line is parallel with what should be the new horizontal reference When the mouse button is released the gel image is rotated 6 6 4 Flipping gels Sometimes images are sc
273. ories and annotations It further describes how to create links to external databases or data sources of any format text file html etc 2 4 6 Matching gels After spots were detected and match sets defined you can match your gel images In other words you find the corresponding protein spots in different gels Chapter 9 introduces you to all the necessary concepts and functionalities for gel matching and the creation of synthetic gels 2 4 7 Analyzing data Chapter 10 offers an overview of the powerful data analysis and classifications tools that are used to study the variations in protein expression among gels or classes of gels The data analysis step may be carried out at two different levels The intra class statistics tools include scatter plots descriptive statistics and factor analysis For inter class analyses the so called overlapping measures and various statistical tests can be used Heuristic clustering can help finding classes 2 4 8 Integrating data You may want to export spot coordinates to a spot excision robot export gel data to a database for example via XML format or import experimental information to be included in annotations The necessary tools are described in Chapter 11 of this manual ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Two dimensional gel analysis 2 4 9 Reporting results You can display information on specific gels and gel components spots matches classes annotations
274. orksheets In this case confirmation messages give the detail of the changes made and you have the choice to save them or not To save changes made to your gels 1 Select the gels for which you would like to save changes 2 Choose File gt Save gt Worksheet or Save All in the menu You may want to save multiple copies of gels or create files of defined regions by using the Save As command This is useful when you do not want to overwrite the previous version of the gel in order to keep track of your analysis or to return to a prior stage Also use this command to crop your gels or to save files in earlier formats Melanie II or Melanie 3 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 123 ED 124 To save gels with a different name or format 1 Select the gels to be saved 2 Choose File gt Save As from the menu 3 For each selected gel enter the destination folder and file name and pick the desired file format from the Save as type list To save gel regions 1 Select regions in the pertinent gels Define a particular region in each gel or use the Shift key to select the same region in all open gels 2 Select the gels for which a cropped region should be saved 3 Choose File gt Save As from the menu 4 For each selected gel enter the destination folder and file name and pick the desired file format from the Save as type list Click on Save 5 ImageMaster asks you if it should save only the selected a
275. ort or histogram Please note that the open gells are displaced to show the selected item in the center of the cell in which the gel is displayed Select Previous You can use this icon to select the preceding object in the report or histogram Again the open gells are displaced to show the selected item in the center of the cell in which the gel is displayed An example of how one can navigate through the lines of an Intra class Report is shown in Figure 5 1 In order to check the matching results for particular matches these matches were selected and a report on matches was generated 68 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Reports 5 I a EN imageMaster 2D Platinum lol x File View Edit Show Select Analyze Reports Tools Window Help 2 2 als Bal o Ari t e ela MES amp 7 Bl vaue Intra Class Report Select on Gels The chosen statistics are Mean 100 and M S D Match ID Mean 100 M S D Separability 4477 1 34843 0 392948 0 516089 4538 0 299013 0 397664 0 556158 4577 1 25715 0 562541 0 945710 4584 0 582289 0 407015 0 479449 4679 1 34106 0 887342 1 05500 4691 1 72898 0 594347 0 735561 Ol Ola Rlalny 3 43268 0 857316 1 15910 0 182636 0 260009 0 496155 0 641371 0 628942 0 978030 0 502048 0 513872 0 785745 2 03374 0 402549 0 433897 b E imageMaster 2D Platinum
276. orts and get a list of all open report windows Help Obtain support online access the official ImageMaster Web site or obtain information about the current version of ImageMaster Keyboard shortcuts Several menu options can be activated by keyboard shortcuts These are indicated at the right hand side of the corresponding menu option A list of all En ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 23 Getting started gt shortcuts is given in Appendix B Please note the logic behind the key combinations Ctrl is used to maneuver gels Shift is used to maneuver spots Alt is used to maneuver annotations Ctrl Shift is used to maneuver matches 3 4 2 Toolbar Objects on the screen can be displayed selected or processed using the various options in the ImageMaster menus However they can also be manipulated with the tools provided in the toolbar The fine points of these tools are explained in other sections of the User Manual but the main features are as follows When the Hand tool is activated you can move your image in thy order to show other parts of it The Magnify tool lets you repeatedly zoom in left click or out is right click on the whole image each time by a factor of two The Region tool is useful for selecting a rectangular area within os an image This region can be used for cropping gels selecting objects within a region or getting a preview zone for spot detection or contrast adjustme
277. ot affected as much by outliers as the Mean Squared Deviation since the differences are not squared e by selecting Half range Size the difference between the largest and the smallest values divided by two is calculated NOTE Notice that absent spots represented by zero values are also considered in the calculation of statistics for both central tendencies and dispersion Examples e The Mean 100 and the Mean Squared Deviation 100 are the most commonly used statistics Figure 10 3 a Notice that the standard deviation ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis is the Mean Squared Deviation multiplied by N where N is the sample size This difference comes from the fact that the standard deviation should be an unbiased estimator e The Median Mean 0 and Mean Absolute Deviation 100 are much more robust to outliers than the statistics above Figure 10 3 b e The Midrange 100 and Half range 100 define an interval that includes all sample values Figure 10 3 c e The Midrange 50 and Half range 50 are known as order statistics and interquartile ranges Figure 10 4 08 0 8 0 8 06 06 0 85 0 4 0 4 0 4 0 2 0 2 0 2 cefbda cefbda cefbda 4206 a 4206 b 4206 c Figure 10 3 Histograms showing the sensitivities of central and dispersion values a Mean 100 and M S D 100 b Median and M A D c Midrange 100 and Half range 100 035
278. other open reports Just select matches by clicking on the corresponding points while using the Ctrl or Shift keys for multiple selections To select all matches in a region hold down the mouse button and then drag the cursor to define an area Use the options in the Select on Gels drop down menu to identify the corresponding spots on the gels and open reports You can also display a new factor projection plot showing only the selected matches by clicking on the Plot Projection icon You can save and print the graphics using the corresponding icons in the Factor Projection Plot window toolbar or copy the plot to the clipboard for use in other applications Information related to the Factor Projection Plot can be found in the Reports drop down menu in the toolbar e The Factor Analysis Report summarizes the variance accounted for by the successive axes expressed as a percent of the total variance The report also lists the coordinates for each gel projected on these axes Figure 10 11 e The Projection Report displays the contribution of each match to the two axes displayed in the corresponding Factor Projection Plot as well as a Quality measure that gives you an appreciation of whether the projection of the match is well represented on the factorial subspace Figure 10 13 e The Intra Class Report for the selected matches shows the individual quantification values for each spot in the match and allows to display related Intra Class Histogra
279. ots in one window In this case all scatter plots will have a fixed size and use the same scales on the X and Y axes The scatter plots are interactive You can click on the points representing the matched spots and select them on the gels and other open reports Just select matched spots by clicking on the corresponding data points while using the Ctrl or Shift keys for multiple selections To select all data points in a region hold down the mouse button and then drag the cursor to define an area Use the options under the Select on Gels icon to identify the corresponding spots on the gels and open reports You can also display a new scatter plot showing only the selected data points by clicking on the Scatter Plot icon As usual you can save and print the graphics using the corresponding icons in the Scatter Plots window toolbar or copy plots to the clipboard for use in other applications Information related to the scatter plots can be found in the Reports menu icon in the toolbar Figure 10 1 e The Gel Report gives information on the gels used in the scatter plots e The Fitting Report displays the slope and offset values of the regression line the correlation coefficient and the number of data points matched spots for each plot e The Scatter Report displays the corresponding spot values in the gels and the error in relation to the regression line for each pair of matched spots This error can be used for example to verify abnorma
280. ountry Region Tel Fax Asia Pacific 852 2811 8693 852 2811 5251 Australasia 61 2 9899 0999 61 2 9899 7511 Austria 01 57606 1619 01 57606 1627 Belgium 0800 73 888 03 272 1637 Canada 1 800 463 5800 1 800 567 1008 Central East amp South East Europe 43 1982 3826 43 1985 8327 Denmark 45 16 2400 45 16 2424 Finland amp Baltics 358 0 9 512 39 40 358 0 9 512 39 439 France 01 6935 6700 01 6941 9677 Germany 0761 4903 490 0761 4903 405 Italy 02 273221 02 27302 212 Japan 81 3 5331 9336 81 3 53319370 Latin America 55 11 3933 7300 55 11 3933 7306 Middle East amp Africa 30 210 9600 687 30 210 9600 693 etherlands 0165 580 410 0165 580 401 orway 815 65 555 815 65 666 Portugal 21417 7035 21417 3184 Russia amp other C l S amp AS 7 095 232 0250 7 095 956 1137 7 095 230 6377 South East Asia 60 3 8024 2080 60 3 8024 2090 Spain 93 594 49 50 93 594 49 55 Sweden 018 612 1900 018 612 1910 Switzerland 0848 8028 12 0848 8028 13 UK 0800 616928 0800 616927 USA 1 800 526 3593 1877 295 8102 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 1 4 2 Sending e mail To provide us with the essential technical and customer information when mailing to imagemaster support ge com please proceed as follows To send an e mail to technical support 1 Choose Help gt Online Support from th
281. p 035 035 035 030 p 030 030 030 025 p025 025 025 beadcf beadef beadef beadcf 3678 a 3678 b 3678 c 3678 d Figure 10 4 Histograms showing the effect of suppressing outliers Midrange and half range values are given for a 100 b 80 c 50 and d 33 10 2 3 Intra Class Histograms Intra Class Histograms and Reports provide valuable tools for looking at unusual matches within a class Analyzing protein expression changes checking spot detection or verifying matching operations are just a few of the numerous potential usages One can also compare spots visually or even navigate through matches At this point it should be noted that Intra Class Histograms and Reports are dual elements they have a reciprocal relationship They are not only complementary tools to interpret the information but are also designed to effortlessly switch between them Starting from an Intra Class Report you can easily display the corresponding histograms and vice versa Histograms are a more visual way to look at matched spots ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 201 Data analysis 202 To display Intra Class Histograms 1 Select the matches to be studied on the gels or on an Intra Class Report 2 Select the gels to be included in the histograms The gels can belong to different classes but the class information will be ignored for intra class statistics 3 Choose Analyze gt Intra Cla
282. pecified reference gel ImageMaster needs to know which positions in the different gels correspond to each other represent the same protein form This is done by defining landmark annotations on both the gel to be aligned and the master reference gel The alignment algorithm then deforms the aligned gel to superimpose those annotations that bear identical labels in the master reference gel and the aligned gel Please see Chapter 8 to find out how to create such annotations NOTE To carry out an alignment annotations do not need to be linked with spots and therefore spots do not need to be detected This is what distinguishes alignment from matching In matching the landmark annotations must be linked to detected spots PART ae man gt anja Sn NR Nr ere wt An per in aq 2 Ss Sy INA Mee 4 r lt i WN SU gt a y SSA aah i H 7 nn ben ante ue u it i p T a or 4 l Y i 4 ef i p SN AL ws a A7 at i ia ae fz k s Pi a b Figure 6 10 Gel a before and b after alignment with the master image The match vectors and Landmark annotations are displayed ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA ImageMaster allows you to choose one of the following alignment functions e Global Authorizes the rotation translation and scaling of the entire gel image to find the best possible correspondence between all the landmarks e Exact Locally warps the image to exactly superimpose al
283. pixel representing approximately 330 x 330 microns of the image area This ProPic image can be analyzed with ImageMaster and spot coordinates in the image can be exported back to the ProPic spot picker in a predetermined file format The ProPic software subsequently translates the image X Y coordinates into ProPic robot X Y coordinates by using a robot map Since the robot mapping process assumes that each X Y coordinate in the image always corresponds to the same position on the robot bed the ProPic image must remain in its original form It can never be resized cropped or rotated The ProPic image is a picture of the current state of the gel from which the spots are to be picked It is not necessarily the image used to determine the spots to pick One can select spots from any analytical image of the same gel obtained from a different resolution system and analyzed with ImageMaster However the analytical image needs to be aligned to the ProPic image so that ImageMaster can export the aligned X and Y coordinates of the selected spots Please keep in mind that proper alignment is crucial at this stage especially for images of old gels that underwent significant shape change since they were originally imaged and analyzed and for gel images that were acquired on a high resolution system To export a spot coordinate file to the ProPic spot picker 1 Analyze your analytical gel image with ImageMaster and select the spots you want to export t
284. played You can do this in terms of different units Image coordinate Percentage and Real value pl and molecular weight Please note that pl_MW annotations have to be defined in order to use the Real value option 6 3 4 Transparency The Transparency mode is used to visualize similarities or differences between the current gels and the specified reference gel Two options exist e Image transparency e Spots overlapped Image transparency With this option sometimes called dual channel display turned on each of the images is displayed in one of two colors red and cyan Figure 6 7 When the pixel colors of the two superimposed gels are added e Overlapping spots appear as shades of gray e Red spots are present only in the current gel e Cyan spots are present only in the specified reference gel e Halos of red or blue around dark spots indicate that the protein is over or under expressed respectively compared to the chosen reference gel ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA This means that the less color you see in the Transparency mode the more similar the gels Of course this is only true if the gels are correctly aligned see Section 6 3 6 and superimposed Et ImageMaster 2D Platinum loj x File View Edit Show Select Analyze Reports Tools Window Help Gels A Spots 0 Annotations fo Figure 6 7 Image transparency Spots overlapped With this option when spots have be
