AIDA 2D Densitometry User's Manual (Windows)
Contents
1. o oooooooooorornmooo 79 Assigning Names to Regions ui ad 82 AIDA 2D Densitometry User 5 Manual Windows Introduction 1 Overview Some methods result in digital images where the signal intensity of certain areas of these images need to be evaluated e g 2D protein gels dot blot assays images of fluorescent multiwell plates autoradiographs of tissue cuts in situ hybridizations etc To enable quantification these areas must be marked by an enclosure of the appropriate shape rectangle circle free drawn object The AIDA 2D Densitometry module is used to evaluate the intensity within two dimensional shapes called regions The signal intensity can be integrated Quantity over the entire region For concentration calibration the intensity 1s assigned to the corresponding area of the region It 1s important to enclose the area of interest e g the spot entirely with the region when using the integration option The integrated signal intensity can be compared between regions relative comparison or normalized by standards in the same image or even in other samples of the same series However it is very important to understand and acknowledge the limits of these evaluations to prevent pitfalls that will lead to results of very limited value Thus some suggestions for practical considerations for the sample preparation and image capturing will be given at the various evaluation section It is important2. 12 AIDA 2D Densitometry User s Manual Windows Table 2 1 Columns Available in the Region Report Window Column Std Quantity quantity unit Std Quantity Recalc Quantity quantity unit Recalc Quantity Std Conc concentration unit Std Conc Recalc Conc concentration unit Recalc Conc Short Description The quantity of the region as entered for the calculation of the standard curve With the Quantity Standards the background corrected Intensity values of the regions are calibrated The percentage of the quantity standard value compared to the reference row in the group of the region A calculation of the quantity in the region based on the current calibration curve The percentage of the quantity for the region determined on the basis of the calibration curve compared to the reference row in the group of the region The concentration of the region as entered for the calculation of the standard curve With the Concentration Standards the background corrected Intensity per Area values of the regions are calibrated The percentage of the concentration standard value compared to the reference row in the group of the region A calculation of the concentration in the region based on the current calibration curve The percentage of the quantity for the region determined on the basis of the calibration curve compared to the reference row in the group of the reg
3. All used standards should be saved for future use This is also true for one time standards because evaluation get frequently re done which means that the standard values are lost unless entered in the list Calibration Curve Pane In the Calibration Curve pane of the Region Calibration toolbox the mathematical calculation of the standard curve can be selected and the result is displayed in the graph at the lower end of the toolbox Calibration Curve Log weighted Regression linear fit PSLinnrrt dormien Corr Coeff f 0 499865 Figure 2 59 Calibration Curve Pane The Calibration Curve drop down menu provides the following functions e Linear Interpolation e Linear Regression e Logarithmic Regression e Log Weighted Regression Calibration Curre Log weighted Regression Linear Interpolation X Linear Regression Logarithmic Regression o Lag weighted Regression Figure 2 60 Selecting Calibration Curve Type Linear Interpolation calculates lines to join each data point 46 AIDA 2D Densitometry User s Manual Windows Linear Regression optimizes a linear graph so that the deviation of the actual points from the curve is minimized Logarithmic Regression is used to determine the standard curve with logarithmic characteristics which is found in many saturating effects e g absorption Log Weighted Regression can be described as linear regression using a double logarithmic scaling Th
4. Log weighted Regression linear fit Calibration Curve Pane dpmicm Corr Coeff rz 0 499865 PSL mrr Figure 2 54 Region Calibration Toolbox Overview To display the Region Calibration toolbox e On the main menu click Evaluation and then click Region Calibration Or e Select the Region Calibration button on the toolbar Using 2D Densitometry 43 raytest Method Pane The drop down menu of the Method control pane is used to specify the calibration method Quantity or Concentration LA Region Calibration Method Figure 2 55 Method Drop Down Menu Selecting Quantity will result in the analysis of integrated intensities of all pixels in the defined region whereas Concentration will compare the average intensity in the regions Standard Pane Using the controls of the Standard pane you can assign standards to selected regions Standard Standard Selection Drop Down Menu _ Tte st Source Array high List of Standard Values AutoAssign Tool H 424000 0 1 112000 0 Manual Assignment Tool t 1 41500 0 1 13350 0 Delete Standard Assignment Button 1 3350 0 Edit Standard Button 4 Figure 2 56 Standard Pane From the Standard drop down menu a predefined list of standard values can be selected The specified standard values are displayed in the pane below the Standard drop down menu Beside this pane there are four buttons AutoAssign Tool This button all
5. Performing 2D Evaluations 67 Figure 3 7 Rotating an Overlay 3 Click the left mouse button again to go back to the relocation mode You might need multiple rounds of adjusting in the beginning 4 After the region is correctly assigned click the right mouse button or press the ENTER key to save it The same strategy is followed for the squares circles and ellipses The ellipses tend to be the most difficult to align The Polygon tool and the Freehand Drawing tool work similar as for known graphic programs 68 AIDA 2D Densitometry User 5 Manual Windows 3 E Y ED Using the Ploygon tool every edge of the new region is clicked whereas using the Freehand Drawing tool the mouse button is hold during the manual encircling Figure 3 8 Using the Polygon Tool To edit these polygons a third mode exists besides the relocation and turning mode In this third mode all handles edges of the region will be displayed and their position can be changed This allows you to reshape the region Figure 3 9 Editing Polygonal Overlays Performing 2D Evaluations 69 Finally the AutoContour tool will connect pixels of similar intensity to encircle an object automatically Figure 3 10 Using the AutoContour Tool To mark a region using the AutoContour tool 1 Select the AutoContour tool 2 Point to a position within the border area of an object that you would like
6. Figure 2 66 Preview Example 22 AIDA 2D Densitometry User s Manual Windows Export and Import In AIDA 2D Densitometry various tables and graphs can be exported to be imported in other documents or programs In addition the calibrations and templates can be exported to be applied to other images The various functions can be found in the Export and Import submenus of the File menu Exporting Templates To export your own evaluation templates 1 On the File menu click the Export menu item and then choose Template from the submenu 2 In the Export Template dialog box that appears type a name for the export file and confirm the action by clicking Save or 1 Click the Export Template button on the toolbar to open the Export Template dialog box Ela Aj eje Figure 2 67 Main Toolbar Export Template Button 2 Type a name for the export file in the File Name text box and complete the export action by clicking Save Export Template 2D Calibration adf Save in a 2 5 ample amp t File name Save as type alba Template Format Cancel Figure 2 68 Export Template Dialog Box Using 2D Densitometry 53 raytest Exporting Region Report To export the Region Report On the File menu click the Export menu item and then choose 2D Region Report from the submenu 2 In the 2D Region Report Export dialog box that appears type a name for the export file 3 Click Save to confirm the
7. 2 49 Select Name List Button This opens the Region Nametables dialog box Region Nametables Rat adf New Modify Remove ile conce Figure 2 50 Region Nametables Dialog Box 40 AIDA 2D Densitometry User 5 Manual Windows Nap ES cH gt Using this dialog box you can create a new list of names by clicking the New button or Region Nametables Rat adf Mametable Editor LA Figure 2 51 Nametable Editor Creating New List of Names Using 2D Densitometry 41 B e edit an existing name table by choosing Modify Region Nametables Rat adf Figure 2 52 Nametable Editor Editing Existing Name Table Actual Region Pane The Actual Region pane provides information about the selected region E Figure 2 53 Actual Region Pane 42 AIDA 2D Densitometry User s Manual Windows Use the Configure button to add or delete categories from the list shown below the Configure button This opens the Column Selection dialog box described in the Region Report section of this manual Region Calibration Toolbox The controls of the Region Calibration toolbox allow you to perform the calibration of the 2D densitometer For the calibration reference regions for the creation of standard curves are necessary on the same image l Region Calibration Method Concentration Method Pane Standard Test Source Array high 1 Standard Pane Calibration Curve
