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Odyssey 3.0 Manual

1. Parameters The Command Line should specify the program to start when the plug in is launched The program and path can be specified by clicking Browse and using the file selection window to find the program Any program that accepts a data file parameter when launched from a command line can be used in the plug in The program must be able to accept data separated by tabs spaces etc The Type list is used to specify what the plug in does and how it interacts with the Command Line and Parameters Each plug in type is described below 200 CHAPTER 10 Reports and Data Export Grid exports a matrix of values for each field specified in the Grid Fields Plug in Template edit by choosing Report gt Grid Fields in Plug in Template Data are arranged in a matrix that matches the features in the grid For example if integrated intensity is one of the exported fields for a grid that matches a 96 well microplate integrated intensity values for each feature in the grid are arranged in an 8 x 12 matrix followed by another 8 x 12 matrix for some other specified field Data are only exported for fields that are selected in the Grid Fields Plug in Type Template window Report is used for plug in reports When Report is selected the Template list is activated so a report template can be selected Only data for the fields specified in the report template are exported When a report template is selected the Parameters a
2. Scan Size 5x5cm 5x 10cm 88k 350k 1 4M 5 7M 5x15cm 132k 525k 2 1M 8 5M 5x 20cm 176k 700k 2 8M 11 3M 5x25cm 220k 875k 3 5M 14 2M 10x10cm 10x 15cm 264k 1 0M 4 1M 10x 20cm 352k 1 4M 5 6M 10x25 cm 440k 1 7M 7 0M 15x15cm 396k 1 6M 6 3M 15 x 20 cm 528k 2 1M 8 4M 15x25 cm 20 x 20 cm 704k 2 8M 20x25cm 800k 3 5M 25x25cm 1 1M 4 4M _JFile size is small enough to scan with Odyssey Software Marginal for Odyssey Software HN scan should be started in using the browser interface Odyssey Software also has limitations on the size of images that can be analyzed The total size of all open images should not exceed 20 25 MB One analysis with two 10MB images will use up most of the memory resources However if your typical image size is 2 MB five separate analyses can be opened Large scans can be cropped into smaller pieces using the browser software if necessary For band sizing applications the resolution setting can be checked by looking at the lane profiles Chapter 5 If the lane profile shows many small jagged peaks on the larger peaks of bands as contrasted with smooth peaks this may indicate the resolution is too coarse These jagged peaks will influence the accuracy of band finding If the small peaks are caused by lack of resolution choosing a smaller resolution value should improve the problem Quality controls scan speed and ultimately how many detector readings
3. Image Size 700 Kbytes per channel Scanner Front Default Values Specity the scan area by pressing the left mouse button and dragging the cursor estimated scan time 6 minutes 49 seconds CHAPTER 2 Starting Scans Naming a Scan and Entering a Description The Name field is not editable at the beginning of a scan The default scan name is filled in automatically according to the naming conven tions in the Application Settings The default name may be blank a sequential name or a time stamp shown below At the end of the scan the default name can be accepted or replaced with a different name before the file is stored on the computer Scanner Console BandSizing User ron Scan Description Name Group ron v Modify Modify The name in the Name field is also the scan name that will be stored on the hard drive of the Odyssey instrument If blank is the current naming convention a time stamp will be used for the scan name on the Odyssey instrument If the default name is replaced with a new scan name at the end of the scan the scan name on the computer will be different than the original scan name on the hard drive in the Odyssey instrument The original scan name can be viewed by choosing Edit gt Scan Description to view the scan description The original name is also listed in the tool tip that is displayed when the cursor is stopped over a scan name in the Scans view of the main Ody