285. population or on the other hand shows all spots present in a series of gels In the first case an average gel image is obtained by averaging the positions shapes and optical densities of the matched spots in a given set of gels This produces an intersection of all gels showing only the spots found in all or at least some of the given images In the second case the unmatched spots are added as well The synthetic gel can then be considered as a spot index or a graphical fusion of protein maps In reality you can choose which spots should be included in a synthetic gel This is because the synthetic gel will contain all selected spots in the selected gels at the moment you create the synthetic image You can therefore filter your spots carefully before generating the synthetic image If spots are matched only one of the matched spots will be present in the synthetic gel Its area corresponds to that of the spot having an area closest to the average of the matched spots while the intensity equals the average intensity of the matched spots For isolated spots included in the synthetic gel the Intensity Area and Vol values of the original spot are assigned The Vol and intensity values are recalculated according to the total Vol and Intensity of the new synthetic image The positions of the spots in the new synthetic gel are based on the positions in the master image and therefore the reference image of the match set The resulting synthet
286. populations of gels is a major innovation over previous versions of the software What is more you can match different levels of populations This is called population matching The countless possibilities that population matching offers are further documented in Chapter 4 The current chapter will concentrate on the matching procedure and the manipulation of matches ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Matches 9 9 2 Matching The prerequisites for matching gels or match sets is that spots are detected the gels or sub match sets to be matched are added to a match set see Chapter 4 and this match set is opened in a worksheet NOTE A DIGE gel is an inherent match set for which the co run images are automatically matched see Chapter 4 to learn how to quickly create match sets for DIGE gels To subsequently match different DIGE gels proceed as with any other match sets NOTE Instead of the reference image the Master is displayed in a MatchSet worksheet As the master and reference images are the same and matches are automatically created between the two the reference image is not displayed by default To show the Reference in the match set choose Show gt Show Reference NOTE The functionalities that are specifically related to editing matches and reporting on matches are only available when working in a MatchSet worksheet 9 2 1 Automatic matching ImageMaster is designed to
287. port xj alaj wlej my vlje if gt Wu gt Bl center x Inter Class Report Val The chosen statistics are Mean 100 and M S D Match ID Max AT1 A_T2 Set Verified Landmark al 4896 0 537666 0 537666 0 2 17240 2 17240 0 699839 i 0 546035 0 132385 0 546035 0 0906696 0 0906696 0 0791606 25 4622 o21arsa o112918 nur lft Ways Figure 8 14 Report on selected matches in selected classes 178 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Matches lt A 9 Matches 9 1 Introduction Matching is a key operation in 2 DE image analysis Basically the image matching algorithm compares gel images to find Matches between related spots that is spots representing the same protein in the gels A match is therefore composed of spot n tuples S1 S2 Sn where S1 is a Spot ID in the first gel and Sn a Spot ID in the last gel To be able to initiate the matching process in ImageMaster 2D Platinum 6 0 gels must be part of a Match Set A match set includes gels or populations of gels in sub match sets that should be compared and therefore matched together Every match set is represented by a Master This master image is created by ImageMaster based on the Reference for the match set chosen by the user Essentially the Master is a copy of the match set reference and contains the same spots which are therefore automatically matched between the two images But once other gels
288. possible to open a workspace project when you don t have access to all the necessary folders and in particular the folder where matches and other data are stored Therefore if a user wants to share his workspace project he has to share his local ImageMaster folder export his project s see Section 4 4 3 or provide a backup of his project or workspace see below NOTE It is good practice to make regular backups of your work using the functionalities described below so that you can recover your work at any time 4 11 1 Backup Restore workspace With the Backup Workspace function a complete workspace can be archived including the projects gels matches master gels reports and other related files This backup is written into a single compressed file with the extension bkp that can be restored when needed Use the Restore Workspace function to later retrieve the backup To backup a workspace 1 Right click on the Workspace Name at the top of the Navigator 2 Choose Backup from the contextual menu ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 3 4 In the Backup Workspace box browse to the directory you want enter a file name and click Save The backup file with the extension bkp is archived To restore a workspace 1 2 Right click on the current Workspace Name at the top of the Navigator Choose Restore Workspace from the contextual menu In the Restore Workspace box browse
289. r The following icons are available from the Workspace toolbar 5 Create a New workspace In the Create New Workspace box specify the Workspace Name Location and Comment By default a workspace file with the extension mws is saved in the ImageMaster Workspaces folder found in the user s My Documents directory amp Open an existing workspace In the Open Workspace box browse the directory where the workspace file is located select its name and click Open Automatically Save the current workspace including changes made to any projects When you close ImageMaster the workspace and projects are also saved Keep in mind that modifications done to images reports or matches are not saved with the workspace To save these changes choose one of the options under File gt Save 4 2 2 Active project The Active Project displays the name of the project from which the active worksheet has been opened This is particularly useful when different projects are contained in the workspace and worksheets have been opened from several of these 36 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace 4 23 Navigator The Navigator displays all files and folders related to a workspace The look and feel is similar to the Windows Explorer file manager There is a hierarchical structure of folders subfolders and files that can be expanded or collapsed dragged and dropped in a new location copied and pasted etc NOTE
290. r Manual 11 0034 38 Edition AA 237 Data integration 238 NOTE Because the XSL stylesheets are specific to the browser you use you will find that different versions both for printing reports and scripts are installed with the software in the Template Report and Template Script folders of the installation directory ImageMaster will therefore ask you to choose the appropriate XSL template each time you print a report script or history Look at which template works with your browser and delete the other one In this way the software automatically opens the remaining file and does not ask you to make a choice 11 2 2 ImageMaster reports The default mode for saving reports in ImageMaster is in XML format Reports saved in this format carry the extension rpt and are restored to their original tabular form on the condition that they were inserted into a Workspace When reopened in ImageMaster these reports can be manipulated sorted customized and edited as with any newly displayed report Outside the ImageMaster program the content of ImageMaster reports can be viewed with a browser However only a color coded raw view of the root and child elements is available generally not very useful The main interest in XML format besides being used directly by the ImageMaster software is that external applications can easily extract necessary data Moreover the files can be converted to other user defined formats Please not
291. r behavior If one of the matches has a low value any interpretation becomes tentative ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Gels that are adjacent on the graph are likely to be similar to each other They may correspond to the same population This is clearly the case in the example above The gels from T1 cluster together below the horizontal axis whereas the gels corresponding to T2 lie above The closer a match is to a given set of gels the more characteristic it is likely to be of those gels That is the more important the match is in determining why those gels are different from other gels In Figure 10 12 for instance one can observe that the matches 4844 4903 4691 4767 and 4839 are close to the gels belonging to T1 The histograms in Figure 10 14 show that these matches have high spot values for the T1 gels and low values for the T2 gels Match 4704 on the other hand is characteristic of the T2 population with higher spot values in the T2 gels In our example the second axis appears very important for separating the gels into two populations It is related to the ratio between the mean spot values in each population of gels The matches in the upper part of the graph have ratios that favor the T2 population while those below the horizontal axis have ratios that favor the T1 gels The Factor Projection Plot is interactive You can click on the points representing the matches and select them on the gels and
292. ram or closing the gel Regularly saving your gels enables you to recover from mistakes although the multiple undo function allows you to return to previous analysis states or to generate gel files with different names Modifications are saved in the ImageMaster 2D Platinum file format unless otherwise specified by the user ImageMaster does not automatically save the changes you make to a gel Instead you can control what data should be saved and when Nevertheless when you close a gel or leave the software ImageMaster alerts you that modifications were made and gives you the possibility to save them NOTE Please note that the Save and Save As functionalities in the File menu apply to gel data in contrast to the Save feature in the Workspace which pertains to the Workspace and Projects and also saves the software options Saving your gels and saving modifications to the workspace see Chapter 4 are therefore two distinct operations The software also allows you to make regular backups of your work that is of the workspace with all the gel images and related files and to restore this data when necessary Find more details about this in Section 4 11 Two options are available from the File gt Save menu e Worksheet allows you to save all the changes made to the gels opened in the active worksheet without giving details or asking for confirmation e Save All allows you to save the changes made to all open gels in all the w
293. rd e The Fitting Report displays the coefficients of the regression function e The Calibration Report displays for each step the step number the measured averaged gray level the calibrated intensity value and the fitting error difference between the curve and the point NOTE Intensity calibration must be performed on a regular basis once a month If you do not calibrate darker material may not be measured in the correct proportions to the lighter material Controlling a calibration The Control calibration mode allows you to verify whether you are using a correct calibration It requires a different specially calibrated step tablet Kodak Step Tablet no 3 for instance which you compare to your previous calibration results So you have a calibration step tablet for everyday use and a specially calibrated control step tablet to verify your calibration periodically To control a calibration 1 Scan your control step tablet Kodak Step Tablet no 3 for instance and import the image file into the ImageMaster software If necessary rotate the image such that the light steps are displayed at the top 2 Choose Tools gt Calibration Tablet gt Control in the menu 3 You are asked to load the calibration to be controlled This calibration could have previously been saved using the Save icon in the Create Calibration window cal or can simply come from a calibrated image file mel such as the calibrated step ta
294. re actually present in all gels or the inclusion of spots that are only present in one or two gels but incorrectly matched to other spots Moreover when selecting matches the program only takes into account the spots that are present in the master gel Any spots absent in the Master are not considered even if they are present in all other gels 9 7 2 Creating synthetic gels Once the spots on your gels have been filtered and only the spots of interest are selected you can create a synthetic gel NOTE Synthetic gels can be created from a MatchSet or Classes worksheet When you work in a Classes worksheet the used Master will correspond to the first common node in the match set structure and the synthetic gel will automatically be inserted at this level To create a synthetic gel 1 Select the spots and gels to be included in the synthetic gel see above 2 Choose Tools gt Gels gt Create Synthetic Gel 3 Specify the destination folder and file name in the Create Synthetic Gel window 4 The synthetic gel is created Figure 9 9 and saved together with the automatically generated matches between the synthetic gel and the master gel ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 193 9 Matches Et imageMaster 2D Platinum lo x File View Edit Show Select Analyze Reports Tools Window Help Fr a nn x A RL s RINE R n er ageee oe o
295. rea Answer Yes 6 8 2 Exporting gels and windows To files Rather than saving your gel images in the ImageMaster file format you may want to export them to a different file format TIFF BMP or PNG In this case the gel images are exported as 8 bit flat rasterized images without any structure This means that gel components such as spots and annotations are saved exactly as they appear on the screen but are no longer recognizable as ImageMaster objects and therefore become part of the image Consequently exported gel images should only be used for presentation purposes and not for further analysis with any software package To export gels or selected gel regions 1 If you want to export gel regions start by defining them with the Region tool Define a particular region in each gel or use the Shift key to select the same region in all open gels 2 Select the gels that you would like to export or the gels for which you would like to export a selected region 3 Choose File gt Export gt Image to File from the menu ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA a en 4 For each selected gel enter the destination folder file name and file type in the Export Image As dialog box 5 Ifa region was defined ImageMaster asks you whether you want to export only the selected area Answer Yes or No You also may want to export a view of the active worksheet To export a worksheet 1 Choose File gt Export gt Wor