8. 3 Release the button Region Calibration 2D Calibration adf Log weighted Regression log v 0 96919 log 3 06108 Correlation Coefficient f 0 999865 Integral a4rea Bkg PSL mm 10000 Conc dpm cnr Figure 2 23 Zoomed Graph To zoom a curve segment 1 Click the Zoom button on the toolbar Window Help Nel Figure 2 24 Zoom Button Main Toolbar 2 Draw a rectangle around the area of the plot you want to see in more detail Region Calibration 2D Calibration adf Region Calibration 2D Calibration adf Log weighted Regression log v 0 96919 log 3 06108 Log weighted Regression log v 0 96919 leg 3 06108 Correlation Coefficient f 0 999865 Correlation Coefficient f 0 999865 E E E E m p a 10 a 10 m RE m 5 i d d I I B d B D D E E 10000 100000 1000000 10000 100000 1000000 Conc dpm cnr Conc dpm cnr Figure 2 25 Specifying the Area to be Zoomed 24 AIDA 2D Densitometry User s Manual Windows 3 Release the button Region Calibration 2D Calibration adf Log weighted Regression log v 0 96919 log 3 06108 Correlation Coefficient f 0 999865 Integral a4rea Bkg PSL mm 100000 Conc dpm cnr Figure 2 26 Zoomed Curve Segment Contextual Menu of the Region Calibration Window The contextual menu of the upper pane of the Region Calibration window contains two commands Scales and Grid Region Calibrat
9. Densitometry User 5 Manual Windows 3 Select the appropriate entry from the list to assign the name to the selected region The list shows the names defined in the current work nametable Region Label Region Name Apply at Regi Select Nam e E ON Figure 3 28 Selecting a Name To change a region name 1 Select the region whose name you want to change 247 ato Figure 3 29 Selecting the Region Performing 2D Evaluations 83 2 Click the appropriate name in the Region Name list Region Label Region Name Apply at Region Creat Select Name List Figure 3 30 Changing Name To assign names automatically upon creation of a region 1 On the Region Determination toolbox select the Region Name tab Apply at Region Creation Select Mame List Figure 3 31 Selecting Region Name Tab 84 AIDA 2D Densitometry User 5 Manual Windows 2 Click Apply at Region Creation Figure 3 32 Checking Apply at Region Creation 3 Select a tool from the Region Creation pane and mark regions sucessively Region Creation Create Vatic Sk Figure 3 33 Selecting Region Creation Tool The names in the Region Name list are assigned one after another Select Mame List Heart Figure 3 34 Assigning Names sucessively Performing 2D Evaluations 85 86 AIDA 2D Densitometry User 5 Manual Windows
10. Export is applied and will be saved with the evaluation data in the adf file The configuration remains valid unless it 1s explicitly changed by the user The automatic export settings are timestamped internally In addition you can add a name and an ID which are included in the Parameter pane of the export file together with the internal time stamp Using 2D Densitometry 59 To create automatic export settings 1 From the Options menu choose Automatic Export The Automatic Export Setting dialog box appears Automatic Export Setting Rat pcb Figure 2 73 Automatic Export Setting Dialog Box The top pane of the dialog box shows the current settings parameters whereas the second displays the current settings for the automatic export of the 2D Region Report Region Calibration Click on the toolbar to switch between the settings pages 60 AIDA 2D Densitometry User s Manual Windows 2 Click Edit Settings The Edit Automatic Export Setting dialog box opens Edit Automatic Export Setting aj 2D Region Report 3 Export an Save List Delimiter _ SN C Mo Tabulator Cancel C Comma Automatic C PromptFor Decimal Delimiter W Parameter Point W Table Header C Comma Setting far Report Columns Intensity Area Bkg M Included in Export Decimal Digits 2 E C Semicolon Figure 2 74 Edit Automatic Export Setting Dialog Box The dialog box provides you with the foll
11. The table in the lower part of the Region Calibration window provides its own contextual menu Select Columns Delete Standards Figure 2 30 Contextual Menu of the Region Calibration Window Table Pane The column headings are equipped with the standard contextual menus described in the Region Report Window section of this manual If you right click on any of the table rows the row s contextual menu opens allowing you to Select Columns for displaying or Delete Standards from the table The following columns are available Table 2 3 Columns Available in the Region Calibration Window Columns Short Description No The number of the region Name The name of the region as it was assigned by the user Quantity Calibration Integral Bkg signal unit The background intensity is subtracted from the calculated intensity of the region to result in the signal intensity of the region For each group of regions the background is determined in the following order the local background area around the region or the local background defined for the group or the general background defined in group 0 whatever is available signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set in the Image Attributes dialog box concentration unit and quantity unit unit of measurement specified by the user through calibration unit quantity or concentr
12. additional Set to 100 item used to set the value of the selected cell to 100 and recalculating the percentages of all other rows in this column accordingly Wi Region Report 2D Calibratio Integral Bkg Integral Area Bkg 5 PSL mrr Group Regions Ungroup Regions Group standards Hide standards Set to 100 Hide sum Show remainder Show total Show header Figure 2 19 Contextual Menu of the Region Report s Cells Using 2D Densitometry 21 Ep raytest Region Calibration Window To open the Region Calibration window Choose Quantity Calibration from the View menu or Double click the miniaturized calibration curve display on the Region Calibration toolbox JER Region Calibration 2D Calibration adf nl xj Log weighted Regression log y 0 9691 9 log 3 06108 Correlation Coefficient 0 999865 E E Bn n 1 a ch B q 1 E 10000 100000 1000000 Conc dpm cnr No Integral Area Bkg Std Conc Recalc Conc PSL mrr dprncrr dpm crr 604 91 1107000 0 1067870 1 255 4 424000 0 438707 8 63 21 112000 0 114080 0 25 41500 0 41100 5 0 13350 13400 1 3950 3908 4 Figure 2 20 Region Calibration Window Example 4 2 T 7 The Region Calibration window is bipartite the upper pane displays the curve of the calibration function the underlying function the correlation coefficient and the x axis intercept The lower one contains a tab
13. by their respective companies THIS PUBLICATION IS PROVIDED AS IS WITHOUT WARRANTY OF ANY KIND EITHER EXPRESS OR IMPLIED INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT THIS PUBLICATION COULD INCLUDE TECHNICAL INACCURACIES OR TYPOGRAPHICAL ERRORS CHANGES ARE PERIODICALLY ADDED TO THE INFORMATION HEREIN THESE CHANGES WILL BE INCORPORATED IN NEW EDITIONS OF THE PUBLICATION raytest MAY MAKEIMPROVEMENTS AND OR CHANGES IN THE PRODUCT S AND OR THE PROGRAMS S DESCRIBED IN THIS PUBLICATION AT ANY TIME Contents 1 Introduction 5 ONCE AE th sie arc ek E E ada da TTE 5 Starine 2D Densitomel 4 0 410 Sd AE 6 2 Using 2D Densitometry 7 Data Windows in 2D DensitoMetty o o ooooooooo eee eens 7 Image Window in 2D Densitometry 0 0 0 00 0 es J Contextual Menu of the Image Window cece eee eee ee 8 mas OYES as 9 Region Report WI Wir A A Ai 11 Data Descriptio pestearen aaea a A EA AS 11 Contextual Menu of the Region Report Window 15 Region Calibration Window sess 22 Graphics Pane of the Regions Calibration Window 23 Contextual Menu of the Region Calibration Window 25 Additional Toolboxes in 2D Densitometry o o o ooooooooooooorn eens 29 Region Determination Toolbox 0 0 0 0 eee eens 29 Region reato Pano onie do duo deste m Zeek tooo d Skea
14. export The 2D Region Report Export Options dialog box appears allowing you to set further option for the export of the result table 2D Region Report Export Options Range Parameter W Table Header Cancel 6 All Rows Selected Rows Cancel f All Columns C Selected Columns List Delimiter Decimal Delimiter Tabulator f Point C Comma System Defaults C Comma C Semicolon Figure 2 69 2D Region Report Export Dialog Box You can specify the Range of the exported rows and columns Table Header All Rows Columns displayed in the Region Report window or only the Selected highlighted Rows Columns in the Region Report window as well as the List Delimiter and Decimal Delimiter in the respective panes of the dialog box Selecting the System Defaults button applies the Windows default settings to List Delimiter and Decimal Delimiter 54 AIDA 2D Densitometry User s Manual Windows In addition the parameter settings for the evaluation can be exported by checking the Parameter option If this option is enabled the following parameter information will be written to the export file Table 2 4 Parameter Export Parameters 1 Row 2 Row Export settings Master nametable Exported Parameter Information Desciption Application name e g Type of exported data e g 2D Region Report Identifier for the export parameter settings Identifier for the master nametable comprise
15. graph in the Region Calibration window Choose Windows Metafile The graph is saved to a separate file in the Windows Metafile format To export the result table of the Region Calibration window 1 Select the Table Ascii File data format 2 Click Save to confirm the export Using 2D Densitometry 57 Qe IN CE 7 y EE The Quantity Calibration Export Options dialog box appears allowing you to set further option for the export of the result table Region Calibration Export Options Range Parameter W Table Header Cancel Jl f All Rows C Selected Rows f All Columns C Selected Columns List Delimiter Decimal Delimiter Tabulator f Point C Comma System Defaults C Comma C Semicolon Figure 2 72 Region Calibration Export Options Dialog Box You can specify the Range of the exported rows and columns Table Header All Rows Columns displayed in the Region Report window or only the Selected highlighted Rows Columns in the Region Report window as well as the List Delimiter and Decimal Delimiter in the respective panes of the dialog box Selecting the System Defaults button applies the Windows default settings to List Delimiter and Decimal Delimiter In addition the parameter settings for the evaluation can be exported by checking the Parameter option 3 Complete the action by clicking the OK button The data are saved to a simple ASCII text file In addition the calibration data can