4. Plat Multi S6VVellPlate Edit Delete Close A grid template can be created using one of the following methods Copy an existing template Select a template in the Grid Templates window click Edit change the grid parameters listed below and click Save As to name the edited template e Click New in the Grid Templates window change the grid param eters and click Save As to name the new template Grid templates can also be saved from the main Odyssey window as follows After placing a grid and adjusting it to match the image leave the grid selected and open the grid properties to save the grid param eters to a grid template Grid properties are opened by right clicking the image and choosing Properties or by clicking the properties button 4 on the left toolbar After opening the properties window click Create Template to open the Modify Grid Settings window with all the parameters filled in to match the grid selected in the Image View window Review the parameters click Save As and name the new template A special type of grid template can be saved by selecting a grid right clicking the image in the Image View window and selecting Save Grid As Template in the popup menu or choose Analyze gt Save Grid As Template This method can be used to visually adjust 125 the grid and save the grid settings after the grid is correctly positioned This method also stores additional grid parameters t
5. 231 System Administration sses 234 Account Ripeos ssesodissrania 236 Managing USES ici c ciietit sees teedeicies 235 Managing Scan Group5S cceeeeee 237 Scanner Information eee 238 Scanner DiagnostiCS oerrinne 238 Scanner Updat ssssecsscscssscnensis 240 Adding and Deleting Scanners 242 Adding Scanners via Auto Discovery 243 Manually Adding Scannets 00 243 Editing and Deleting Scanners 244 Chapter 13 Calculation Descriptions Derivation of the Mathematical EXPV SSIONS x 3ccsvcesecevecsdcesvecssecesceseesvecsdeess 245 Definition of Terms 245 ASSUIMPUGOIS T E sense 246 Integrated Intensity and Integrated Pixel VOUE sereine ae n A E 246 Odyssey Calculations 249 Number of Pixels Pixel Area and Shape a E E E E 249 Background sxsssosasaisvs sasatcieessbssvaentoaeseests 249 Raw Integrated Intensity ce ee 250 Integrated Intensity ssiri 250 Average Intensity ccccccseeeeeeeeeseens 251 Trimmed Mean ccceeeceeeeeereeeeeneeeees 251 Peak INtenisity visssssieescasvessecpsacacaestaaevees s 251 Minimum Intensity ccccccesecseeeeeees 251 Signal to Noise Ratio s s s 252 CONCENt ALON cece scale scesseeaceessiessccccbescses 252 Probability zenean enan 252 Molecular Weight nessieesesssssrissss 253 Percent Saturation 2 c sc csteesceesteesceeseees 254 Percent Response for ICW Assays 254 Z Factor Calculations naiinis 256 Index x
6. 79 Displaying Band Background FIUOTESCENCE 2 ececeseeseceeeeeeeoneeteneees 79 Displaying Lane Background FIUOTESCENCE sseni 80 Displaying Lane Profiles With Background Fluorescence REMOVE ccccceereeeees 80 Controlling Band Finding Using the Lane Profile Window esssi icscsesesscesneegedeshvivseeats 81 Comparing Lane Profiles 0 cee 82 Normalizing Bands in Lanes 83 Normalization Procedure 83 Creating and Using Lane Templates 85 Saving a Lane Template cee 85 Placing Lanes Using a Template 86 Deleting a Lane Template 86 Using the Application Settings 0 0 86 Profile Wicthiircisncss euiar 87 Total Width ssciriisirensunoinnesa 88 Band Finding Threshold ee 88 Display Migration cece eee 88 Lane Colobinae 89 Displaying Band Quantification as a Percentage cireres 89 Image View Display Settings for Lanes 89 Chapter 6 Band Sizing Checking the Application Settings 91 Checking the Display Migration Settings 93 Band Sizing in Single Channel Mode 94 Switching Image Channels 00 95 Using Size Standard Sets eee 95 Creating Size Standard Sets 96 Editing Size Standard Sets eee 98 Deleting Size Standard Sets 98 Using Size Standard Sets eee 98 Applying Standards to the Image 100 Adding MW Lines One at a Time 100 Adding MW LINES sssrinin 1