296. receding element one or more times PO 1 matches P01 P001 P0001 atches the preceding element zero or more times PO 1 matches P1 PO1 POO1 P0001 n Matches the preceding element exactly n times PO 3 1 matches P0001 but not PO1 or POO1 n Matches the preceding element at least n times PO 2 1 matches P001 P0001 but not P01 n m atches the preceding element at least n times but not more than m times PO 1 3 1 matches PO1 P001 and P0001 but not P1 or P00001 The characters between parentheses form a subexpression P 24 matches P24 P2424 P242424 Matches the expression before or after the vertical line Mostly used within a subexpression P ab cd 1 matches Pab1 and Pcd1 A circumflex outside a bracket expression anchors the element it starts with to the beginning of a string such an element can match only a sequence starting at the first character of a string A ec matches ecoli ecoli_eftu but not eftu_ecoli 164 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Annotations 3 Syntax Description Example A dollar sign outside a bracket ecoli matches ecoli expression anchors the element eftu_ecoli but not ecoli_eftu it terminates with to the end of a string such an element can match only a sequence ending at the last character of a string Table 8 1 Regular
297. recommended to create spots by using the automatic spot detection algorithm in ImageMaster and to avoid manual editing as much as possible At most spots should be manually separated where necessary In spite of these words of warning you still have the capabilities to create modify or delete selected spots in ImageMaster NOTE Spot editing is not allowed on aligned gels because image warping may deform spots and on DIGE gels For edited spots the Saliency value becomes Zero ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 7 7 1 Editing spots In ImageMaster spot editing is done directly on the image although one needs to be in the special spot editing mode To modify spots 1 Select the image you want to work on 2 Choose Show gt Spots gt Shape gt Outlined to see the spot borders more easily It is also recommended to show any hidden spots Show gt Spots gt Show All in order to prevent the addition of already existing spots and thus generating overlapped spots 3 Choose Edit gt Spots gt Edit Enabled in the menu a check mark next to the option indicates it is activated The icon of the Spot tool in the main toolbar also changes to reflect the fact that spot editing is activated Figure 7 4 4 Select the spot you would like to edit with the Spot tool 5 Carry out the desired spot modifications as described below 6 Todeactivate this option choose Edit gt Spots gt Edit Enabled E
298. reen but also from your hard disk The main advantage of deleting gels from within ImageMaster is that associated matches are suppressed as well To delete existing gels 1 Select the gels to be deleted in a worksheet 2 Choose Tools gt Gels gt Delete from Disk in the menu 3 All selected gels and their associated match files are closed and removed from your hard disk 6 6 2 Renaming gels Since the ImageMaster Workspace checks the consistency between the gel Name and Gel ID it is highly recommended to rename your gels at least those ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA that have already been opened and saved with ImageMaster from within the program By doing so you avoid inconsistencies in the workspace To rename your gels 1 2 6 6 3 Select the gels to be renamed in a worksheet Choose Tools gt Gels gt Rename from the menu Enter a new gel name for each selected gel The gels are automatically renamed gel legend workspace and file name Rotating gels When images are scanned in the wrong orientation they can be rotated to correct for this You can typically rotate your gels by 90 180 and 90 degrees But free rotation can also be applied To rotate an image 1 2 Select the gel to be rotated in a worksheet Choose Tools gt Gels gt Rotate from the menu and select the desired option 90 CW 180 90 CCW Free Enter a new file name and destination folder f
299. reen rectangle corresponding to the visible gel area 6 3 3 Scrollbars Scrollbars in blue and grey are given on the right and bottom edges of each image The size of a scroll box indicates the proportional amount of the used area of the image that is visible in the cell The position of the scroll box indicates the ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 87 ED 88 relative location of the visible area within the image These scrollbars enable you to e Change the zoom factor of the image The cursor of the mouse changes to a double sided arrow icon when placed over the respective scroll box Click the mouse on the edge of either end of the scroll box and change the length of the box The image zooms in or out depending upon if you make the scroll box shorter or longer The other scroll box scales accordingly By clicking on the blue square box at the intersection of the two scrollbars you can reset the image to its full image size e Move the visible area of the gel up down left or right Simply place the cursor of the mouse over the scroll bar click the mouse and move in the direction you want It is possible to hide the scrollbars by deselecting View gt Scrollbars gt Show in the menu If you want to view a specific area of your gel image choose View gt Scrollbars gt Adjust In the Adjust Visible Area window set the exact horizontal and vertical start and end coordinates of the area to be dis
300. rennen a De 90 H hand tool hardware requirements help menu online user manual heuristic clustering histograms INter claSsS sti coke cA bite ok A rl E E N ds a N al inter class and intra class_ intra class _ history copy to new script navigator print refresh save http link I ImageMaster 2D Elite 2D Platinum 5 0 DIGE modules window images close depth format resolution select stack import gel data to XML _ installation intensity normalization inter class histograms overlapping measures report specify SUGISUCS ihe TE AEM Re Sahl TAN ed ee ido bene male eA hain coe BER intra class histograms normalize report isoelectric point calibration K kKeUDOCKGSHORCUES Tsien ee Een 23 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Index add copy delete display duplicate import modify report select landmark annotations category legend link objects to external data M magnify tool master image match add delete display hide report select sets show show ID statistics report vectors matching annotations automatic landmarks start matchsets _ add gels in workspace copy and paste create in workspace master image open propertie
301. requirements Coworkers can look or work with the same data but organize their workspace differently The Workspace is essentially symbolic Changes to the workspace do not affect images reports matches and master gels that are saved on your hard disk However the information in the workspace project is necessary to reconstruct the match sets and retrieve the matches This is the reason why it is important to regularly save the workspace saves the projects and why the workspace is saved by default when closing the software 4 2 Workspace window Click the Workspace Workspace tab below the ImageMaster toolbar to display the Workspace window The window Figure 4 2 contains the Workspace Toolbar the Active Project the Navigator and File Details ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace 35 es Workspace xi Dell Project Bacteria b 5 Sonja s workspace 1 projects MatchSet 5 8 Bacteria Bacteria 1VA A_T1 a a Gels A DIGE Gels Bacteria 1 A 4_T1 A Bacteria 1 A 4_T1 GB Bacteria 1AW_T2 MatchSets Dei Bacteria 1 AVA_T2 BA Bacteria 1 A A_T2 AB Bacteria 1 B B_T1 GG Classes Bacteria NE B_TI EM Bacteria 1 Bacteria 1 B B_T1 Bacteria 1 B B_T2 Bacteria 1 B B_T2 E er Bacteria 1 B B_T2 Eh E Reports E Documents Figure 4 2 Workspace window a Toolbar b Active Project c Navigator and d File Details 4 2 1 Workspace toolba
302. right hand side of the menu item Menu Usage File Perform basic functions such as close save import export and print You can also exit ImageMaster View Modify the worksheet layout or name Change the arrangement of images in a pane Edit Undo redo the last operations show a history of operations or edit add modify or delete specific gels spots annotations or matches Show Change the way gels are displayed show hide specific spots annotations or matches or modify their physical properties shape color or visibility of IDs Select Select specific gels spots annotations or matches based on their ID value content or other properties Analyze Compute differences and similarities between gel images data analysis based on robust statistics factor analysis statistical tests and clustering techniques All reports containing calculated properties are found in this menu Reports Display information on selected gels spots labels annotations categories or matches and use the navigation tools in the reports to view the data on your gels The three dimensional 3D view is treated as a report and is therefore located in this menu Tools Remove or rename gels on the hard disk create new gel images from selected ones flip rotate and scale gels or create synthetic images run scripts manage calibrations and change ImageMaster settings Window Display pixel and or spot information open zoom windows or mouse selection rep
303. rinciple a project will include images that are visually comparable in a straightforward way Very dissimilar gel images due to differences in staining or tissue for example should better end up in different projects Images that are technically related such as multiplexed or DIGE gel images are an exception Since such images come from the same gel at least their spot positions are directly comparable 4 4 1 Creating a project To create a project 1 Right click on the Workspace Name at the top of the Navigator 2 Choose New Project from the contextual menu 3 Inthe Create New Project box specify the Project Name Location and Comment Click OK By default a project file with the extension prj is saved in the ImageMaster Workspaces folder found in the user s My Documents directory If you want to save the file in a different folder then browse and open the folder 4 The Project Name and default folders appear in the Navigator The project file is automatically saved A project is saved whenever you click Save in the Workspace toolbar You can remove a project from the current workspace by right clicking on the Project Name and choosing Delete from the contextual menu This does not delete the project file from your hard disk Therefore the project can be inserted into another workspace ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 39 40 4 4 2 Inserting an existing project To insert an existing project 1 R
304. rnet It is the responsibility of the user to acquire the database licenses if needed In particular the PROSITE and SWISS 2DPAGE databases are copyright and all commercial users of these databases are required to purchase a database license from Geneva Bioinformatics GeneBio SA Please contact GeneBio at info genebio com for more information Geneva Bioinformatics GeneBio SA Avenue de Champel 25 CH 1206 Geneva Switzerland mageMaster 2D Platinum uses the DeCyder co detection algorithm 2005 General Electric Company All rights reserved mageMaster 2D Platinum uses the TIFF library 1988 1999 Sam Leffler and 1991 1999 Silicon Graphics Inc All rights reserved mageMaster 2D Platinum uses software developed by the Apache Software Foundation http www apache org 1999 2003 The Apache Software Foundation All rights reserved Cy CyDye Ettan Typhoon DeCyder ImageMaster LabScan and i i mageScanner are trademarks of GE Healthcare Ltd GE tagline and GE www a mersha m biosciences com monogram are trademarks of General Electric Company SYPRO is a trademark of Molecular Probes Inc GE Healthcare Macrovision is a registered trademark and FLEXIm is a trademark of Amersham Biosciences AB Macrovision Corporation ExPASy is registered by the Swiss Institute of Bi n k t 30 Bioinformatics Microsoft Windows PowerPoint and Microsoft Internet JOr g atan Explorer logo are trademarks of Microsoft Corporation Netscape N
305. rom the menu 3 Blue vectors are shown between matched spots in the displayed gel and the master gel 4 You can hide the match vectors again by choosing Show gt Matches gt Hide Vectors x rn a oF 5 Figure 9 4 Matched spots in matched gels are linked by blue vectors 9 4 2 Show hide match IDs You can display Match IDs for selected spots on selected gels Figure 9 5 Obviously the gels must be matched to the Master SSS ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 187 9 Matches To display Match IDs 1 Select the spots and gels you would like to display the Match ID for 2 Choose Show gt Matches gt Show ID from the menu 3 To hide the Match IDs again possibly in specific gels only select the desired gels and choose Show gt Matches gt Hide All ID EN imageMaster 2D Platinum lol x File View Edit Show Select Analyze Reports Tools Window Help 4513 isio 4526 Misa Gels a Spots ho Annotations E Figure 9 5 Match IDs for selected spots on selected gels 9 5 Adding deleting matches Sometimes it may be necessary to manually add matches This can be the case when the matching algorithm needs a few input matches or when you would like to add or change matches after the automatic matching procedure 9 5 1 Adding deleting matches To add a match 1 Select a spot in the master image and the corresponding spots in the other images you can select se
306. rsor The exact cursor position is indicated by the green cross Bl imageMaster 2D Platinum E File View Edit Show Select Analyze Reports Tools Window Help BIEUSHIONA Figure 6 5 Zoom Window used to localize the visible gel area represented by a green box in a larger view of the gel The exact cursor position is indicated by a green cross 86 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Overview option When you zoom in on a gel it can be helpful to have an overview of where the region is localized on the full image Figure 6 6 This overview enables you to easily locate and move to any region you want on your gel To display and use the Overview option 1 Choose View gt Overview in the menu If Overview is checked then the option is activated 2 The overviews appear in the lower right corners of all images in the Display Zone The green rectangle in the overview corresponds to the current view of the gel 3 Drag the green rectangle to another position to display a new region 4 To deactivate this option choose View gt Overview Overview is no longer checked in the menu This is the default setting EN imageMaster 2D Platinum loj x File View Edit Show Select Analyze Reports Tools Window Help Gels B Spots 0 Annotations jo Figure 6 6 Overview option activated In the lower right corner of all images in the Display Zone is a small overview of the entire gel with a g
307. rt The lower this number the higher the probability is that the means of the two samples are different Knowing this value and the sample size you can easily look up the probabilities in a Mann Whitney table ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Caution should be used when analyzing the results of a Mann Whitney test First the assumptions are often violated This is the case for example when spots are completely absent in one of the classes in that case you have repeated values of 0 Moreover if you have small samples the Mann Whitney test is meaningless In fact if the total sample size is seven or less the test always gives a probability of finding different means in the case of identical populations greater than 0 05 no matter how little the samples differ Kolmogorov Smirnov test The Kolmogorov Smirnov test tries to determine if two data sets differ significantly In other words it is used to test whether or not two samples may reasonably be assumed to come from the same distribution It has the advantage of not making an assumption about the distribution of the data and is frequently preferred over the Mann Whitney rank sum test where there are many ties repeated values Note however that this generally comes at a price Other tests for example the t test may be more sensitive if the data meet the requirements needed for that test The assumptions of the Kolmogorov Smirnov test are e The probabil