16. radio button in the Format pane The options in the Print pane enable or disable the printing of the calibration curve Graphic und or the calibration table Table Print In AIDA there are two ways for printing your image and evaluation data e Choose Print from the File menu Or Use the Protocol Print pane on the Publishing Layout toolbox The Print command of the File menu allows you to print your data on the fly without permanently storing the layout printing settings whereas the Protocol Print pane provides you with a complete set of printing features which includes saving the setting with the data file for reasons of reproducibility Choosing the Print command from the File menu opens the Print dialog box allowing you to configure the printout to contain only the overlays results and graphs that are currently needed Click the checkboxes for the various items to turn them on or off Select the number of copies you want to print Print 2D Densitometry 2D Calibration adf Printer EPSON EPL S700L Advanced at LPT1 Cancel Printer Edges left 2 0cm EJ top 20m E m Y Header tight 2 0cm E bottom 200m v Parameter Parameter and Image Options iv Image whole image overlays 4 Region Report portrait Y grayscale M Region Calibration ean ee Iv scales m v overexposed C 100 ES Ed H Region Report Options Figure 2 64 Print Dialog Box 50 AIDA 2D Densitomet
17. the Config Table Header button the table header name for the columns Integral Integral Area Quantity can be configured The following three buttons are used to group ungroup and renumber in this order groups and regions Every area can only belong to one group Grouped areas will get a common summary line at the end of all integrated regions in the region report Groups can be formed by selecting multiple regions and clicking the Group button Selected regions can be removed from their group by selecting the Ungroup button Using the Renumbering Tool all groups are renumbered in sequential order This avoids gaps in numbers which are caused by ungroup processes Using 2D Densitometry 35 Region Label and Name Pane The Region Label and Name pane is used to control the labeling and name of regions Region Label Region Name hove Label Font None i Lih Name C Free Choice Configure ENTM Select Mame List Figure 2 39 Region Label and Name Pane Region Label tab You can turn off labeling None radio button select a number as current label Number radio button or display the name of the region instead Name radio button Alternatively you can select a label freely from the column list displayed after clicking the Free Choice option and then clicking the Configure button The contents of this list is identical to the one from the Columns Selection dialog box cf the Region Report pane
18. to encircle example above heart of a rat cut autoradiography press the mouse button and shift around carefully within the object until the correct border is selected 3 Release the mouse button On release the border is marked and can be edited as usual You can cancel any overlay region editing action anytime you want by simply pressing the ESC key The numbering of the regions can be configured using the Region Label and Name controls of the Region Determination toolbox for details refer to the Region Label and Name pane in the Region Determination Toolbox chapter of this manual Adjust the settings to your experimental design and personal preferences 70 AIDA 2D Densitometry User 5 Manual Windows Using Arrays Matrices It can be helpful to use matrices to evaluate regular patterns of regularly shaped regions e g slot or dot blots multiwell plates etc To create a matrix 1 Mark the first region of the regular pattern in question using the appropriate controls of the Region Creation pane on the Region Determination toolbox 2 Accomplish the region creation by clicking the right mouse button or alternatively pressing the ENTER key 3 Click the Create Matrix button on the Region Creation pane to specify the appropriate matrix layout for the region pattern Matrix options Dimensions Lines Cancel Columns Center distance vertical es mm horizontal min Nu
19. 4 Options Menu Opening the Image Overlays Dialog Box Using 2D Densitometry 9 After choosing Image Overlays from the Options menu the Image Overlays dialog box opens allowing you to specify the type None Number Name Free Choice font font size and font style bold italic roman typeface on white background for labeling For details see the appropriate sections below Image Overlays LZRTEST PEB Figure 2 5 Image Overlays Dialog Box 10 AIDA 2D Densitometry User s Manual Windows Region Report Window All information about the evaluation of the assigned regions can be found and configured to the specific needs in the Region Report The Region Report window is shown by clicking this button E in the Region Determination toolbox or by choosing 2D Region Report from the View menu Bi Region Report 2D Calibration adf BEES No Type Area Integral Integral Bkg Integral Bkg Integral amp rea Bkg Recalc Conc Std Conc mme FSL PSL FSL ram dpro cr dprn cr 1 Std 72 3 4357457 4305 8 62 58 604 94 10946991 1107000 0 5 std 723 eens 6276 aso ses 50902 133500 fsa 7231 2291 1900 oar 268 58756 39500 T Bkg 723 aaa oof omj omj sa Sum 4335 70074 8 esesta 10000 1611 eae Figure 2 6 Region Report Window Data Description Available Columns in the Region Report Table 2 1 Columns Available in the Region Report Window Column Short D
20. B raytest AIDA 2D Densitometry for Windows User s Manual ec m CIE O 7 TO 8 Ore O E Or Q2 Part Number 2003 01 1 0 2003 raytest Isotopenmebger te GmbH Benzstr 4 D 75334 Straubenhardt Germany All rights reserved This product or document is protected by copyright and distributed under licenses restricting its use copying distribution and decompilation No part of this product or document may be reproduced in any form by any means without prior written authorization of ray test and its licensors if any RESTRICTED RIGHTS LEGEND Use duplication or disclosure by the government is subject to restrictions as set forth in subparagraph c 1 11 ofthe Rights in Technical Data and Computer Software clause at DFARS 252 227 7013 and FAR 52 227 19 The product described in this manual may be protected by one or more U S patents foreign patents or pending applications TRADEMARKS AIDA AIDA Image Analyzer AIDA Annotation AIDA AIDA Array Metrix AIDA Array Compare AIDA Multi Labeling AIDA ID Evaluation AIDA ID Densitometry AIDA 2D Densitometry AIDA Easy Array AIDA Whole Body Autoradiography AIDA ID Thin Layer Chromatography and AIDA 2D Thin Layer Chromatography are trademarks or registered trademarks of ray test in the United States and may be protected as trademark in other countries All other product service or company names mentioned herein are claimed as trademarks and trade names
21. Bkg button on the Integration pane of the Region Determination toolbox This opens the Local Bkg Settings dialog box Local Bkg Settings F Frame shape None Rectangle C Ellipse Frame width a cancer Figure 3 17 Local Bkg Settings Dialog Box 3 Enter the distance in this example in pixels around the assigned region 74 AIDA 2D Densitometry User 5 Manual Windows The resulting regions around the assigned areas will be used to determine the background signal and subtract it from the integration results Figure 3 18 Regions with Marked Local Background Areas To define a background for all other region shapes 1 Select more than one region and group them using the contextual menu of the Region Report window 2 Select one of the member of the region group and select the Background option on the Integration pane of Region Determination toolbox If a region group does not contain any background region the background regions of group 0 are applied for the region by default Performing Calibrations The signal intensity of a region can be compared with the signal intensity of other regions relative comparison or normalized by standards in the same image or even in other samples of the same series For the normalization calibration it is necessary to define reference regions for the creation of standard curves To assign standards 1 Select the Manual Assignment tool from the Region C
22. Region Calibration respectively from the View menu Image Window in 2D Densitometry All data are evaluated from the Image window Although it might not always be on top it is always open The title bar of the Image window shows the full file path of the image E f raytest Manuals Y 3 Final EE Im FE x Figure 2 1 Image Window Example You can copy the content of the Image window to the clipboard using the Copy Content command of the Edit menu See the Export Image section in the AIDA Image Analyzer Basic Concepts and Features User s Manual for further explanation Contextual Menu of the Image Window In 2D Densitometry the contextual menu of the Image window looks slightly different depending on the type of overlay you have chosen Single Selection If you have selected a single overlay region only the contextual menu that opens after right clicking the respective object provides you with the possible commands for this specific selection mode in addition to the standard editing functions like Cut Copy Paste Delete and Duplicate These include commands for labeling regions and setting integration limits Delete Duplicate Matrix Change name Change group name MameTable i Move label Set as background Set as standard Set integration limits Figure 2 2 Contextual Menu Single Object Selected Multiple Selection If you have selected multiple overlays regions the resulting cont