7. Background 0 35 0 41 0 14 0 29 lo 85 Export Path C OdysseytOdyssey_Projects TutoriahTiterScamGrid AnalysisStats txt Browse The Count column indicates the total number of features selected for a given channel For the statistics shown above 20 features were selected in both the 700 channel and 800 channel channels were overlaid when 20 features were selected 206 CHAPTER 10 Reports and Data Export To export the statistics in tab delimited text format click Browse to select the path and click Export The path can also be typed in the Export Path field Printing an Image View Images displayed in an Image View window can be printed by choosing File gt Print Image View To scale the image so the printed image fits the current page size click Scale to Fit Page in the Print Image View window To print the image at actual size click Actual Size To constrain the printed image so it prints at a certain height or width click Set Size for Image Width or Height select Width or Height and enter the size to which the image should be constrained Print Image View Print the visible portion of the current image view The aspect ratio of the image is always maintained Clipping of the image may occur if dimensions are too large y Print View Mode O Actual Size O Set Size For Image Width or Height Width Ld Print Scan Parameters with image
8. 58 Flip or Rotate to New Analysis 00 59 Crop to New Analysis eseeeceeeee 60 Crop to Multiple Images eee 61 Exporting SCANS sareren nsan 64 BaCkUp iaeiy eissiscccespivielscsisetesstesteatele nE 64 Chapter 5 Creating Lanes and Finding Bands Before YOU Bep iMrs onan 65 Creating or Opening an Analysis 65 Single Channel vs Overlaid Image Channels camanan n n 65 Creating the First Lane eee 66 Finding Straight Lanes 0 cece 66 Finding Curved Lanes ceeeeeee 67 Moving and Resizing Lanes 68 MOVING Lanese ninisi 68 Linked AMOS isisao 68 Changing Lane Width siesessssonus 69 Changing Lane Height 0 cee 69 Changing Lane Shape ceeeee 69 Copying and Pasting Lanes eee 70 Copying Multiple Lanes wo eee 71 Using the Paste Special Command 71 Deleting Lanes ssssssesscesessseeeeenes 71 Creating Multiple Lanes cece 72 Verifying Band Finding 0 eee 73 Too Many Bands ssesssresiniresrosineis 74 Not Enough Bands e cesses 74 Fine Tuning Band Finding 74 Verifying Band Markers Are Centered 74 Verifying Bands Are Fully Enclosed 75 Refinding Bands sserisrisisresrosinrosi 76 Using the Lane Profile Window 76 Understanding the Lane Profile 77 Displaying Band Centers eeeee 78 Displaying Band Boundaries
9. 117 Features are added along a straight or curved line that starts from the center of an existing feature and ends at a point where the user double clicks the mouse The first step is to add the first feature and select it Since this feature is one end point of the imaginary line connecting the image features it should be on the left right top or bottom side of the line After selecting the first feature click the multiple features tool H or choose Analyze gt Add Multiple Features Add Multiple Features Create equidistant features along a center line from the parameters and steps below m1 Verify and update the fields below Number of features Gis Equally space features Horizontally O Vertically Shape base name Spot Extension start number Create names ordering Leftto right Name example Spot_1 m2 Draw a center line on the image for new features The current selected feature is the starting point of the feature center line Click Continue and create a straight or curved center line as follows Straight line Double click the end point to generate features Curved line Single click at inflection points and double click the end point C Dont show this dialog at the start of Add Multiple Features action Goto Application Settings to enable this dialog again In the Add Multiple Features window start by specifying the number of features to add and whether the
10. Printer Setup Printer WLI COR_001 MARCOMM_HP8100 Orientation Portrait Page Setup Change Printer Preview Cancel 207 Scan parameters resolution etc can be printed with the image by selecting Print Scan Parameters with Image Click Page Setup to select portrait or landscape page orientation To change printers before printing click Change Printer The Preview button displays the image as it will appear on the printed page After making changes click Print to send the image and all annota tions to the printer The visible portion of the image is printed with all current features including annotations brightness and contrast changes zoom etc Any portion of the image that is scrolled outside of the window boundaries will not be printed For pseudo color images the pseudo color legend will be appended to the right side of the image Note If the Application Settings Image View Display have been used to enlarge the text display area in order to prevent annotations from being truncated at the edge of the image this extra text display area is included when an image view is printed assuming the extra text area is visible in the Image View window Viewing and Printing the Scanner Log Choosing Report gt Scanner Log displays the scanner log of the Odyssey Imager From this window the log can also be printed The scanner log is useful for diagnosing problems LI COR technical support may request a copy of the