308. rts gt Match Statistics Report from the menu 3 A Match Statistics Report is displayed 9 6 3 Intra class report Intra class Reports contain information about matched spots in a class of gels Match ID central tendency dispersion spot values coefficient of variation range ratio separability etc In Chapter 10 you can find in depth information about the Intra Class Report and its functionalities Figure 9 8 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Matches ex A xl alal le a l le Pla ule m gt B vau Intra Class Report Vol The chosen statistics are Mean 100 and M S D Match ID Mean 100 M S D Variation Ratio 100 Separability A_T1_Gel1 A_T1_Gel2 342 4456 0 0365684 0 0293886 0 803662 11 0250 0 0452889 0 0538708 0 0630224 0 07 343 4823 0 197289 0 128024 0 648916 10 4074 0 163236 0 305891 0 304164 0 3 344 4607 0 216323 0 113875 0 526411 8 93871 0 104266 0 239972 0 344238 0 3 345 4594 0 131266 0 0848866 0 646676 8 30224 0 0686879 0 159803 0 205525 0 2 0 177527 0 103055 0 580505 0 260546 0 0385055 0 122954 0 0858610 0 698320 0 191090 0 241444 Figure 9 8 Intra Class Report on matched spots in selected gels 9 7 Synthetic gels With the Create Synthetic Gel feature you can create a composite gel image that contains only representative spots of a
309. ry you can perform a calibration of the image capture device within ImageMaster using calibration step wedges or calibration strips see Section 6 5 6 2 Selecting gels and gel regions Most of ImageMaster s functions can only be performed on selected gels in the active worksheet Gels are selected by clicking on their legends using the Select menu options or via a report Gel regions are defined using the Region tool Legends Each image has a legend with its name in the upper left corner The color of the legend indicates whether the image is selected green or red for a master image or not gray or pink for a master image In order to select an image click on its legend Use the Shift or Ctrl keys to make multiple selections Select menu ImageMaster allows you to select gels based on specific criteria Choose Select gt Gels in the menu and select one of the following options e All Selects all gels that are found in the active worksheet The shortcut for this option is Ctrl A e Aligned Selects only the aligned gels e Inverse selection Reverses the selection of gels ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Reports When you double click on gels spots annotations or matches in a report or use the Select on Gels icon in the report toolbar the related gels are automatically selected in the active worksheet This means that when you double click on a spot in a Spot Report the gel containing th
310. s reference image measure histogram Melanie menu bar min area me Molecular weight calibr tion u ala an La 142 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 283 Index 284 N next whe node locked license 18 normalization 106 111 204 0 615 lt 1 EEEIEE IEE AAL SAIE BERERURTENTESTONINDENE PEES PTET SEOSTE ENESE E S REEE EY E SEN 208 P pI_MW category pinned population matching predefined label categories previous profile projects add create insert open properties VIEW an protein databases query pseudo colors Q GuUeryidatabases sic susieck ks telat ade shin ch tel eee tes E E a obeleed salt on 156 R Fedora nee Coe As sevice oe Se aca cae oe 251 reference NDS acne betes aan as Gab a Mee mare fee aa er A yes See hast dent LEN region tool regions regular expressions FEDOME Serris 3D view add to workspace create folder in workspace export gel gel calibration gel description IDs inter class open statistical tests toolbar EOS HSM HE oa eae ca cal ha alee acta ccs ata Eee ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Index representative spots restore workspace S saliency scatter plot report script create navigator PECVIOUS Ta ea nee ieee coca banner 68 selection box export import set category setup wizard
311. s or scatter plots are examples of files that can be added to the Documents folder So are spreadsheet files text files Microsoft Word Microsoft PowerPoint files and many more Workspace Project folders Operation a Gels PMN wa Detecting afmacnses K I Matching afose K Analysing afres FC Pa Exporting sa Documents Figure 4 1 Image analysis from start to finish in the Workspace The gels in the workspace have icons that indicate what stage in the analysis procedure your image has reached Table 4 1 Table 4 2 You can distinguish DIGE gels from non DIGE gels see which gels have been detected and matched and tell which gels are used as reference for matching 34 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Icon Meaning D Non detected gel A Detected gel Matched gel reference gel By A g g Table 4 1 The gel icons indicate what stage in the analysis procedure your image has reached Non DIGE gels Icon Meaning BR Non detected DIGE image DIGE reference image e g standard pool BR Detected DIGE image DIGE reference image e g standard pool BB Matched DIGE image DIGE reference image e g standard pool Table 4 2 The gel icons indicate what stage in the analysis procedure your image has reached DIGE gels The beauty of the ImageMaster workspace is that you can customize it according to your own
312. s worksheet The matches that were found to be characteristic for one of the classes are shown in an Inter Class Histograms window You can learn more ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 215 Data analysis 216 about Inter Class Histograms in Section 10 3 3 They display for each class formed the central tendency blue horizontal line and the dispersion interval red vertical line To find out which gels compose each class designated with a letter you can select the Gel Report item from the Reports icon NOTE The matches that stay selected on the gels after the heuristic clustering analysis are those that were considered significant in the analysis and are therefore present in the Inter Class Histograms Plot The resulting classification can be visualized in a dendrogram which represents the classes and their gels in a descending classification tree Figure 10 16 To plot such a dendrogram choose Analyze gt Heuristic Clustering gt Plot By using the icons from the toolbar in the Heuristic Clustering Plot window the dendrogram image can be saved lin png bmp or tif format printed or copied to the clipboard for use in other applications 10 3 Inter class statistics 10 3 1 Specifying classes To identify protein expression variations between populations of gels you need to specify what gels belong to which population by creating classes There are several possibilities for doing
313. s analysis steps in this chapter Although the three options associated with these features namely Undo Redo History and Script may seem quite independent they in fact share a number of common interface elements and properties that are described below 12 1 1 Action descriptors Each operation carried out in ImageMaster is described by a generic name called Action Descriptor The Action Descriptor is used in Undo Redo History and Script to identify a specific operation so that you can act on it for example undo the operation or copy it from a history to a new script Nearly all operations executed are displayed in the Action Descriptor list which you can find in the Undo Redo History and Script windows However there are some exceptions to this depending upon the option used These exclusions are specified in the ensuing sections 12 1 2 Displayed actions The number and type of operations included in the Undo Redo lists and History can become very large and diverse thus making it difficult to find the actions of interest when deleting or copying to a new script for instance In order to reduce the number of displayed actions four major types of operations were defined e Edition Covers the functionalities available under the Edit menu this kind of operation renders permanent modifications to the image data e Selection Comprises the options from the Select menu These allow the selection of specific objects or the refinemen
314. s is a complex statistical technique whose comprehensive description is beyond the scope of this manual For more information please see Appendix C for references In the following paragraphs you learn how to perform a factor analysis and how to interpret the results Your attention is also directed to some critical points of this analysis To carry out a factor analysis 1 Click the Factor Analysis icon in the Intra Class Report toolbar Figure 10 10 2 Ifany matches are selected the program asks whether you want to use only the selected rows or not Please see further on in this section to judge which of the options is most applicable 3 The program displays a Factor Analysis Report with the lines corresponding to the axes that can be drawn in the Factor Projection Plot Select 2 axes to be displayed in the plot the first two ones generally 4 Click the Plot Projection icon in the Factor Analysis Report toolbar ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis 209 Data analysis 5 Adialog box lets you choose the number of matches to be displayed Please note that the selected number does not influence the factor analysis results It just improves the clarity of the plot by reducing the number of visible matches 6 The Factor Projection Plot is displayed It is completely resizable when in floating mode You can drag the corners or borders of the window to make it smaller or larger Interpreting
315. s matches between the spots in the images You can easily add a DIGE gel to your MatchSets folder or insert it into an already existing match set To create a match set from a DIGE gel 1 Select one or more DIGE gels not the individual images in the DIGE Gels folder Use the Shift or Ctrl keys to make multiple selections Right click on one of the gels Choose Create MatchSet from the contextual menu In the Add DIGE in MatchSet box select the match set you would like to add your new match sets to In the Create MatchSet box specify a Name and Comment for each match set to be created By default the name of the DIGE gel is used Click OK The new match sets appear in the MatchSets folder and the tick mark in the gel image icons indicates that matches exist between the images belonging to a same DIGE gel ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace Alternatively you can drag and drop or copy and paste your DIGE gel to the MatchSets folder to create the corresponding match set 4 6 3 Adding gels to a match set There are different ways to add gels to a match set To use contextual menus to add gels to a match set 1 Select one or more images in the Gels folder Make sure spots were detected and saved Use the Shift or Ctrl keys to make multiple selections Right click on one of the images 2 Choose Add in MatchSet from the contextual menu 3 Inthe Add Gels in MatchSet box type in a name if yo
316. s possible to differentiate one class from the others according to a match ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Reports Histograms Displayed value Inter Class Report E x ula Slo 3 sje eli m u B Re z Inter Class Report Val The chosen statistics are Mean 100 and M S D B 0 917304 0 917304 0 917304 1000000 1000000 1000000 0 691993 0 691993 0 691993 0 606663 0 606663 0 606663 0 779785 0 779785 0 779785 Figure 10 23 Inter Class Report As usual the icons in the Inter Class Report toolbar allow you to save print or export your report and navigate through the data on the gels The Settings icon is used to customize your table display for example show the annotations in the master gel for all matches listed in your table You can also create additional label categories in the master gel and update any modifications made to labels by using the Annotate and Update Gel buttons The items in the Histograms icon are used to produce an Inter Class Histogram or an Inter Class Intra Class Histogram Finally you can generate related reports with the Reports icon e The Gel Report item can be used as a legend for the classes and their gels e The Intra Class Report displays an Intra Class Report see Section 10 2 4 on the selected matches for a selected class e The Report from Selection function creates a new report that only contains
317. s with a question mark or equal sign If you would like to use the SWISS 2DPAGE for instance with the basic query engine you should copy the sequence http www expasy org cgi bin get ch2d entry 4 Paste the copied information into the External Engine field To link to a database that is not federated or that does not contain 2 DE data 1 Directly query the database you would like to link your spots to until you find a specific protein or other entry For example you might display the entry for the protein structure 1BMT in the well known Protein Data Bank PDB 2 Check in your browser whether you can delete parts of the Web page address without losing the database entry In the example of the PDB the original address http www rcsb org pdb cgi explore cgi pid 72461033478448 amp page 0 amp pdbid 1BMT can be shortened to http www rcsb org pdb cgi explore cgi pdbld 1BMT 3 Copy the address of the corresponding Web page in your browser without including the accession or identification number of the current entry Generally this address consists of the database HTTP address and query engine followed by a question mark or equal sign In the example above you would copy the string http www rcsb org pdb cgi explore cgi pdbid 4 Inthe Create Category box paste the copied information into the External Engine field Analogous to an HTTP query where the content of a label is transmitted to a CGI script on
318. se file or if the license file was e mailed 10 Restart the computer 3 2 5 Test the eLicense After installation of ImageMaster downloading and placing the license file start ImageMaster on all computers where the program is installed to test that the license file can be found see below When using a floating license a FLEXIm License Finder window will be displayed asking to specify the License Server System or License File Choose the first option and then enter the name of the computer on which the license server is installed Click OK Note that depending on the license acquired ImageMaster 2D Platinum DIGE or ImageMaster 2D Platinum you will or will not be able to use the DIGE functionalities in the software 3 3 Starting and exiting ImageMaster ImageMaster starts like any other software by selecting Start gt Programs gt GE Healthcare gt ImageMaster 2D Platinum from the Windows menu You can also double click the mageMaster icon Figure 3 2 on the desktop The ImageMaster splash page appears while the software is loading You can click on the logo to make it disappear ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 21 Getting started a Figure 3 2 ImageMaster icon Select File gt Exit in the menu to close the program You can also click on the Close button in the upper right corner of the program window ImageMaster prompts you to save any modifications made to gel images and matching data before
319. se that are linked to a spot These annotations are marked by a square at their basis and have the same coordinates as the spot to which they are linked that is the spot s center of gravity In addition the annotations are automatically selected when the linked spot is selected and vice versa Figure 8 1 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA NOTE To create a spot linked annotation just click somewhere within a spot during creation see below To decrease the chance of missing a spot when clicking on the gel you can display spots with the Outlined shape rather than the Crossed option To create an annotation 1 2 5 6 Click on the Annotation tool in the ImageMaster toolbar Double click on the pixel or spot in the gel where the annotation should be located In the Create Annotation by Click box enter the name of a new category or choose one of the existing categories by clicking on its name When a new category is created the Create Category box appears Please see Section 8 3 1 to find out more about the parameters requested Type the desired label in the next dialog box and click OK when finished The annotation is created and its label is displayed on the gel Labels can be added to an existing annotation In fact one annotation may have many labels although it can only contain one label from each category If one spot contains several proteins it may need to carry differ