23. alibration toolbox 2 Click on a region Performing 2D Evaluations 15 The Region Calibration dialog box pops up and prompts you to enter the standard value und unit of measurement Region Calibration Region no 11 Standard value 3 125 Unit na Cancel Figure 3 19 Entering Standard Values and Unit of Measurement In the example shown below the regions R6 to R11 are a two fold dilution series of a standard Region R5 was assigned as a background region Region R1 to R4 are various dilutions of a sample that should be analyzed ees Os e On Os Os OM Ol Figure 3 20 Region Calibration Example 3 Enter the appropriate standard value und click OK to assign it to the region 76 AIDA 2D Densitometry User 5 Manual Windows In the Region Calibration toolbox the entered standard values are listed below the Standard drop down menu LA Region Calibration Method Concentration Standard user defined Calibration Curve Logarithmic Regression Iv 0 0 origin FSL hg Corr Coeff f 0 999887 Figure 3 21 Region Calibration Toolbox With Entered Standard Values Displayed The list of standard values can be saved for later use as a standard list Standard values from saved lists can be used to assign all standards by merely clicking at each region subsequently 4 Select a Calibration Curve type from the Calibration Curve drop down menu The type of the calibration
24. ated without the unwanted signal The intensity borders can be assigned via the Change Integration Limits dialog box lower limit upper limit A 70 197 a u Full Range Use colors for integrated area non integrated area Figure 2 37 Change Integration Limits Dialog Box 34 AIDA 2D Densitometry User s Manual Windows The intensity histogram is used to set the lower left and upper right intensity levels that will be used for the integration This is done by dragging the vertical black lines from the right and left end of the histogram to the desired position or by entering the appropriate values in the designated fields Lower Limit Upper Limit As a consequence the pixels that have a lower intensity or a higher intensity than the designated integration area peaks on the left or right of the margins shown in the example will not be used for integration The choice of coloring the Integrated Area or the Non integrated area to visualize the effect can be selected in the Use Colors for field Report Pane The Report pane of the Region Determination toolbox is used to activate and configure the report that summarizes the integration results Region Report Button Renumbering Tool Ungroup Button Config Table Header Button Group Button Figure 2 38 Report Pane Clicking the Region Report button activates the display of the report same as the Region Report function in the View menu With
25. ation unit of measurement depending on the argument Using 2D Densitometry 27 Table 2 3 Columns Available in the Region Calibration Window Columns Std Quantity quantity unit Short Description The quantity of the region as entered for the calculation of Recalc Quantity quantity unit Concentration Calibration Integral Area Bkg signal unit length unit Std Conc concentration unit Recalc Conc concentration unit Difference unit Rel Deviation Deviation unit Rel Error the standard curve With the Quantity Standards the background corrected Intensity values of the regions are calibrated A calculation of the quantity in the region based on the current calibration curve The background intensity 1s subtracted from the calculated intensity of the region and this signal intensity is divided by the area The concentration of the region as entered for the calculation of the standard curve With the Concentration Standards the background corrected Intensity per Area values of the regions are calibrated A calculation of the concentration in the region based on the current calibration curve The difference without sign between the entered standard and the recalculated value The relative difference with sign of the entered standard and the recalculated value compared to the standard value 7 Deviation Std Conc Std Quantity The difference with si
26. be exported to an AIDA Calibration file that can be imported into another image 58 AIDA 2D Densitometry User s Manual Windows Automatic Export of the Region Reports Region Calibrations AIDA s Automatic Export option allows you to export 2D Region Reports and or Region Calibrations automatically when you save an evaluation You can specify preferences for the range of the table columns to be automatically exported as well as export settings which are independent from the current work configurations of the table s The naming of the export files follows the scheme Evaluation Name Region Report txt or Evaluation Name Region Calibration txt 1 e if the name of the evaluation file is example adf the Region Report export file has the name example Region Report txt and the Region Calibration export file is named example Region Calibration txt Existing export files are overwritten without prompting the user However a backup file of the previous export file version is created Automatic Export always exports the entire result table Restricting the export to selected rows 1s not possible The enabling of the automatic export option as well as its settings are evaluation specific attributes which are valid until they are explicitly changed using the Preferences or Automatic Export options of the Options menu or by importing an AIDA template If you create a new evaluation the current default configuration for Automatic
27. curve can be selected on the basis of theoretical considerations or simply to fit the observed situation The resulting calibration curve can be immediately evaluated and the r value will give a parameter to judge the fit should be next to 1 5 Turn on the 0 0 Origin option 1f useful Performing 2D Evaluations T Linear Regression y 1 1 Ex 4 91 Correlation Coetioen f 0 9991 Iriercept on sens 9 74 Intanziy Bkg a u 10 0 an al s ED 7D Quantity ral Conc Figure 3 22 Calibration Graph Example As mentioned above a correlation coefficient value of 1 identifies a perfect correlation a value of 0 indicates full independence of values Good standard curves should have value very near to 1 78 AIDA 2D Densitometry User 5 Manual Windows Using Nametables Nametables are used to name regions for reference purpose Creating and Editing Work Nametables Work nametables are used to assign names to regions Any user can create a new or edit an existing work nametable To create a new work nametable 1 On the Region Determination toolbox click the Select Name List button Region Label Region Mame Move Label Font i Mone js 2 C Number IXETTHTTIHIET Name C Free Choice Configure ERN Figure 3 23 Region Determination Toolbox Select Name List Button Performing 2D Evaluations 79 The Region Nametables dialog box appears showing the current
28. d of the unique AIDA internal indentifier and an import identifier and name Image File Parameters for Fuji BAS Scanner Parameter file Image data file Original filename Scan date Imaging plate type Scanner Sensitivity Latitude IP Number Project Sample Exposure Comment Digital Resolution Image size Pixel size Scan operator Evaluation File Parameters Evaluation file Image parameter file path pcb or inf Image raw data file path img Name defined during the scan process Date and time of scan Type of the imaging plate used Scanner type e g Fuji BAS 5000 Sensitivity parameter of scan Latitude parameter of scan ID of IP plate used entered during the scan process Project name entered during the scan process Sample name entered during the scan process Exposure time of the plate entered during the scan process Image and evaluation comments combined Digital resolution of image Width and height of image Spatial resolution of image Operator of the scan process Evaluation file path adf Using 2D Densitometry 53 Table 2 4 Exported Parameter Information Parameter Desciption Evaluation date Date and time of evaluation Evaluation operator Operator of evaluation The actual image file parameters depend on the data file format and version E GRE GRE GRE GRE GRE GRE GRE GG Gb GRE HB GRE AE db db GR Aida Whole Body Autoradiography GLP 2D Region Report Export set
29. e result is a better optimization of the curve fit for the smaller values instead of a better fit for the higher values Linear Regression If you choose Linear Interpolation Linear Regression or Logorithmic Regression you can select the 0 0 Origin option to force the curve through the origin For Log Weighted Regression select the Linear fit option to force the curve to a linear function The correlation coefficient Corr Coeff of the estimation is shown below the curve A value of 1 identifies a perfect correlation a value of 0 indicates full independence of values Good standard curves should have value very near to 1 Double clicking on the graphics pane opens the Region Calibration window see the Region Calibration Window section of this manual for details Sometimes a warning will be displayed below the graphics pane stating bkg not determined to indicate that no background region has been assigned yet It can also indicate the lack of a local background for some regions Using 2D Densitometry 47 raytest Publishing Layout Toolbox in 2D Densitometry In AIDA 2D Densitometry the standard AIDA Publishing Layout toolbox is expanded by two additional buttons for specifying the print options for the Region Report and Region Calibration 3 Als 9 12 Publishing Layout Annotate LIMB Protocol Print re Print Preview E Page Layout Edges top 2 em EN bott