11. ODYSSEY INFRARED IMAGING SYSTEM User Guide Version 3 0 losciences End User License Agreement For LI COR Odyssey Software IMPORTANT READ CAREFULLY This LI COR End User License Agreement EULA is a legal agreement between you either an individual or a single entity Licensee and LI COR Inc having a principal place of business in Nebraska Licensor or LI COR for the LI COR software identified above which includes computer software associated media printed materials and online or electronic documentation SOFTWARE PRODUCT By installing copying or otherwise using the SOFTWARE PRODUCT you agree to be bound by the terms of this EULA If you do not agree to the terms of this EULA do not install or use the SOFTWARE PRODUCT you may however return it to LI COR Inc for a full refund SOFTWARE PRODUCT LICENSE THIRD PARTY SOFTWARE The SOFTWARE PRODUCT contains third party software Third Party Software which require notices and or additional terms and conditions Such required Third Party Software notices and or terms and conditions are located in the Odyssey Software Help System and are made a part of and incorporated by reference into this EULA By accepting this EULA you are also accepting the additional terms and conditions set forth therein THE SOURCE CODE VERSIONS OF THIRD PARTY ORIGINAL CODE ARE AVAILABLE UNDER THE TERMS AND CONDITIONS OF EACH THIRD PARTY LICENSE ANY WARRANTY MADE AVAILABLE UND
12. Cell 417 7ANIR S ann nial 25417A5 00 Mi nT gt Report Fields Template Feature_Data v Launch Pugh The Report View menu choice is disabled until image features are selected and only data for selected features are displayed in the table The report template selected in the Template drop down list deter 184 CHAPTER 10 Reports and Data Export mines the fields displayed in the table the sort order and the default export path If the current template is not what is needed change the template by clicking Edit which is exactly the same as choosing Report gt Report Field Templates Click Print to print the data to a printer of your choice or click Export to save data in a tab delimited text file See Printing and Exporting Reports below for details The Launch Plug in button displays a list of available plug in reports After selecting a plug in and clicking Launch data are transferred from the Report View to the plug in and the plug in performs the task it was designed for Launch Plug In List Microsoft Excel for Lanes Consult Plug in Reports below for troubleshooting tips regarding plug ins Printing and Exporting Reports The Report gt Print and Report gt Export menu choices are functionally the same as opening the Report View and clicking the Print or Export button respectively Important Both the Print and Export menu choices on t
13. Edit Feature Properties ObjectType Rectangle D 578 Name 25 ng Standard Description l Rectangle Name or description annotations are not displayed unless they are enabled in the Application settings see Chapter 11 in order for them to be displayed 141 Renaming Multiple Features Multiple features can be simultaneously renamed by selecting the features and choosing Analyze gt Rename Selected Shapes Features are named with a prefix base name and a numbered extension see Name Example in the window below Names are added to features in the order specified left to right right to left top to bottom or bottom to top Rename Selected Shapes Rename parameters Shape base name Spot Extension start number Create names ordering Left to right Y Name example Spot_1 Ok cances Adding Text Annotations To add an annotation click the text tool T and then click once on the image where you want the upper left corner of the text to start Annotation Text Properties Annotation Text Gel 80972 C include Border Around Text r Annotation Font Color white Fort Dialog Font Size 14 v Fonts Example Ca Cone 142 CHAPTER 7 Drawing Features on Images Note If feature names or annotations are truncated at the edge of the image use the Application Settings to extend the te