320. ser defined category The label contents can be any textual data numerical values or a Boolean value The user can also decide that a label within a given category is unique As shown in Figure 8 1 each annotation is composed of a basis which can be a square or a cross depending on whether the annotation is attached to a spot or a pixel a flagpole by default displayed in cyan and a flag that consists of a set of colored labels 82 Predefined label categories Some predefined label categories are provided with the ImageMaster software To help you visually identify the various label categories they are automatically created using different background colors as indicated below after each category name ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 151 8 Annotations 152 e Ac pink This category is provided to hold the protein s accession number AC taken from a user defined database and can be the link to ImageMaster s remote database query engine When such a link is defined double clicking on a label of this category displays the corresponding protein entry in the selected database with the default Internet browser see Section 8 3 1 e Landmark blue This category is used to mark pixels or spots in the gels as reference points for the purpose of gel alignment or matching and for the calculation of corresponding locations between gels Two annotations are considered as referring to the same point in differ
321. sh to review any of these skills please refer to the documentation that came with your computer Convention Use Bold Highlights different options or parameters that are inherent to ImageMaster 2D Platinum It also draws attention to newly discussed terms Italic Highlights menu options window names icon names file paths and folder names Italic gt Italic The arrow indicates a menu choice For example you would choose the italicized menu title and then click on the italicized option NOTE Highlights important comments issues or tips Shift Designates holding down the Shift key Ctrl Designates holding down the Control key Left click Means clicking the left mouse button Right click Means clicking the right mouse button Double click Means clicking the left mouse button twice Drag Signifies positioning the cursor on an object and holding down the left mouse button while moving the mouse Table 1 1 Conventions used in the ImageMaster documentation 1 4 Getting further assistance GE Healthcare provides technical and scientific support for the ImageMaster software Please contact us if any problems arise with the installation or use of the program Our support team is happy to help you 1 4 1 How to contact us E mail us at imagemaster support ge com ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Introduction Introduction Support numbers C
322. sing this configuration you can compare at any moment class A with class B A_T1 with A_T2 A_T1 with B_T1 or even A with B_T2 Still using match set Bacteria 1 as a basis you can also create a class structure as in Bacteria 1B Figure 4 8 This class contains two sub classes T1 and T2 that each hold the gels corresponding to a specific treatment It essentially allows you to compare T1 and T2 Another option is to use match set Bacteria 2 when defining your classes but with the same class configuration as in class Bacteria 1 In this way you can easily use the classes A and B and the subclasses A_T1 A_T2 B_T1 and B_T2 for various comparisons However the results can be more or less different from the first case discussed above where the Bacteria 1 match structure serves in the analysis NOTE To see from which match set a gel in a class is derived you can look at the MatchSet column in the File Details at the right side of the workspace window Figure 4 9 It is also there that you can verify if gels belong to the same match set necessary for statistical comparisons ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace 55 E Del Project None Ga Classes Name MatchSet a ee B A_T1_Gel3 mel Bacteria 1 A 4_T1 7 Saar M A_T1_Gel2 mel Bacteria 14A_T1 M A_T1_Gel3 mel EB A_T1_Gel1 mel Bacteria 1 A A_T1 E A_TI_Gel2 mel EB B_T1_Gel1 me
323. splayed 4 Click on the Create Labels icon in the Gel Description Report toolbar 5 In the Gel Descriptions box click the Add button 6 Inthe Add Category box enter the name of a new description category and click OK 7 Set the Category Attribute to Text Number or Boolean 8 Continue creating new categories until you are finished 120 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 9 To suppress an erroneous or unwanted category select it from the Categories list and click the Delete button 10 Click OK when you are finished The Gel Description Report now displays a column for each description category You can directly add new information in the appropriate cells or edit existing data Just double click in a cell to start typing your information When finished a simple click in any cell ends the editing mode The modified cells appear in dark green when the corresponding line is selected or in gray when not selected Figure 6 20 An asterisk in the report s window title reflects the fact that changes were made to it xi S m p Descriptions for selected Gels Filename Sample Staining Operator ECOL Ecoli Silver Technician 1 94 0005 Ecoli Silver Technician 2 Figure 6 20 Gel Description Report Gray or dark green cells were modified and need to be updated on the gel Subsequently the Update Gel icon should be used to make the modifications definitive and to
324. ss gt Histograms 4 Inthe pop up list select the value type Intensity Area Vol to be displayed 5 Choose the desired statistics central tendency and dispersion values see Section 10 2 2 in the subsequent dialog box Figure 10 5 shows histograms for some selected matches The sliders at the bottom and the right side of the window allow you to see other matches If many matches are selected you may have to use the little page slider at the bottom right corner to see the matches displayed on additional pages Inthe histograms the vertical orange bars correspond to the spot values the blue horizontal line represents the chosen central tendency and the red lines delimit the range defined by Central value Dispersion Central value Dispersion The Match ID displayed at the bottom of each histogram allows you to keep track of the matches you are analyzing The histograms can of course be selected as with any other ImageMaster object and saved printed or exported to other software by using the corresponding icons To select a histogram simply click on it To select many histograms use the Shift or Ctrl keys After having selected the corresponding histograms you can also re select matches on the gels by using the Select on Gels icon Navigating through the matches is accomplished using the Select Next and Select Previous buttons Other selection possibilities for example Select on Gels Reports Select from Gels are a
325. ssociated with the Select on Gels icon Please see Chapter 5 for their detailed description ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis 10 E Reports Sort by similarity Displayed value Intra Class Histograms x Bia rie vet Value Histograms on Matches Vol The chosen statistics are Mean 100 and M S D Range extremes Central tendency Gel index Match ID Adaptive gradations KE F Sorted values Page slider Figure 10 5 Intra Class Histograms Additional information on the gels and matches is available from the Reports drop down menu in the toolbar Figure 10 5 e The Gel Report is a legend for the gel index on the histograms e The lntra Class Report item displays the numerical values corresponding to selected histograms e The Report from Selection function creates a new histogram window that only contains the histograms that are selected in the active window NOTE You can use the Intra Class Report to change the order of your histograms sort your data in the report and then re display the corresponding histograms or to refine your histogram selection see Section 10 2 4 In addition to the functions explained above ImageMaster provides the following options in the histograms window to enhance the visual comparison of protein expression changes within matched gels Be ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 203
326. st be defined by the user The number of characteristic spots is determined by the Sensitivity parameter This defines the gap between two classes the difference between the highest spot value in one class and the lowest value in a second class Figure 10 15 The smaller this parameter the smaller the accepted difference between the class intervals in a match Additionally the number of spots considered in the classification is higher Sensitivity Match Interval Class 1 Interval Class 2 min max min max Figure 10 15 Sensitivity parameter To search for classes with the heuristic clustering algorithm 1 Select the matches possibly all to be considered in the classification 2 Select the gels to be classified The gels can belong to different classes but the class information will be ignored for the heuristic clustering 3 Choose Analyze gt Heuristic Clustering gt Do Clustering 4 Inthe pop up list select the value type Intensity Area Vol to be considered 5 Inthe subsequent dialog box define the Classification Level the Minimum and Maximum number of classes and the Sensitivity parameter Depending on your computer capacity this could take time especially when the maximum number of classes is high and many matches and gels were selected When the heuristic clustering process is finished ImageMaster automatically creates default classes in the workspace and opens them in a new Classe
327. t as it is the case for DIGE Histograms see Section 7 10 2 If you display a DIGE Histogram from the contextual menu in the workspace and then select DIGE Report from the Reports icon in the DIGE Histogram tool bar all spots will be included To include only selected spots select spots in two gels and choose Analyze gt DIGE gt Report 150 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Annotations 3 8 Annotations 81 Introduction Individual pixels and spots in a gel image can be labeled with annotations The annotations are used in functionalities such as alignment matching or flagging spots with their particular characteristics protein name database accession number and so on Annotations can also be used to mark spots with common characteristics Finally annotations offer the possibility of linking and associating gel objects to external query engines or data sources of any format text html spreadsheet multimedia database located locally or on the Internet This annotation is attached bo a pixel cross basis 70 1 PO6994 IMDH_ECOLI his annotation is attached j to a spot square basis Figure 8 1 Annotations are composed of an annotation basis square or cross an annotation flagpole and a set of labels flag An annotation is defined by its position on the gel X and Y coordinates and its set of labels Each label belongs to a predefined or u
328. t such as previewing spot detection parameters adjusting gray levels viewing three dimensional plots of spot intensities cropping etc ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 11 Two dimensional gel analysis 12 Spots Spots represent the proteins on the gel They can be automatically detected by ImageMaster or manually edited by the user Spots can be quantified Their intensity area and volume are computed Annotations and labels Spots or individual points pixels can be marked by annotations An annotation is defined by its position and its set of labels It contains relevant information that is separated into label categories predefined and or user defined categories The predefined categories in ImageMaster include pl_MW holds standard pl and MW values for a pl MW calibration Ac contains database accession numbers Landmark defines tie points for gel warping and matching Set marks spots that share the same properties and Comment keeps user information Moreover an unlimited number of user defined categories can be added Labels can also contain direct links to other files Web sites and query engines 2 3 2 Spot and gel relationships Matches A match represents the relationship between corresponding spots in different gels It connects the same protein in the different gels It is composed of a spot n tuple S1 S2 Sn where S1 is a spot in the first gel and Sn a spot in the last gel
329. t correspond to the selected objects on your gels Please note that ImageMaster scrolls up or down in the report so that at least one selected line becomes visible 5 2 4 Displaying related reports Next to some report specific icons such as those linked with the Histograms Measure Histograms or the Factor Analysis functions which are discussed in the pertinent sections of this manual you may find the following button In most cases it allows you to create a Report from Selection that is a new report that only contains the selected lines or histograms from the active report Thus you can create a new report only containing objects that fit your specific criteria without first having to reselect the items on the gel ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Reports EEE Intra Class Report x a flS e pay elo A wl vae Select on Gels Intra Class Repc Select on Gels Reports The chosen stati KEN ee Variation Ratio 100 Separability 4 Refine by Value Only keep selected the rows for which the Ratio 100 value verifies W gt 3 11 I ss Report x als Blo m s P MESI B Value 7 A Loy Intra Class Report Vol The chosen statistics are Mean 100 and M S D Match ID Mean 100 M S D Variation Ratio 100 Separability a 4825 7 00284 1 57735 0 225245 2 16653 3 43268 0 857316 0 249752 1 98882 0 502048 0
330. t is best to return to the Unloaded mode as soon as possible To load the raw image data 1 Select the gels 2 Choose Edit gt Gels gt Raw Image gt Load from the menu 3 No visible changes occur in the gel but you may now display the raw pixel values in the Cursor Information window To unload the raw image data 1 Select the gels You can also choose Edit gt Gels gt Raw Image gt Select Gels with Loaded Image in the menu 2 Choose Edit gt Gels gt Raw Image gt Unload in the menu 3 Thememory used to store image data is liberated and the raw pixel values are no longer available To load the raw image data every time a gel is opened 1 Choose Tools gt Options in the menu 2 Inthe Options window click on the General tab 3 Check the Keep image in memory box 4 The raw pixel values for all images are stored in memory by default The color depth of your computer screen go to the Start gt Settings gt Control Panel gt Display in the Windows menu for more details affects the memory used by ImageMaster to display gels For example a display using 16 million colors 24 bits requires three times more memory than one using 256 colors 8 bits Therefore you can also optimize memory usage in ImageMaster by adapting the color depth of your computer screen 6 4 2 Cursor information At any given time the Cursor Information window can be used to display pixel values such as the X and Y coordinates or pl a
331. t of an existing selection Once selected you can perform further actions on the chosen objects e Display This type of operation is typically found in the View Analyze Report and Window menus It generally displays new windows for example reports modifies the Display Zone layout or stack settings or manages the visual relationships between images for example alignment e Show The features available from the Show menu belong to this type of operation They act as a kind of visual filter or can be considered as non permanent modifications to the gels because nothing is saved with the image file All ImageMaster functions are classified into one of these types so that three different filter criteria can be applied to the items in the Action Descriptor list ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 249 Safety control and automation es These filter criteria and the type of actions they include are listed in the table below Edition Selection Display Show All X X X X Without Display X X Only Edit X Each of these filters has a specific context e Allis interesting if you want to list all types of operations carried out during your work session e Without Display is particularly useful when generating scripts Modifications to the display move image etc are not important and often cannot be scripted e Only Edit is used when you want to track permanent chang