30. eminder M Region Report 2D Calibrati Ls Group Regions Ungroup Regions Group standards Hide standards Hide sum Show remainder Show total Show header Figure 2 12 Contextual Menu of a Single Region Report Row Using 2D Densitometry 17 If multiple rows are selected the contextual menu contains only the grouping commands applicable in this context Hi Region Report 2D Ca Mo Grp Mame Group Regions Decana Ungroup Regions Group standards Hide standards Figure 2 13 Contextual Menu of Region Report Rows Multiple Selection The contextual menus of the TYPE NAME STD and 9 o cells provide additional menu items for their respective context If a single row is selected the contextual menu of the Type cells contains the additional commands Set as Normal Set as Background as well as Set as Total for changing the region s type T Region Report 2D Calibratio Type Area Grp Name Integral Group Regions Ungroup Regions Group standards Hide standards Average Regions Set as normal Sebas background Set as total Hide surn Show remainder Show total Show header Figure 2 14 Contextual Menu of the Region Reports Type Cell Single Selection 18 AIDA 2D Densitometry User s Manual Windows AS SEE If multiple rows are selected the contextual menu of the Type cells contains the additional command Average Regions which inserts an additional row for the av
31. erage values of regions The three above mentioned menu items for changing the region type are unavailable in this context and therefore displayed in gray ni Region Report 2D Calibratio Grp Name Type Area Integral Group Regions Ungroup Regions Group standards Hide standards Average Regions Set as normal Sebas background Set as total Figure 2 15 Contextual Menu of the Region Reports Type Cell Multiple Selection The contextual menu of the Name cells contains the additional command Change Name for renaming the selected region Hi Region Report 2D Calibration adf Type Area Group Regions Ungroup Regions Group standards Hide standards Change name Hide sum Show remainder Show total Show header Figure 2 16 Contextual Menu of the Region Reports Name Cell Using 2D Densitometry 19 The additional menu item Set Standard of the Std row cells opens the Region Calibration dialog box allowing you to change the standard value and unit for the selected region P Region Report 2D Calibration ad Area Std Conc Grp Mame Integral dprncrr Group Regions Ungroup Regions Group standards Hide standards Set standard Hide sum Show remainder Show total Show header Region no 1 Unit Figure 2 18 Region Calibration Dialog Box 20 AIDA 2D Densitometry User s Manual Windows The contextual menu of cells containing percentages provides an
32. escription No The number of the region Grp The group number to which the region belongs 0 no group selected for this region Name The name of the region as it was assigned by the user Grp Name The name of the group assigned by the user to which the region belongs Type The type of the region e g bkg background Int Limits Integration limits for the region Position length unit The position of the region as determined by x and y coordinates in the image signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set in the Image Attributes dialog box concentration unit and quantity unit unit of measurement specified by the user through calibration Using 2D Densitometry 11 y Be see 9 A LISS x Table 2 1 Columns Available in the Region Report Window Column Position X ength unit Position Y ength unit Area length unit Area Integral signal unit Integral 9 6 Integral Bkg signal unit Integral Bkg 96 Integral Area signal unit length unit Integral Area 96 Integral Area Bkg signal unit length unit Integral Area Bkg 9o Short Description The position of the region as determined by x coordinate in the image The position of the region as determined by y coordinate in the image The calculated area of the region The percentage of t
33. etry User s Manual Windows The regions can be renumbered if necessary by activating the Renumber function This function opens the Renumber Regions dialog box The numbers are displayed subsequently in a combo box and can be assigned to the desired regions by simply clicking on them Renumber Regions Mext region number to be assigned E Click on a region to assign the number Choose Done to end renumbering Figure 2 46 Renumber Regions Dialog Box The Region Name tab controls are used to assign names to the various regions To assign names to regions 1 Select a region on the image 2 Click the Region Name tab Region Label Region Name Apply at Region Creation Figure 2 47 Region Name Tab Common names can be entered via a text box into a list that can facilitate the designation of redundant names Negative Control Positive Control Standard Background etc 3 Type a name in the text box or select one from the list below the text box 4 Click Assign Using 2D Densitometry 39 5 Optional Click the Apply at Region Creation option to assign the names in the list to any newly created region one after another in the order of their appearance in the list Figure 2 48 Selecting Apply at Region Creation Checkbox To edit the list of names e Click the Select Name List button below the Region Name tab Stomach M Apply at Region Creation Figure
34. extual menu is considerably reduced compared with the single selection contextual menu showing only those commands available in this selection mode The provided menu items form a subset of the functions available in the single selection mode Delete Duplicate Matrix Change name Change group name Set as background Set as standard Figure 2 3 Contextual Menu Multiple Objects Selected 8 AIDA 2D Densitometry User 5 Manual Windows If no region is selected only the basic editing functions of the standard contextual menu described in the AIDA Image Analyzer Basic Concepts and Features User s Manual will be accessible Note All of the functions in these contextual menus are also accessible through different means menus buttons toolboxes windows They will be explained in the respective sections of this manual However it is often more convenient to activate the function through the readily accessible background menu This will make repetitive and routine tasks faster and more convenient Image Overlays You can specify the label type and font you want to apply to the overlays lanes and bands using the Image Overlays command of the Options menu Options View Window Help Image Attributes Image Overlays Preferences Configuration Path Image Search Path Autosa ing Overlay Drag Mode Overlay Colors Create User Registration Figure 2
35. ey da 31 TALES ao Ranes oo RE e 33 REPO Paca a AS A de ved defen 35 Region Label and Name Pane 0 eee nee 36 Actual R gion Pane co as dardos 42 Region Calibration Toolbox 6 206 402 5048 4508 5840085 a GO 43 Method Pale vu cats uode Wish Seite ee wht M ess reo aaa boat oi nh hom a acdc tan Sy a 44 Standard Pate sacs dmt dale acia o acd aie Nae Daa M 3 Bone dow dite ace 44 Calibration Curve Pane 29 o Le dao Soke ave cores deti deve Soap ate det anata eed 46 Publishing Layout Toolbox in 2D Densitometry 0 0 0 0 cee ees 48 Print Options for Region Report 0 00 00 cc eee eens 49 Print Options for Region Calibration 0 0 0 0 0 eee eee 49 PEC AAA aol Ws OS SE OS Ree ae ore ORB eee ore ee 50 loi tn aa eine aces aa eee nae STANDS 51 111 iv Exportand Importa vu od pes ecrire p eed ad Aa 53 Exportins Templates coeur Ro RUSO RE seres 53 Exportinp Region REDOR osos hed eee eee ES ddr 54 Exporting Region Calibration llle 56 Automatic Export of the Region Reports Region Calibrations 59 Importing Region Calibration se ERR SR A AR REPOS RES 63 Performing 2D Evaluations 65 ASSIONING RESIGNS 5x3 xe xou ADO S dad adu bre A P RM RES 65 Using Atrays Matrices os dence Po eL ROS pr A te e RD Hed 7 Defining a Background Signal o ooooooooooonno eens 74 Performing Cab LOOSE ee es 75 Usina NaMe DIES e me ag ed oe Bm 79 Creating and Editing Work Nametables
36. gn between the entered standard and the recalculated value Recalc Conc Recalc Quantity Std Conc Std Quantity The relative difference without sign of the entered standard and the recalculated value compared to the standard value Difference Std Conc Std Quantity signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set in the Image Attributes dialog box concentration unit and quantity unit unit of measurement specified by the user through calibration unit quantity or concentration unit of measurement depending on the argument 28 AIDA 2D Densitometry User s Manual Windows Additional Toolboxes in 2D Densitometry In addition to the standard AIDA toolboxes Display Control and Publishing Layout the following toolboxes become available after selecting the 2D Densitometry mode Region Determination Region Calibration You can access these additional toolboxes from the Evaluation menu or by clicking the appropriate button on the toolbar Sci Options View Display Control Publishing Layout v Region Determination Region Calibration Figure 2 31 Mapping between Evaluation Menu Entries and Toolbar Buttons Region Determination Toolbox The controls of the Region Determination toolbox allow you to define regions in the image for signal intensity evaluation To display the Lane amp Peak De
37. he region area compared to the reference row in the group of the region e g the sum of all evaluated areas in a group The calculated intensity of the region The percentage of the intensity of the region compared to the reference row in the group of the region The background intensity 1s subtracted from the calculated intensity of the region to result in the signal intensity of the region For each group of regions the background is determined in the following order the local background area around the region or the local background defined for the group or the general background defined in group 0 whatever is available The percentage of the background corrected intensity of the region compared to the reference row in the group of the region The intensity per area The percentage of the intensity per pixel of the region compared to the reference row in the group of the region The background intensity is subtracted from the calculated intensity of the region and this signal intensity is divided by the area The percentage of the background corrected intensity per pixel of the region compared to the reference row in the group of the region signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set in the Image Attributes dialog box concentration unit and quantity unit unit of measurement specified by the user through calibration