14. New MW Value 170 Add MW Line Select MA Std Set Select a Std Set v Apply Std Set Edit Mode Add Points O Modify Lines O Modify Points Snap to Lane X Snaps Size Lines to Selected Lane Bands Apply to Both Cancel When lines are snapped to the bands control points are added and MW lines are drawn as described above for MW standard sets After the MW lines are snapped to the bands the lines must be applied to the image using the Apply to Both or Apply buttons see Applying Standards to the Image above Editing Molecular Weight Lines On images where fragments had differential migration rates during electrophoresis bands may smile frown or be slanted For band sizing to be accurate molecular weight lines must follow the contour of the gel whatever shape that might be The point at which the MW line crosses the centerline of the lane is assigned the same molecular weight as the line Therefore if the MW line crosses the centerline 105 too high or too low band sizing will be inaccurate Odyssey software has tools to move and reshape MW lines in the Edit size standards window the same window used to add the MW lines Moving Whole Lines Whole lines may need to be moved because all points on the line are too high or low To move a line set the Edit Mode in the Edit Size Standards window to Modify Lines as shown below Edit Size Stds for 800 TIF Add MWY Line or Select MVV Set New MW Valu
15. Sort Ascending O Sort Descending 2Z Factor normalizing to 700 channel 0 73 Neg Control StdDey 28 20 mean 439 02 Pos Control StdDev 5 14 mean 73 68 Neg Cotl Neg Cntl Neg Cntl Neg Catl Pos Cntl Pos Cntl Pos Cntl Pos Cntl Pos Cntl Recalculate Change Params If the ICW export settings Settings gt ICW Export are configured to include header information in reports the Z factor values will be included in reports generated by clicking Print or Export 3 Chapter 10 Reports and Data Export This chapter discusses the standard reporting features of Odyssey software ICW reports which are available in the optional In Cell Western Module are discussed in Chapter 9 Report Table View Analysis data for selected features grids etc can be viewed or exported in table format by choosing Report gt Report View i Report for Scan TiterScan Analysis Grid Analysis2 Name ILI Counts Shape Area Channel Concentr Raw Int Cell A1 9971 27 20 02 700 nja 347100 00 A Cell A2 15808 25 20 02 700 nja 50285 00 Cell A3 54199 11 20 02 700 nja 1886671 00 Cell 44 55266 79 20 02 na 1923837 00 Cell AS 55675 87 20 02 na 1938077 00 Cell 46 55053 35 20 02 na 1916407 00 Cell A 59187 42 20 02 nja 2060314 00 Cell 48 57359 96 20 02 nja 1996700 00 Cell 49 63498 94 20 02 na 2210398 00 Cell 410 58424 77 20 02 na 2033766 00 Cell A11 64512 07 20 02 na 2245665 00
16. if necessary to switch between channels until the channel with the molecular weight marker bands is displayed Using Size Standard Sets After lanes are defined and bands are edited the molecular weight MW of each MW standard band needs to be assigned Molecular weights are assigned by adding one MW line for each MW standard band MW lines when properly placed connect all the points of equal MW on the gel Thus each intersection between a MW line and the centerline of a lane represents the same MW For gels where the bands slant or smile the MW line must be reshaped to follow the contour of the gel as described later in this section Note Odyssey requires an equal number of MW standard bands in each standards lane MW sets must also have exactly the same number of standards as there are bands in the standards lanes MW weight lines can be added individually as described later in this chapter or in groups that are stored as molecular weight sets For repetitive scanning with the same molecular weight standards molecular weight sets save time and eliminate having to enter each molecular weight standard for every image Creating MW standard lines individually is more useful when a certain set of standards is going to be used for a only few gels 95 96 CHAPTER 6 Band Sizing Creating Size Standard Sets After lanes are created and bands are found MW sets can be created by choosing Settings gt Size Standard Sets Size