332. t one of the color palettes in the Colors list and or invert the gray levels by checking the Invert box 4 The modifications are applied to the defined regions in the selected gels 5 Ifyou are satisfied click OK to apply the changes to all gels 6 Togo back to the default gray levels choose the Gray option from the Colors list 6 4 5 Profile It is sometimes quite difficult to judge whether spots should be split whether they are saturated or not or whether they have some other problems Sometimes spots may have a so called donut structure with low intensities in the center compared to the borders It is very important to identify such problems as they will lead to incorrect spot quantitation However such errors are difficult to detect because the human eye is not very sensitive to different shades of gray The Profile function can help in such cases Horizontal and vertical sections on the gel at the position of the mouse cursor show the intensity variations in the two directions Figure 6 14 The Profile visually clarifies the intensity changes in the gel and can thus assist in making decisions Please note that the major advantage of the Profile feature compared with the 3D View see below is that it can be used within the selected image during spot editing and so on Thus you have information at your disposal about the third dimension spot intensity without having to open an additional window as is the case with the 3D View
333. tags enabling the definition transmission validation and interpretation of data between applications and organizations XML files can be viewed in the latest versions of Internet Explorer version 5 or higher Netscape version 6 or higher and Mozilla version 1 4 or higher However as XML was designed to describe data and not to display data it does not look like a web page An XML document contains color coded root and child elements A plus or minus sign to the left of the elements can be clicked to expand or collapse the element structure If you want to view the raw XML source you must select View gt Source from the browser menu XML does not use predefined tags as is the case for HTML Therefore the browser does not understand the meaning of the tags and does not know how to display the XML document Therefore XSL extensible Stylesheet Language stylesheets must be used in addition to the XML document to transform the XML into the sort of document that is recognized by the browser This is the case when tabular reports or a history or script are printed from ImageMaster The software uses the XSL stylesheets located in the Template folder of the ImageMaster installation directory to print attractive documents If you are familiar with XML and XSL you can even create personalized templates for printing Notice that XSL stylesheets can also be used to convert an XML file into another XML file ImageMaster 2D Platinum Use
334. tection on DIGE images 1 Go to the DIGE Gels folder in the Workspace window 2 Select the DIGE gels to be detected Right click on one of the gels 3 Choose Detect from the contextual menu 4 The images corresponding to the selected gels are loaded and opened in a new worksheet 5 Inthe DIGE Spot Detection window enter an estimation see below of the Number of Spots present on the images Click OK 6 A status window appears showing the progress of the spot detection Depending on your computer resources the spot detection process can take some minutes ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 133 134 7 The spots appear on the gels Alternatively if your DIGE gels are already opened in the Display Zone then you can select the gels and go to Edit gt Spots gt Detect Enter an estimation for the Number of Spots to be detected in the DIGE Spots Detection window and click OK 7 4 2 Spot detection parameter When detecting DIGE images you must enter an estimation of the Number of Spots present on the images It is recommended that this value be overestimated to compensate for the detection of non proteinaceous spots on the image e g dust particles which can subsequently be excluded from the analysis using spot filtering see Section 7 5 2 to learn more about spot filtering with the Refine by Value feature If all the spots have not been identified the spot detection process can be repeated with a higher num
335. tems in the label field e Ashort descriptor that will be the visible part of the label e A keyword indicating the type of link http file text e Alink content which contains the information necessary to establish the link or the content of the link in the case of a text link To indicate that a label is linked its short descriptor is followed by three dots When you double click on such a linked label you do not enter the editing mode as usual but the link Web page file or text is automatically opened with the appropriate software Alternatively to open any linked label choose Show gt Annotations gt Linked Data in the menu Note that you can define links in any label category but you can only have one link per label Http link You can link spots or pixels to specific Web pages A double click on an http linked label will launch your default Internet browser and automatically call the corresponding Web page You can for instance create a direct link to the ExPASy Proteomics Server Figure 8 3 In this case the label content should contain the string http followed by the address of the Web page File link You can link spots or pixels to software files Double clicking on a file linked label launches the specified file with the default system application associated to the file extension The linked files can be placed in a specific directory which is defined by choosing Tools gt Options in the menu and b
336. they do not correspond to the whole spot which is difficult to define The reason for this lies in the quantification methodology described in Section 7 3 3 7 3 1 Procedure To detect spots efficiently we recommend previewing the spot detection results on a few small image areas drawn with the Region tool before or during spot parameter optimization so that you can decide whether the parameters should be adjusted or not Each change in one of the spot detection parameters is immediately reflected in the selected region Figure 7 1 except if you turn the Auto Preview option off in the Detect Spots window In this case you need to click the Preview button to refresh the spot detection preview The advantage of manual previewing is that it is quicker to make simultaneous changes to more than one parameter Once you find the optimal parameters you can detect all spots on the selected images Please note that you can already view certain properties of spots for example Intensity Volume or Saliency in the preview mode while the spots are not yet detected permanently Do this by displaying the Cursor Information window see Section 7 9 and moving your cursor over a spot or by selecting a spot and displaying a Spot Report see Section 7 9 This is particularly useful for entering a valid Saliency parameter see Section 7 3 2 Obviously the Detect Spots window must remain open while doing this ImageMaster 2D Platinum User Manual 1
337. this The first one requires that you already know the populations in your set of gels This is the case for example when you are comparing gels from healthy tissue extracts with those from disease associated samples You can then define the classes yourself Classes are created in the Workspace see Chapter 4 When you have no preliminary knowledge of the populations in the set of gels you can implement a heuristic clustering analysis see Section 10 2 6 and possibly draw conclusions from a factor analysis study see Section 10 2 5 When doing heuristic clustering classes are automatically defined and you can analyze them with the tools described below 10 3 2 Overlapping measures As for the intra class analysis the spot values of each protein class can be summarized by two statistical descriptors which are the central tendency and the dispersion see Section 10 2 2 In addition to these descriptors ImageMaster computes overlapping measures between the class intervals which are defined by the ranges Central value Dispersion Central value Dispersion These ranges quantify the overlap between the spot values from two classes and thus gauge how different the protein expression changes between the classes really ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis 10 E are These measures of overlap take into account both the difference between the central tendency in each population and the dispersion
338. tion AA Annotations 3 EEE er e In Region Shows all annotations in the area selected with the Region tool This option is handy when all annotations were previously hidden e For Spots Shows all the annotations linked to selected spots This feature is particularly useful when all annotations were previously hidden e Only Selected Shows only the selected annotations and hides all the other ones e Hide Selected Hides the selected annotations er ones Piss 00 Mye l METK_ECO 5 20 Mw 43000 500 1 15 20 43000 H A his protein is a component of the EUCINE ISOLEUCINE VALINE THREONINE transport system LIVI_ECOLI ar 06999 P2_ECOLI 4 a m er w1 5 20 43000 a g Ar ae 4 m p j s his protein is a component of the F j EUCINE ISOLEUCINE VALINE ws THREONINE transport system 4 e F b Figure 8 7 a All categories and labels are visible b Only the Ac pl MW and Comment categories are visible and only for particular spots To show or hide specific categories 1 Select the gels for which you would like to change the category visibilities 2 Choose Show gt Annotations gt Visible Categories from the menu 3 Inthe Set Categories Visibility box the check boxes of the visible categories are selected
339. tion from a gel Note that this can also be done for gels that were already calibrated when you imported them into ImageMaster To remove a calibration from a gel 1 Select the gels from which you want to remove the calibration 2 Choose Edit gt Gels gt Reset Calibration in the menu 3 You are asked to confirm your choice 6 5 2 Spot normalization Spot normalization is a kind of internal calibration that makes the data independent of experimental variations between gels caused by conditions such as differences in protein loading or staining In ImageMaster this simply implies the use of the relative Intensity Intensity or relative Volume Vol to quantify and compare the gel spots By definition er ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 111 ED 112 3 i here Intensity is the calibrated intensity ONY x qoo Where ntensitys Is AE n intensity of spot S in a gel containing n gt Intensitys spots S 1 i Vol Vol x 100 where Vols is the volume of spot S in a n gel containing n spots gt Vols s 1 These measures take into account variations due to protein loading and staining by considering the total intensity or volume over all the spots in the gel This means that in a gel where globally the spots are darker than in another image the majority of spot volumes is higher However the bulk of Vol should be similar to those in the compared image at least for gels with similar spot patterns
340. to a list of scripts opened during the current work session and scripts that were previously saved in the default ImageMaster Scripts folder in the user s My Documents directory Save the script You are given the choice of saving only the selected actions Scripts saved in the default ImageMaster Scripts folder in the user s My Documents directory can be accessed directly using the Open icon aa Print the script Choose to print only the selected actions Note that before printing the script is first displayed in your default Internet browser The XSL stylesheet located in the Template Script folder of the ImageMaster installation directory is used to transform the XML formatted script into an attractive document find more details about XML and XSL in Chapter 11 You can then use the print option in your browser to get a paper copy Cut the selected actions from your script uw Copy the selected actions for subsequent pasting in a script fe Paste the previously copied actions from any History or Script window below the currently selected action 4 Run the script Be sure to select all the images that should be run with the script The originally encoded script sometimes needs to be modified because you forgot to do an operation would like to suppress an action or want to adapt some parameters to a specific set of gels The following tools and possibilities are available for modi
341. to compare protein expression changes between different gels Therefore two major requirements must be met e First automatic spot detection needs to identify a maximum number of proteins while minimizing the number of artifacts incorrectly detected as spots and especially reducing the number of undetected proteins This requires finding a good compromise between the sensitivity and the specificity of the spot detection algorithm If the automatic spot detection results are not fully satisfactory manual spot editing can be attempted for non DIGE gels e A second prerequisite is that the differential expression values are accurate and reproducible The goal is not to measure the absolute protein abundance of each spot in an image Within a gel there is no quantitative relationship between the measurable amounts of protein in different spots This is because proteins are colored with dyes that bind differently to different proteins What is important however is that the relative quantities of the same protein spots in different gels are correctly evaluated The spot detection and quantification methods in ImageMaster were specifically designed for this purpose You will learn more on how this is done in the following pages In ImageMaster 2D Platinum 6 0 two different spot detection algorithms are implemented The Melanie ImageMaster algorithm is used for non DIGE gel images DIGE images are co detected using the algorithm created by the D
342. together Figure 10 8 It must be noted that when using raw spot values very abundant spots predominantly influence the similarity criterion To prevent this it is better to use the Sort by Similarity option in combination with normalized spot values see above Figure 10 8 Histograms displayed according to their shape similarity 10 2 4 Intra Class Report Similar to the Intra Class Histograms the Intra Class Report describes numerical data from matches i e their matched spots as well as their corresponding ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 205 Data analysis statistical values In addition to the central tendencies and dispersion quantities described earlier Section 10 2 2 other measures for the analysis of matched spots within a class of gels are available in the Intra Class Report Figure 10 9 r Additional values V Coef Variation I Range ratio I Separability L Figure 10 9 Additional values that can be added to the Intra Class Report The Coefficient of Variation Coef Variation is the dispersion divided by the central tendency It measures the relative variability of the spots in a match by correcting for the magnitude of the data values thus giving a measure that has no units When you choose the Median and Mean Absolute Deviation statistics this measure is also known as the Coefficient of Dispersion The Range Ratio is the maximum value divided by the smallest value in th
343. tomatically takes them into account for the matching procedure In this case you just have to choose Edit gt Matches gt Match Gels from the menu When the annotations belong to any other category you must first match the annotations to generate starting matches Once this is done you can run the automatic matching algorithm again by choosing Edit gt Matches gt Match Gels from the menu You must keep the existing pairs when asked for it To match annotations 1 Select the gels for which annotations should be matched 2 Choose Edit gt Matches gt Match Annotations from the menu 3 Inthe pop up dialog box select the desired category to be matched 4 ImageMaster creates matches between identical labels of the chosen category 5 You can now run the automatic matching procedure 9 3 Selecting matches 9 3 1 Menu options Selecting matches consists of selecting their corresponding spots You can select particular matches by using the items in the Select gt Matches menu ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 183 9 Matches 184 By ID Allows the selection of matches based on their Match ID For spots Selects all spots matched with selected spots in the selected gels All Selects all spots matched with spots in the master image Refine Selection Refines an initial match selection based on the presence or absence of matched spots in the selected gels see below for more details Inverse Selectio