38. ing dialog box appears Cancel 20030114 154529 4399 Delete rat 2003 01 14 Figure 2 76 Select Automatic Eport Setting Dialog Box 2 Select a settings set and click Select to complete the action 62 AIDA 2D Densitometry User s Manual Windows To delete a settings set 1 Select the set you want to delete in the list of the Select Automatic Export Setting dialog box 2 Click Delete Importing Region Calibration 1 To import the calibration curve from another image e g taken at the same time with the same BAS screen or from a second gel handled at the same time and captured under identical conditions use the Import command of the File menu The Region Calibration Import dialog box appears allowing you to specify the calibration file cal Region Calibration Import 2D Calibration adf Look in E 20 5 ample amp t 20 Calibration cal File name 2D Calibration cal Files of type ADA Calibration Cancel Figure 2 77 Region Calibration Import Dialog Box Using 2D Densitometry 63 64 AIDA 2D Densitometry User s Manual Windows Performing 2D Evaluations 3 Assigning Regions To define regions for signal intensity evaluation it is important to set the Image Attributes in the Options menu first see the AIDA Image Analyzer Basic Concepts and Features User s Manual If these settings are not correct further image evaluation will not result in meaningfu
39. ion signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set in the Image Attributes dialog box concentration unit and quantity unit unit of measurement specified by the user through calibration Using 2D Densitometry 13 The following additional rows can be found or created exclusively in the Region Report Table 2 2 Additional Rows in the Region Report Sum Displays the scientifically correct sum for the selected column e g Intensity Area means 2 Intensities X Areas Different groups get individual sums Ttl Total Displays the values of the selected column of Ttl region in the group For group 0 the Ttl region is the entire image Remainder Ttl Sum Quantity results in italics indicate that they lay outside of the calibration curve values and therefore are prone to significant error You can select the number and type of columns you want to display using the contextual menu of any column in the window see below To change the column order 1 Click the heading of the column to be moved 2 Press the mouse button and drag to the desired position in the table 3 Release the mouse button EN Region Report ZD Calibrat Integral Bkg Sete EAE dn IPSL mmr EN Region Report ZD Calibrat Integral Bkg HEER TE de PSL em 604 34 Integral Area Bkg Integral Bkg PSL mr 5 Figure 2 7 Changing Colu
40. ion 2D Calibration adf Log weighted Regression log v 0 36313 leg 3 06108 Correlation Coefficient f 0 999865 Integral a4rea Bkg PSL mm 10000 100000 1000000 Conc dpm cnr Figure 2 27 Contextual menu of the Region Calibration Window Graphics Pane The Scales command allows you to configure the graphical output After clicking the Scales command the Data Scaling dialog box opens Here the Minimum and Maximum value of the x Concentration and y axis Intensity can be selected In addition you can Using 2D Densitometry 25 turn on logarithmic representation and automatic scaling of either axis separately by clicking the appropriate option Logarithmic and Automatic below the Minimum and Maximum spin boxes Data Scaling he X Conc dpmicm E Minimum Maximum 3 555 1 277 700 i Logarithmic W Automatic _Cancel Integrali amp rea Bkg PeL mrr Minimurn Maximum 237 1665 ES 27 2 M Logarithmic v Automatic Figure 2 26 Data Scaling Dialog Box Region Calibration Window The Grid command opens the Grid dialog box which allows you to lay grids over both axis that are an extension of either the big or the small ticks on the axes Grid X Vertical Mone C Big Tics C Small Tics Harizantal Mone C Big Tics C Small Tics Cancel Help Figure 2 29 Grid Dialog Box 26 AIDA 2D Densitometry User s Manual Windows
41. ions can also be grouped together By Lines or By Columns if they represent multiple independent analysis points for example Integration Pane The Integration pane of the Region Determination toolbox is used to define the background calculation for the integration of regions Any region that is marked with the Background option turned on is taken as an example for the background signal Multiple background areas can be assigned and will be averaged Integration Background n Local Ekg Integration limits Figure 2 35 Integration Pane To assign a frame around the active region as a local background select the checkbox next to the Local Bkg button The general background calculated by the Background function is ignored for regions that have a local background assigned to them This function is not advised for blurred spots of usual 2D gel applications because part of the Using 2D Densitometry 33 actual sample might be mistaken for local background Clicking the Local Bkg button opens the Local Bkg Settings dialog box where a shape and size in pixels can be selected for the local background frame Local Ekg Settings Frame shape C None C Rectangle Frame width e m Figure 2 36 Local Bkg Settings Dialog Box The Integration Limits option of the Integration pane allows you to integrate only those pixels that lie within a defined intensity range Thus a region that has some noise dirt on it can be integr
42. l results Image Attributes L RTEST PCB Image Interpretation Image specific Fixed interpretation defined by image format Cancel Length Unit Figure 3 1 Setting Image Attributes To enable signal intensity evaluation the regions of interest must be marked by an enclosure of the appropriate shape In the beginning you might find 1t difficult to exactly encircle a region of interest Some experience is needed to get this done on the first attempt 65 To select regions of interest 1 Click the appropriate button here circle in the Region Creation pane of the Region Determination toolbox to select a shape alternatively click the circle symbol on the toolbar 22 Region Determination Region creation FEE Create matrix Greate mati PAE E k Figure 3 2 Selecting Circle Tool 2 Position the mouse pointer over the top left edge of the region in the image press the mouse button and drag the mouse to the lower right edge of the region to be assigned Figure 3 3 Marking Region 3 Release the mouse button 66 AIDA 2D Densitometry User 5 Manual Windows Figure 3 4 Adjusting Region Shape 5 Click in the middle of the region to adjust the position of the entire region Figure 3 5 Adjusting Region Overlay Position To rotate a region overlay 1 Click the left mouse button to switch to rotation mode Figure 3 6 Activating Rotation Mode
43. le listing the standards their given and recalculated standard valuesetc Additional columns can be displayed Just right click on the table heading and choose the Select Columns command from the contextual menu that appears The Columns Selection dialog box opens allowing you to select the desired columns from the list 22 AIDA 2D Densitometry User 5 Manual Windows Graphics Pane of the Regions Calibration Window The curve shown in the upper part of the Region Calibration window in this example displays the background corrected intensity per area as a function of the quantity concentration of the standard For a more detailed view of the plot you can activate the zoom function To zoom one of the plot s axis 1 On the toolbar click the Select button Window Help AO Figure 2 21 Select Button Main Toolbar 2 Click and drag one of the axis with the left mouse button pressed Region Calibration 2D Calibration adf loj xi Region Calibration 2D Calibration adf Log weighted Regression log y 0 9691 9 log 3 06108 Log weighted Regression log y 0 9691 9 log 3 06108 Correlation Coefficient Y 1999865 Correlation Coefficient Y 0 999865 m E E E 7 a EL 10 E 10 a a s 5 n T pu n E E E 1 T 1 5 E E 3 10000 100000 1000000 10000 100000 1000000 Conc dpm cnr Conc dpm cnr Figure 2 22 Clicking and Dragging the X Axis Using 2D Densitometry 23
44. lumns using the Select Columns command see below To specify the decimal digits for the value output in a column choose Settings of from the contextual menu and enter the value for the decimal digits in the dialog box displayed Column Integral Bkg Lx Decimal digits OK Cancel Figure 2 10 Setting Output Format for Columns Use the One lined Header Two lined Header command to reduce or expand the column heading by one line the second line indicates the units of measurement To select deselect the columns that should be displayed in the Region Report 1 Choose Select Columns A dialog box appears displaying two lists Show Columns Hide Columns 2 Select the columns you want to show or hide and click the appropriate arrow symbol to transfer the items between the two lists 16 AIDA 2D Densitometry User s Manual Windows 3 To deselect a particular item just click it again Columns Selection XX Show Columns Position X mm Integral 9 Position Y mm Integrali amp rea PSL mrm Integrali amp rea 95 Intedqral Area Bkud 95 Integrali amp rea Bkg PSL mrn Recalc Conc 95 Recalc Conc Hpmicrm Std Conc Std Conc dprmi crr came Figure 2 11 Columns Selection Dialog Box In addition each cell of the Region Report has its own contextual menu attached providing commands for grouping of regions and standards as well as displaying hiding of the special row types Sum Total R
45. mbering options bylines FT alternatin C by columns Automatic region grouping C bylines none C by columns Figure 3 11 Matrix Options Dialog Box 4 Enter the number of lines 2 in this examples and columns 6 in this examples as well as the distances x and y direction between the center of the regions Performing 2D Evaluations 71 This will be easy for scanners just measure the original and enter the actual distance but difficult for camera images trial and error However the work is worthwhile because all regions will be drawn and assigned at the same time and the same array can be used for another image even if the size of the image is somewhat different from the original one II T Figure 3 12 Matrix Creation To save the matrix 1 Choose Template from the Export submenu of the File menu 2 Type a name for your matrix template in the text box of the dialog box that appears e g slotblot atf You can apply your saved template to a similar image from the same kind of application by selecting the Template command from the Import submenu and then choosing the appropriate template from the displayed file list in the dialog box that appears The matrix region will be displayed in one single frame which can be moved and reshaped as if it would be only one region a con Ca Figure 3 13 Matrix Creation Completed You can relocate the matrix template by dragging the frame in the appro
46. mn Order 14 AIDA 2D Densitometry User s Manual Windows To change the column width 1 Position the mouse pointer over the border of the column whose width you want to change 2 Click and drag the border in the appropriate direction 3 Release the mouse button Wi Region Report 2D C No Type P Std 1 T Region Report 2D E Mo Type 1 Std Hi Region Report 2D 0 No Name Type Area Figure 2 8 Changing Column Width Contextual Menu of the Region Report Window Each column in the Result Table has a contextual menu attached To open the contextual menu right click on the column heading gray area Bl Region Report ZD Calibratia Mo Grp Mame Type f wv Sort ascending by Ma Sort descending by Mo Select Ma Hide Ma One lined Header Select Columns Figure 2 9 Contextual Menu of the Region Report Window Using 2D Densitometry 15 With the first two commands of the contextual menu you can sort the table of the Region Report by any value column in ascending order A to Z or zero to 9 or descending order Z to A or 9 to zero Just choose the appropriate menu item Sort Ascending by Sort Descending by The Select Unselect commands of the contextual menu are used to select deselect a column to export The column heading of selected columns are highlighted To hide a column select the Hide command from its contextual menu You can recover hidden co
47. of this manual You can change the position of a region label using the Move Label button to increase visibility To move a label to a desired destination 1 Select the region with a mouse click EZ raytesb Manuals Y 3 Final Release Aida d ES x Figure 2 40 Selecting Region 36 AIDA 2D Densitometry User s Manual Windows 2 Click the Move Label button on the Region Label and Name pane of the Region Determination toolbox Region Label F Figure 2 41 Move Label Button The label of the previously selected region turns its color from magenta to red EZ f raytest Manuals Y 3 Final Release Aida a X Figure 2 42 Activated Label 3 Drag the label to its designated location ES f raytest Manuals Y 3 Final Release Aidan mi Ed Figure 2 43 Moving Label Using 2D Densitometry 37 4 Confirm the action by clicking the right mouse button or alternatively by pressing the ENTER key on your keyboard ES fi raytest Manuals 3 Final Release Aida P m E Figure 2 44 Label Repositioning Confirmed You can select the Font type and Size of the characters as well as their font style bold B italic I and roman typeface on white background using the Font button The Choose Font dialog box is displayed allowing you to specify the font options for the label Choose Font xj 10 0 I Pt 1 1 prin IB ZA Comcel Figure 2 45 Choosing Font Properties 38 AIDA 2D Densitom
48. om UNE cm Region Report Options Button Region Calibration Options Button right 2 0cm HH a Header file path M page number M current date Footnote MR author M last change time M software version Figure 2 61 Publishing Layout Toolbox Overview and Toolbar 48 AIDA 2D Densitometry User s Manual Windows Print Options for Region Report With the first one you can configure layout setting for the Region Report window in the Protocol Print pane Protocol Print FE Print Preview EA Region Report Formal Portrait C Landscape El Print 4 Table Figure 2 62 Print Options for the Region Report Window Here you can select the paper orientation Portrait or Landscape by clicking the appropriate radio button in the Format pane In the Print pane the scope for the print output can be specified To turn on printing of the Region Report table click the Table box Print Options for Region Calibration The second additional button of the Publishing Layout toolbox is used to configure the layout setting for the Region Calibration window Protocol Print FE Print Preview E Region Calibration Formal Portrait C Landscape Print M Graphic W Table Figure 2 63 Print Option for the Region Calibration Window Using 2D Densitometry 49 raytest You can select the paper orientation Portrait or Landscape by clicking the appropriate
49. on Polygon Tool This Tool allows you to draw an enclosure by clicking at multiple subsequent points surrounding the object and finalizing the encircling with a double click Freehand Drawing Tool Click at a start point and drawing the outside shape of the object while holding down the mouse button On release the object is closed automatically The areas can be changed in size by moving the handles around The corner handles are used for total size change the side handles are used for size changes in the direction of the handle Clicking in the middle of the object switches to the turning mode as outlined above A third click will switch to the re shape modus where all edges of the object are shown with an individual handle Using 2D Densitometry 31 raytest The AutoContour Tool connects pixels of similar intensity to encircle an object automatically 1 After activating the function click on the border area of a region within the image that you would like to encircle and drag the mouse pointer slowly to the side of the object 2 Try to drag the cursor around with the mouse button pressed until the area is completely encircled 3 Release the mouse button 4 Remember to press ENTER or the right mouse button after the area has been correctly assigned To place multiple identical objects on top of the image use the Create Matrix button to open the Matrix Options dialog box This function is useful for the anal
50. owing option Export on Save Automatic enables the automatic export and generates the export filename automatically from the evaluation filename No disables the automatic export If you select Prompt for you will be prompted for an export data filename when you save an evaluation Parameter enables disables the export of the result table s parameter pane Table Header enables disables the export of the table header List Delimiter allows you to select a delimiter for the columns Decimal Delimiter allows you to select a decimal delimiter format Settings for Report Columns select columns subsequently and click to select or deselect the Included in Export option to include exclude the respective column Depending on the column you selected you can specify additional settings in this example the Decimal Digits for the exported values 3 Select the appropriate options and click OK to complete the action Using 2D Densitometry 61 Saving Automatic Export Settings 1 On the Automatic Export Setting dialog box click Save Setting The Save Automatic Export Setting dialog box opens Save Automatic Export Setting Rat pcb Delete Figure 2 75 Save Automatic Export Setting Dialog Box 2 Type a unique name and an ID for the settings and click Save to confirm the action Selecting Automatic Export Settings 1 On the Automatic Export Setting dialog box click Select Setting The Select Automatic Export Sett
51. ows you to subsequently assign all values in the list by clicking with the mouse on the appropriate region Thus the first value 1107000 0 is assigned to the first clicked region the second value 424000 0 to the secondly clicked region and so on 44 AIDA 2D Densitometry User 5 Manual Windows oo AX lt Z y MAN E Manual Assignment Tool The second button allows you to assign any value to the clicked region by entering the appropriate value and unit for the selected region in the Region Calibration dialog box that appears Region Calibration Region no 4 Standard value 41500 Linit Figure 2 57 Region Calibration Dialog Box Delete Standard Assignment Button By clicking this button all calibration values of selected regions can be canceled Edit Standard Button The last of the four buttons is used to enter new lists of standard values Clicking this button opens the Quantity Standards or Concentration Standards dialog box which is used to enter a new list or to modify an existing one To create a new list name the list in the Standard text box and specify 1ts Values Unit and Precision displayed fraction digits Quantity Standards Standard Unit ng Precision co E Default value 50 Add Modify Delete ane Figure 2 58 Quantity Standards Dialog Box The Add Modify and Delete buttons are used to alter lists in the same box Using 2D Densitometry 45 raytest
52. priate direction Sl C58 Figure 3 14 Relocating Matrix Template Jil Ezgi 12 AIDA 2D Densitometry User 5 Manual Windows To rotate the template just double click it and drag one of the handles that appear sd m dd HAL i L Figure 3 15 Rotating Matrix Template Finally accomplish your adjustments by right clicking the matrix or alternatively pressing ENTER PP op Tr WE d o L Figure 3 16 Matrix Template Adjustments Completed dM Performing 2D Evaluations 73 Defining a Background Signal It is advisable to determine the background signal before quantifying the signals Since in many applications the background can vary considerably from location to location the possibility to assign an individual local background around a region can help to evaluate the signals Note that local backgrounds can be defined for rectangular and ellipsoidal regions only It should also be noted that the background around the region must be clearly separated from the signal If e g a 2D gel spot is assigned it is likely that the outer borders of the signal come to lie under the background area because they are very difficult to see for the human eye To define a local background region rectangular and ellipsoidal regions only 1 Mark the respective image areas using the Region Creation tools of the Region Determination toolbox 2 To aktivate the local background function click the Local