344. ts To create a script 1 Choose Tools gt Script gt New from the menu 2 Code your new script by copying specific actions from a history or other script using the tools described above 3 Save your new script using the corresponding icon in the Script window toolbar To run an existing script 1 Choose Tools gt Script gt Run from the menu 2 Browse the directory where the script file is located and select its name 3 ImageMaster may ask you for approval to run the script see below Answer Yes 4 Thescript runs immediately stopping at each operation that was set as a user input action or when an illogical or impossible operation is ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 257 Safety control and automation encountered for example Select All Matches when images were not matched To require confirmation before running a script 1 Goto Tools gt Script gt With Confirmation in the menu If With Confirmation is checked then the option is activated This is the default setting 2 Todeactivate this option choose Tools gt Script gt With Confirmation With Confirmation is no longer checked in the menu To runa recently used script 3 Goto Tools gt Script gt Recent in the menu 4 Choose one of the scripts from the list only possible if scripts were used or created during the current work session 5 The script runs immediately ImageMaster may ask for confirmation SSS 258 ImageMaster 2D Pl
345. tual menu to open the classes 3 Each class is opened in a separate pane in the newly created Classes worksheet 4 7 5 Statistical analysis Once classes are opened in the Display Zone you are ready to analyse your gels and calculate Intra Class or Inter Class statistics See Chapter 10 to learn more about data analysis 4 7 6 Class properties The Name and Comment are properties of classes The Comment describes and or explains the content of the class It serves as a useful source of information to which a coworker is referred or as a reminder when reviewing old work You can add or modify the Comment or Name while viewing the Properties of a class Note that you cannot modify the name of an open class ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Workspace SSS To edit the Comment or Name of a class 1 Right click on the class 2 Choose Properties from the contextual menu 3 Inthe Class Properties box add or modify the Name or Comment Click OK 4 The changes to Properties are saved 4 8 Working with reports The Reports folder manages all tabular report files with the extension rpt that you produce in ImageMaster Refer to Chapter 5 to learn more about reporting 4 8 1 Creating a subfolder You can create folders and subfolders in the Reports folder in order to better organize your saved reports To create a subfolder 1 Right click on the Reports folder or a subfolder 2 Choose Create Folder from th
346. ty to link spots on gel images to protein data in 2 DE or other databases All you have to do is input the appropriate query format database address and query engine in the External Engine field of the new label category and enter valid database accession numbers AC as labels When you subsequently double click on a label of such a category ImageMaster opens your default Internet browser and launches an HTTP query that takes the form of a Web page address http www expasy org cgi bin nice2dpage p P02649 The elements are e http www expasy org database HTTP address e cgi bin nice2dpage pl database query engine e P02649 database accession number This example relates to a query of the SWISS 2DPAGE database on the ExPASy Proteomics Server using the nice2dpage query engine As a result the entry for Human Apo E corresponding to the protein with accession number P02649 is returned in your browser ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA To set the database HTTP address and query engine 1 Inthe Create Category box click on the URL button located next to the External Engine field 2 The page http www expasy org ch2d 2d access html detailing a list of federated 2 D PAGE databases and servers is displayed This list provides the required database query formats address and query engine 3 Copy the desired database address and query engine The necessary information generally end
347. u click on The coordinates of this center point are found at the lower right corner of the image The angles of rotation in the vertical and horizontal directions ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 103 ED 104 as well as the zoom factor are displayed at the right side of the 3D View window Please note that vertical rotation is limited between 0 and 90 you cannot look under the gel Below the 3D image the Lighting check box enables you to switch off the light sources that ensure a proper three dimensional aspect of the image The result is that the gel will appear like a normal gel image when it is looked at with a vertical angle of 90 flat position and that the individual pixels are visible NOTE The pixel gray level is overlaid on the 3D image the top of the peaks is therefore always darker This also means that you can combine the 3D View with the various color palettes available through the Adjust Contrast feature The second box at the bottom of the 3D View window called Spots Overlay should be checked when you want to display the spot positions on the view Spots are shown with outlines corresponding to the spot borders or as crosses indicating the spot centers To choose one of these options you should go the Spot Shape item under the Show drop down menu of the toolbar The 3D View module in ImageMaster also enables comparative viewing of corresponding regions or spots in di
348. u must answer Yes 6 Atthe end of the procedure ensure that the Launch GE Healthcare Software Licensing Server box is checked and click Finish This automatically launches the license server dialog LMTOOLS 7 Click the Config Services tab and ensure that all options are set as in Figure 3 1 Note that the paths shown C Program Files GE Healthcare eLicense server are valid if the default installation is made ee ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 19 Getting started LMTOOLS by Macrovision Corporation http www macrovision com lol x File Edit Mode Help Service License File System Settings Utilities Start Stop Reread Server Status Server Diags Config Services Borrowing Service Name GE healthcare eLicense server E Configure Service Path to the Imgrd exe file m Files GE Healthcare eLicense server lmgrd exe Browse Path to the license file m Files GE Healthcare eLicense server Licenses Browse Path to the debug log file m Files GE Healthcare eLicense server lmgra log Browse View Log Start Server at Power Up Use Services Figure 3 1 Configuration in the Config Services tab in LMTOOLS 8 Click the Save Service button even if you made no changes in the previous steps 9 Close LMTOOLS 3 2 4 Collecting and placing an eLicense file Next an eLicense file should be collected and placed in the appropriate folder on your computer In the case of a node locked license the lice
349. u want to place the gels in a new match set Or choose an existing match set from the list Click OK 4 Ifa new match set is created specify the reference gel in the following Add Gels in MatchSet box and click OK 5 The gels are added and the reference gel is tagged To copy and paste gels to a match set 1 Select one or more gels in the Gels folder or MatchSets folder Use the Shift or Ctrl keys to make multiple selections Right click on one of the gels 2 Choose Copy from the contextual menu 3 Right click on the destination match set 4 Choose Paste from the contextual menu 5 The gels are added To drag and drop gels to a match set 1 Select one or more gels in the Gels folder Use the Shift or Ctrl keys to make multiple selections 2 Left click on the gels a Hand cursor appears 3 Drag and drop the gels into the destination match set 4 6 4 Copying match sets Match sets can be copied for use in another match set configuration Saas ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 51 52 To copy a match set 1 Select one or more match sets in the MatchSets folder Use the Shift or Ctrl keys to make multiple selections 2 Right click on one of the match sets and choose Copy from the contextual menu Right click on the destination match set and choose Paste from the contextual menu 3 The copied match sets appear in the designated location with one or several characters appended to their names match set
350. uality and reproducibility Careful experimentation remains the only way to guarantee significant end results This includes refining protocols for the sample preparation of each starting material pre fractionating the sample using precise and correct experimental conditions during 2 D separation and protein staining or labeling employing larger gel formats or narrower pH ranges and so on Image acquisition parameters also play a very important role 2 2 Differential gel electrophoresis 2 2 1 Technique Conventional 2 DE techniques require the separation of each sample on an individual gel in order to compare protein abundance in different samples This one sample per gel approach introduces a high level of system variation to the data such as the variation that arises from differences in protein uptake into the first dimension strip second dimension gel running etc This high level of system variation can outweigh the often subtle induced biological changes that the experiment is intended to detect for example differences that are caused by a disease state drug treatment or life cycle stage To further complicate this problem it is also necessary to separate the induced biological changes within an experiment from the inherent biological variation In other words the differences between two individual animals cultures or plants that are present irrespective of the applied experimental conditions To achieve this multiple sample repl
351. ulates the relative intensity Intensity and relative volume Vol for each spot These are normalized values that remain relatively independent of irrelevant variations between images By definition intensity Intensity 499 where Intensitys is the calibrated l n f intensity of spot S in a gel containing n gt Intensity spots S 1 Vol Vol lt 100 where Vols is the volume of spot S in a n gel containing n spots gt Vols S 1 er ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 131 These measures take into account variations due to protein loading and staining by considering the total intensity or volume over all the spots in the image This means that in an image where globally the spots are darker than in another image the majority of spot volumes is higher However the bulk of Vol should be similar to those in the compared image at least for gels with similar spot patterns Please note that although the Vol is a rather efficient measure for evaluating protein expression differences between gels the Intensity is not as relevant to use EN imageMaster 2D Platinum lo x Fie View Edit Show Select Analyze Reports Tools Window Help Mosar aIedSHOMA Gels ft Spots jo Annotations jo p Sr Y 589 Z 4367 Figure 7 2 Spot quantification The 3D View in ImageMaster reflects the spot shape and volume of what will effectively be quantified The spot outline correspo
352. um User Manual 11 0034 38 Edition AA 235 Data integration 236 1D and 2D calibrations 1D calibration steps are converted to annotations of the pl_MW category For pl calibrations the labels contain the pl value followed by a space and the number 1 indicating that no MW information was available for this annotation For MW calibrations the labels contain the number 1 indicating that no pl data was available followed by a space and the MW expressed in Daltons Da Using this data ImageMaster 2D Platinum automatically displays computed pl and MW values for all the spots in the gel and any matched gels 2D calibration information available from the Protein List in ImageMaster 2D Elite is also converted to annotations of the pl_MW category when proteins are assigned to spots In this case the labels contain the pl value followed by a space and the MW expressed in Da Protein lists Information from the Protein Lists in ImageMaster 2D Elite is converted to spot annotations The pl and MW values are transferred to annotations of the pl_MW category see above Accession numbers appear under the Ac category Annotations Annotations from ImageMaster 2D Elite are converted to annotations of category Annotation Please note that annotation formats such as font style size color etc are lost 11 1 3 From Twain compatible scanners ImageMaster can also acquire images directly from TWAIN compatible scanners You must indicate
353. umber for example P02649 The database HTTP address and query engine are entered as constraints to the annotation category Go to the Categories tab in the ImageMaster Options accessible by choosing Tools gt Options in the menu and type them in as one string in the External Engine field for example http www expasy org cgi bin nice2dpage pl A list of federated 2 D PAGE databases with the required database query formats can be found at http www expasy org ch2d 2d access html 11 4 2 Querying the database The database accession number for example P02649 is entered as a label of the particular category linked to a spot It identifies the protein corresponding to that spot When you subsequently double click on the label while the Annotation tool is selected ImageMaster opens your default Internet browser and launches a query that consists of the concatenation of the HTTP address query engine and accession number for example http www expasy org cgi bin nice2dpage pl P02649 As a result the entry for the protein with the given accession number opens in your browser Figure 11 4 In the case of the above example this would be the entry for Human Apo E Gels 11 4 3 SWISS 2DPAGE master gels SWISS 2DPAGE master gels or reference maps contain annotations for all proteins corresponding to entries in the SWISS 2DPAGE 2 DE database Each annotation contains the accession number in the Ac category of the datab
354. ure 10 12 shows the Factor Projection Plot obtained when the first two axes in Figure 10 11 were selected This plot displays the projection of each match red or green cross and each gel blue vector on the two factorial axes In the present example only the 20 most significant matches are displayed on the projection plot If all matches were shown one would find that many of them cluster around the origin of the graph This illustrates that the majority of matches i e protein spots are not significant in classifying the gels The further away a spot is from the origin the more important it is likely to be in terms of characterizing the gels To find a possible meaning for a given factor one should first identify the matches that largely contribute to this factor The Factor Projection Report Figure 10 13 a available through the Reports item in the Factor Projection Plot is used for this purpose When sorting the matches in this report according to their contribution to the first axis one discovers that matches 4825 4715 4704 4898 and 4784 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 211 Data analysis 212 appear at the top of the table Interestingly these matches are those with the highest relative volumes as found from the Intra Class Report that is accessible in the Factor Projection Report toolbar In fact the first axis is generally correlated with protein abundance xij a alal ole Eyl e SM