53. rint Preview command of the File menu opens the Preview dialog box which allows you to specify the layout settings for the preview The options presented here are the same as in the Print dialog box and those selectable from the various panes of the Publishing Layout toolbox However they are not stored with the data file Using 2D Densitometry 51 The Preview option of the Publishing Layout toolbox allows you to preview all pages as configured in the Publishing Layout toolbox before they are actually printed Clicking the Preview button opens the Preview window directly because all layout setting are already made through the Print Protocol pane E Aida Whole Body Autoradiography GLP Preview Rat adf File Edit Evaluation Options View Window Help f Close Page 1 of 2 Page 2 of 2 Whole Body Aulradiography GLP Monday February 12 2001 10 50 10 PM Whole Body uloradlography GLP Monday February 12 2001 10 50 10 PM es Program Fllez raylez hAlda Image Analyzer dev 3 10 O28 Example z Ralaitimodited Page 1072 es Program Files raylez h lda Image Analyzer dev 3 10 O28 Example z RaladYimodited Page 2012 Parame kr Ali Trall Comiguraton Awil Trall 20 Region Reporl Alvor Sys lem Adminis tater Auha Sys lem Adminis rater Las Charged Monday Februar 12 2001 10 43 40 PM Lar Changed Monday February 12 2001 10 43 40 PM Alda Development Version 3 10 028 Alda Developmeni Version 3 10 023
54. ry User s Manual Windows The margins of the printout can be selected in the Edges part of the dialog box The Header button displays the following dialog box Page Header and Footer x OK Iv file patti V author IV page number v lastchange time Cancel IV current date v software version Figure 2 65 Page Header and Footer Dialog Box Here the Header and Footer of the report can be configured to contain various image and evaluation specific data The Parameter data overlays the Image Region Report and Region Calibration information can be selected in the left part of the Print dialog box In the right part of the dialog box Parameter and Image Options you can select Whole Image or Window Content from the first drop down menu Landscape or Portrait format can be selected from the second drop down menu The printing of Overlays Grayscales Scales and Overexposed can be selected The printout can be automatically fitted to the page or the relative size of the image representation can be selected in In the Profile Options pane you can specify to print the Current Selection or all profiles as well as the paper orientation Landscape or Portrait for the printout Preview As for printing in AIDA there are two ways of previewing pages before they are actually printed Using the Print Preview command from the File menu or Using the Preview button on the Publishing Layout toolbox The P
55. termination toolbox On the main menu click Evaluation and choose the Region Determination entry Or Click the Region Determination button on the toolbar Using 2D Densitometry 29 eue ojala create Matix Bar round rer WEG Region Creation Pane Integration Pane Report Pane Region Label and Name Pane Actual Region Pane Figure 2 32 Region Determination Toolbox Overview 30 AIDA 2D Densitometry User s Manual Windows Region Creation Pane The Region Creation pane of the toolbox is used to mark various areas in the image Rectangle Tool Square Tool Region Creation ojoje Ellipse Tool Circle Tool Freehand Drawing Tool Create Matrix Polygon Tool AutoContour Tool Figure 2 33 Region Creation Pane Overview Areas of different shapes rectangles squares ellipses and circles can be marked To create a region 1 Click on the symbol of the desired shape 2 Click at the upper left corner of the area hold down the mouse button and drag it to the desired lower right end of the shape 3 Release the mouse button The area can be reshaped by moving the handles m Clicking within the area changes the handles to this symbol now the object can be rotated around its center Clicking within the area again changes the handles back When the overlay has received its final shape and tilt right click or press the ENTER key to save the object and go on with the next acti
56. that all comparisons of samples to standards should be validated Evaluations get more accurate with more standard values which should be well scattered around the intensities to be examined When in doubt 1t might be a good idea to check the reproducibility of known results Including and evaluating multiple dilutions of samples will also make quantitative estimations safer Starting 2D Densitometry To select the 2D Densitometry mode 1 Click the Evaluation button in the toolbar A dialog box appears offering you a list of available evaluation modes Change Evaluation Mode Available Evaluation Modes 1D Evaluation 10 Thin Layer Chromatography 2D Thin Layer Chromatography ls Annotation Array Matrix Crystallography Material Science Multi Channel Display Whale Body Autoradiography Figure 1 1 Changing Evaluation Mode The list entries reflect the installed AIDA application modules 2 Select the 2D Densitometry list item und click OK Note This evaluation mode is only available if you purchased the AIDA 2D Densitometry module 6 AIDA 2D Densitometry User s Manual Windows Using 2D Densitometry 2 Data Windows in 2D Densitometry In addition to the AIDA standard data windows Image and Histogram the following data windows are available in the 2D Densitometry mode Region Report Region Calibration You can access these data windows by choosing the appropriate entry 2D Region Report
57. ting 20021104 110236 3896 Till Default Setting 04 11 2002 Master nametable 20021104 142426 7666 Master Name Table 2002 08 16 35 Parameter file i Aida Demo Rat inf Image data file i Aida Demo Rat img Original file rat Scan date Tuesday April 20 1993 4 02 43 AM Imaging plate type 20 40 Scanner Fuji BAS 1000 Sensitivity 10000 Latitude 4 Digital resolution 256 8 bit per pixel Image size 348 940 69 60mm 188 00mm Pixel size 200um 200um Evaluation file i Aida Demo Rat adf Evaluation date Monday November 04 2002 3 22 42 PM Evaluation operator System Administrator Administrator Figure 2 70 Example of Exported Parameter Information 4 Complete the action by clicking Ok The data are saved to a simple ASCII text file Exporting Region Calibration To export Region Calibration Data 56 1 On the File menu click the Export menu item and then choose Region Calibration from the submenu AIDA 2D Densitometry User s Manual Windows oo Z y E 2 In the Region Calibration Export dialog box that appears type a name for the export file and specify the data format Region Calibration Export 2D Calibration adf Save in Cx 2D Sample e amp er E3 2D Calibration cal File name 2D Calibratiori2 cal Save as type AIDA Calibration L ancel Windows Metafile Table ASCII File AIDA Calibration Figure 2 71 Region Calibration Export Dialog Box To export the
58. work nametable s list of names and a dropdown list with the names of all existing work nametables Region Nametables Rat pcb a oo Modify Remove List af Mames Region Nametables Rat pcb Remove Cancel Figure 3 24 Region Nametables Dialog Box 2 Click New The Nametable Editor dialog box appears 80 AIDA 2D Densitometry User 5 Manual Windows 3 Type a name for the work nametable in the Title text box and a list of names in the List of Names text box Mametable Editor xi Title work nametable List of Names Cancel Type in your list of names in the edit box an the right each name on a line Type in a title for this list in the edit box above Figure 3 25 Nametable Editor Dialog Box 4 Click Accept to confirm the changes To modify a work nametable On the Region Determination toolbox click the Select Name List button The Region Nametables dialog box appears 2 Click Modify The Nametable Editor dialog box appears 3 Edit the entries in the in the List of Names list box 4 Click Accept to confirm the changes Performing 2D Evaluations 81 Assigning Names to Regions To assign names to regions 1 Select a region a A ms Figure 3 26 Selecting a Region 2 On the Region Determination toolbox select the Region Name tab Region Label Region Name M Apply at Region Creation Figure 3 27 Selecting Region Name Tab 82 AIDA 2D
59. ysis of multi well plates or dot blots were signals of identical shape and distance have to be analyzed OR OS Qf of os ox o 03 04 C96 O4 Oa Ogis Ora Ciis Ces Cuz Oto Oria Oro Ora C ait options M xl Cis Ope e Oa Opes Okso Ops Ordo Okas C Dimensions Ope Ces Chio Opto Opa Og Okas Okaa Okas C Lines os DO 0009 columns Cancel Oksi OM Orea Oros Orea Cher Crea Crea C OH O OW OM OM og Of p Center distance OM ON ON Obs Cus Cus Caf OM Oa C vertical B pixel horizontal pixel Numbering options hy lines FT alternatin C by columns d Automatic region grouping C by lines none by columns Figure 2 34 Matrix Options Dialog Box With Application Example Dimension In the Dimension pane of the Matrix Options dialog box you can enter the number of objects to be created in x Lines and y Column direction of the matrix Center Distance In the Center Distance part of the dialog box enter the Vertical and Horizontal distance mid to mid between objects in the matrix 32 AIDA 2D Densitometry User 5 Manual Windows Numbering Options The regions in the matrix can be numbered By Lines from left to right or By Column from top to bottom Clicking the Alternating option results in numbering the first line from left to right the second line from right to left and so on The Alternating numbering is also available for columns Automatic Region Grouping The resulting reg
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