355. ust select the desired type of normalization Value Relative Ratio or Normalized in the Displayed value list Figure 10 10 A number of standard functionalities are available from the Intra Class Report toolbar see Chapter 5 for details Selected lines use Shift or Ctrl keys for multiple selections can be saved printed or exported to other software You can view your data on gels or related reports using the selection and navigation buttons The Intra Class Report supports the display and editing of annotation content and therefore offers the Annotate and Update Gel functions These functions enable you to add new categories and to propagate label modifications to your master gels Finally you can customize the content of the Intra Class Report by using the Settings option Factor Analysis Histograms Update Gel Annotate Reports Settings Displayed value Intra Class Report Data analysis BIS ao sole P ule Oo Ble Intra Class Report Vol The chosen statistics are Mean 100 and M S D Match ID Mean 100 Variation Ratio 100 a Separability A_T1_Gel1 A_T1_Gel2 4456 0 0365684 0 0293886 0 803662 11 0250 0 452889 0 0538708 0 0630224 0 07 343 4823 0 197283 0 128024 0 648916 104074 0 163236 o soseg o 304164 03 a4 asor 0 216323 0 113875 0 526411 8 93871 0 10
356. veral spots in a given image if you want to create a multiple match Use the Shift or Ctrl keys to make multiple selections 2 Choose Edit gt Matches gt Add Match from the menu 3 The new match is created 188 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Matches ex To delete a match 1 Select the spots for which matches should be deleted 2 Select the gels from which the corresponding matches should be deleted Use the Shift or Ctrl Keys to make multiple selections 3 Choose Edit gt Matches gt Delete Match from the menu 4 The matches for the selected spots are deleted NOTE To undo the addition or deletion of matches you should not only select the gel for which the operation should occur but also the Master 9 6 Reports on matches 9 6 1 Match report ImageMaster can display a Match Report It shows a list of matched spots in the selected gels The first column in such a report displays the Match ID that is the Spot ID in the master image The following columns display the corresponding Spot IDs in the matched gels that were selected for the creation of the report Figure 9 6 To display a Match Report 1 Select the spots that should appear in the Match Report 2 Select the matched gels you would like to include in the report 3 Choose Reports gt Match Report from the menu 4 You get a list of spot IDs for all matched spots among the selected gels x ale lol s lele m el Bl
357. view button 6 Adjust the detection parameters In particular optimize the Smooth parameter to detect all real spots and split the overlapping ones Subsequently filter out the noise by changing the Saliency and Min Area values See below for more details on spot detection parameters 7 When you are satisfied with the preview click OK to detect all spots in the selected gels using the parameter values you have set Please note that you can still change your gel selection at this point 8 The spot shapes are displayed on the images You may want to save the current detection parameters so that they can be applied to other gels at a later time To do this click the Save icon in the toolbar of the Detect Spots window Enter a file name and click Save in the Spot Detection dialog box ImageMaster saves the detection parameters with the extension dpm By default they are saved in the ImageMaster Menus folder in the user s My Documents directory Parameter sets saved in this default folder are always available from the Open icon in the toolbar of the Detect Spots window 7 3 2 Spot detection parameters Spot detection parameters are best adjusted in the following order e Smooth First set the Smooth parameter It fixes the number of times ImageMaster will smooth the image before detecting spots using a smooth by diffusion algorithm The Smooth parameter should be optimized to detect all real spots and split as many overlapping spots as poss
358. w menu ImageMaster allows you to move gels based on specific positions Choose Show gt Gels gt Move in the menu and select one of the following options e Same location All gels in the active worksheet are moved to the same position with the same magnification as the selected gel ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA e Up Scroll up in the selected gels e Down Scroll down in the selected gels e Left Scroll to the left in the selected gels e Right Scroll to the right in the selected gels e Top Left Display the top left of the selected gels e Top Right Display the top right of the selected gels e Bottom Left Display the bottom left of the selected gels e Bottom Right Display the bottom right of the selected gels 6 3 2 Zoom gels There are several ways to enlarge or reduce the view of gels You can use the Magnify tool Show menu options Zoom window Overview option or scrollbars see Section 6 3 3 Magnify tool To zoom in or out on a gel 1 Click on the Magnify tool in the toolbar Figure 6 2 2 Tozoom in repeatedly click the area of the gel where you want to see details 3 Tozoom out right click repeatedly on the gel To move all gels in the active worksheet to the same position with the same magnification hold down the Shift key while zooming in or out on one of the gels Show menu ImageMaster allows you to zoom in and out on gels based on specific factors Choose Show
359. with a spot annotation and the other with a pixel annotation as long as the annotations are centered on the markers To create a pick list 1 Identify the two reference markers on your image 2 Zoom the image to better see the left reference marker 3 Check if the marker is detected as a nice round spot If it is not select the spot s on the marker and delete by choosing Edit gt Spots gt Delete from the menu 4 Click on the Annotation tool in the main toolbar 5 Double click on the marker spot or if such a spot is not present on the pixel that is in the middle of the marker 6 Select the Comment category 7 Enter IR1 as the label text 8 Ifan annotation attached to a pixel is not in the center of the reference marker you can move it to the appropriate position Do this by clicking on its basis cross and holding down the left mouse button while dragging the annotation to its new position 9 Move your gel to see the second right reference marker ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data integration 10 Repeat the procedure but enter the label text IR2 this time 11 Once the two markers have been annotated select spots to pick Figure 11 3 12 Choose File gt Export gt Spots to Picker gt GE Healthcare Ettan 13 For each gel you will be asked to save a pick list in text or XML format only the text file can be read by the Ettan Spot Picker 14 The Ettan Spot Picker can now read
360. work in conjunction with several specialized 2 DE databases but is particularly compatible with data from the SWISS 2DPAGE database that is accessible over the Internet If your computer has access to the World Wide Web you can remotely query and retrieve protein data related to spots on your gels NOTE The ImageMaster software provides access to several databases on the Internet It is the responsibility of the user to acquire the database licenses if needed In particular the PROSITE and SWISS 2DPAGE databases are copyrighted and all commercial users of these databases are required to purchase a database license from GeneBio No license fee is charged to academic users for non commercial use For questions about obtaining a license subscription for the PROSITE and SWISS 2DPAGE databases please contact GeneBio www genebio com 11 4 1 Setting the database A detailed description of how to query a remote database through the Internet is given in Chapter 8 In short these queries use the label holding a protein accession number taken from a 2 DE database of a particular category linked to a specific database to interrogate the database The HTTP query must be composed of e The database HTTP address for example http www expasu org ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 245 Data integration 246 e The database query engine for example cgi bin nice2dpage pl e The database accession n
361. y Fall Workspace 3 Ole ge Project Bacteria a Gels MatchSet A DIGE Gels Bacteria NAM_TI A a B Bacteria 1 A 4_T1 BA Synthetic gel ATI AT2mel Bacteria 1au_T1 Gl MatchSets Bacteria 1 A MA_T2 A_T2 H Bacteria 1 y Bacteria 1 A A_T2 A_T2 EB A Rat asctena naa 12 A 72 M A_T2_Gelt mel BY A_T2_Gel2 mel E A_T2_Gel3 mel eet ATI PR A_T1_Gell mel By AT1_Gel2 mel E A_T1_Gel3 mel BY Synthetic gel ATI ATZ fo B ces A Bacteria 2 a Classes 3 Bacteria 1 BGA FA Synthetic gel ATI ATZ Gels p Spots b Annotations jo Figure 9 9 A synthetic gel Synthetic gel AT1 AT2 and its six contributing gels The classes A_T1and A_T2 were originally opened in a worksheet to create the synthetic gel Therefore Master_A was used for the matching first common node in the match set structure see File Details and the synthetic gel was automatically inserted at this level in match set A 194 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Data analysis 10 Data analysis 10 1 Introduction To study the variations in protein expression among a series of gels the gels should be matched together be part ofthe same match set placed in classes and opened in a Classes worksheet Please refer to Chapter 4 to learn more about the workspace and the definition of classes Data analysis can be carried out at two different levels Protein expression changes wit
362. y description suji F P02649 by accession a number by clicking on a spot by author General information about the entry by serial number View entry in original SWISS 2DPAGE format by full text search Entry name APOE_HUMAN SRS Primary P02649 accession number Entered in SWISS 2DPAGE Release 01 February 1995 in Last modified in Release 13 December 2000 Name and origin of the protein Description Apolipoprotein E Apo E Gene name s APOE From Homo sapiens Human TaxID 9606 Eukaryota Metazoa Chordata Craniata Vertebrata Euteleostomi Taxonomy Mammalia Eutheria Euarchontoglires Primates Catarrhini Hominidae Homo References 1 MAPPING ON GEL MEDLINE 78094420 PubMed 271964 NCBI ExPASy EBI Israel Japan H la Internet N Figure 11 4 The SWISS 2DPAGE entry for human Apo E Entries from this database contain full protein names bibliographic references annotations such as protein function pathological variations and the pl and MW of the related spots on the 2 DE maps It also includes cross references to numerous other databases ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 247 Data integration 248 ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Safety control and automation 12 Safety control and automation 12 1 Introduction Discover how ImageMaster lets you monitor the operations carried out on your images and automate variou
363. y level histogram in Figure 6 11 The histogram displays the frequency with which each gray level from 3524 to 28530 occurs in the gel image A_T1_Gel1 The low gray levels at the left of the histogram corresponding to the background occur very often whereas the high gray levels at the right of the histogram corresponding to the darkest spots are much less frequent The red line indicates that the minimum and maximum gray levels 3524 and 28530 respectively are remapped by the default linear mapping The vertical axis for the red remapping graph corresponds to the 256 screen gray levels Image Display Settings x g E Choose the color palette and setthe contrast mapping options Colors Gray gt T Invert O Image A_T1_Gell mel Unit Value v I Only in Region 3524 28530 3524 0 28530 Bending 4 E gt 0 es Figure 6 11 Adjust contrast function However you can choose other mapping functions to accentuate small faint spots ImageMaster offers two ways to change the default gray level mapping and thus improve the visual display of the gels You can define the minimum and maximum gray levels i e look at only the light orthe dark regions in the images Do this by decreasing the size ofthe gray level range that is to be remapped linearly to the screen gray levels To accomplish this move the left or right borders of the slider that is found below the histogram function Once the size interv
364. y setting the Annotations folder in the Database tab In this case you only need to give the name and file extension when creating the link Alternatively you can link labels with files located anywhere on your system You should then include the complete file path when referencing the file ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA 159 8 Annotations A ExPASy Proteomics Server Microsoft Internet Explorer 1E File Edit View Favorites Tools Help Back A A Qsearch Favorites media 3 D S fi a Address httosiwwww expasy org Go Links SiteMap Search ExPASy Contact us Search Swiss Prov TrEMBL E for Go Clear ExPASy Proteomics Server The ExPASy Expert Protein Bioinformatics SIB is dedicz Enter label text for category lt Comment gt PAGE Disclaimer Referenc Swiss Institute of d structures as well as 2 D This is a link to ExPAS http Awww expasy org Internet P10413 his is a link to ExPASy Show Label lol x Hlal AA composition 12 5 14 1 Edit Label Show content of text link Additional info text FUNCTION Molecular chaperone Has ATPase activity SUBUNIT Homodimer Oligomerizes at 65 degrees Celsius SUBCELLULAR LOCATION Cytoplasmic PTM Phosphorylated SIMILARITY Belongs to the heat shock protein 90 family Enter label text for category lt AAComposition gt
365. y understand the functionalities explained below You can refer to the following chapters to learn more about these ImageMaster objects 6 5 1 Intensity calibration Instead of displaying and using the gray levels taken directly from the image capture device you can assign real world values often the optical density OD to the measured pixel values The advantage of this is that the range of gray values is the same no matter what the original pixel depth Some image capture devices automatically perform this type of calibration When they do ImageMaster generally reads the calibrated intensity values from the saved files For example images acquired with the GE Healthcare ImageScanner using the LabScan 5 0 software are already calibrated ImageMaster 2D Platinum User Manual 11 0034 38 Edition AA Displaying calibration information You have different ways of finding out if your images were calibrated and for viewing the calibration information e Choose Reports gt Gel Report to view the calibration function Calibration and calibration units Unit for the selected gels If nothing appears in these columns the gels are not calibrated e Choose Reports gt Gel Calibration gt Report to display a report including the calibration function the name of the step tablet used the name of the person that did the calibration and the calibration date e If your gel has been calibrated with LabScan 5 0 you can select the gel a
366. zing gels ee 106 6 5 1 Intensit calbra tioN en a a ceca snctess 106 6 5 2 Spot normalization 3 6 5 3 Intensity normalization using a scatter plot ueeueeee 112 6 6 Processing gel Soei ennn ra E E EA NE 114 6 6 1 Delete fron diS Rense aein ea e a a 114 6 6 2 Renaming GelS wns cena a N E A 114 6 6 3 Rotating gels 6 6 4 FMD DING GES inaa aN 6 6 5 Scalind gels aus een 6 6 6 Inverting gray levels 6 6 7 Cropping gels 6 7 Reporting on gels 119 6 7 1 GOL TED OM ne scat RSS Rai 119 6 7 2 Gel deschiption report nsne na 120 6 7 3 Gel calibr tion Feport uuaussenganennnsesenaunemannaan 122 6 8 Saving exporting or printing IMAGES nnnensnennenen 123 6 8 1 Saving gels N 6 8 2 Exporting gels and windows 124 6 8 3 PEihting gelseu n nn 125 Spots 7 2 I Introd etionizesmiiaseeeeepiinisresteeungienneuhgniitee 7 2 SPOTID S ineens E RA 7 3 Detecting spots in non DIGE gels ui 73 1 ILANA E Ee ed 7 3 2 Spot detection PAFAMETtELS seesessessssssssssscsscssscssseeceeecessssssssesssssssnsnanneeee 7 3 3 Spot QUANTIFICOLION cecssssssssssssssssssssssssssssssesssescssssseseesecssssssssssssssssususunsssessees 7 4 Co detecting spots in DIGE gels o cceeeccssssssssssssssssseeessssseccssssssssnseeeess 7 4 1 Proced ra p 7 4 2 Spot detection parameter 7 4 3 Spot guantificati nzsaunenakniaeninar are 7 5 Selecting Spots 75 1 SPOt 100 PERE E SE 7 5 2 Select MOM yoe E E OE NRO ImageMaster 2D Platinum

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