17. When plug ins are no longer needed they can be deleted from the list in the Plug in List window by selecting them and clicking Delete Troubleshooting Plug in Reports The most common problem is that the path to the analysis program receiving the data Excel etc is not correct If the program is not found when the plug in is launched an error message is displayed To change the path use the following procedure 1 Choose Plug in gt Manage Plug ins 2 Select the plug in to be edited from the list and click Edit 202 CHAPTER 10 Reports and Data Export 3 Make sure the path in the Command Line field is the correct path If not use the Browse button to find the program Programs generally have a EXE file name extension EXCEL EXE etc Edit Plug In Name Aicrosoft Excell For Lanes Command Line 2s Microsoft OfficeOFFICE11 EXCEL EXE Parameters Save Cancel 4 Click Save and run the plug in again Another common problem is having the wrong Field Separator character selected in the Application Settings An incorrect field separator can result in all data for a given row appearing in a single spreadsheet cell To change the field separator choose Settings gt Application and then choose Report from the Settings List Graphing Data Start by selecting the features to include in the graph If both image channels are displayed data for both channels will be included in the i
18. cee 210 Changing Image Display Style 212 Adjusting Image Curves oo 213 Using the Intensity Adjustment Curve 214 Using the Histogram cceeeeeeeees 217 Cropping Rotating and Flipping Images 219 Magnifying the Image cece 219 Zoom Functions on the Toolbar 219 Keyboard Shortcuts c ceeseeeeeeeeee 220 Over laid IMa BES onas 221 Aligning Mages ssassn 221 Changing to Grayscale Image Display IA EE E A E 223 Changing to Color Image Display Style 223 Changing to Pseudo Color Image Display StYlOsssacssncevssevonsencesestsnessns tos casnsscdeevneces tes 223 Switching Between Image Channels 223 Displaying a Second Image View WINKOW wp saccosencarnettnsacssdeseusseunneatesatssaenets 224 Hiding Image Annotations s s s 224 Using the Application Settings to Display Labels issecssisncscestcsoteveonerthsaepstevensdedeberaweees 225 Changing Font Specifications 226 Displaying Data in Tool Tips 227 Using the Image View Display Settings 227 Setting the Default Sensitivity for New MAPES uspe u E EEEE iS 227 Changing Image Colors From Red Green sssssesseseseserrrrsrserererersseseeee 228 Extending the Text Display Area Around IMAGES arpitana iaa 228 Chapter 12 User Accounts and Settings Application Settings 0 0 c cee 229 User Administration cece 230 Changing Your Own Password 230 Managing Your Own Scan Groups
19. multiplied by the area per pixel Area per pixel a resolution x 10 Field Definition Sat Number of saturated pixels divided by total number of pixels multiplied by 100 to give a percentage Project Name of project containing the lane Scan Name of scan containing the lane Analysis Name of analysis containing the lane Total 1 1 Percent of total integrated intensity of all bands in a lane Raw Int Intensity See description in Chapter 13 Integrated Inten sity Integrated intensity Normalization Factor A multiplier between 0 and 1 used to normalize each lane in the reference channel Normalized l l Normalized Integrated Intensity 195 196 CHAPTER 10 Reports and Data Export Plug in Reports A variety of plug in reports can be accessed via the Plug in menu A description of each plug in is given below Plug in Name Plug in Type Plug in Description Feature Data Report Report Sends data for all selected features to Excel The data sent for each feature is dictated by a report template named Feature_Data which can be modified to change the report Only data for selected features are included in the report Microsoft Excel for Grid Sends grid data to Excel Choose Grids Report gt Grid Fields Plug in Template to control which data are exported Microsoft Excel for ICW Sends response data and integrated ICW intensities
20. Channels Intensity Scan Area cm X Coord Y Coord Origin width Height Size Adjust Image Curves Alter Image Display Image Size 700 Kbytes per channel Progress bar Scan time remaining 4 minutes 48 seconds At the bottom of the Scanner Console window the status line indicates the time required to finish the scan The progress bar indicates the percentage of the scan area that has been scanned In the message area the message System Cooling may be displayed initially which indicates that the detectors in the laser microscope are being cooled to their operational temperature If no fluorescence is displayed where it is expected click the Alter Image Display button to adjust the image see Chapter 11 If bands are just dim use brightness and contrast adjustments If there are no bands move the Linear Manual Sensitivity slider auto adjustments off The Adjust Image Curves window can also be used to make similar adjustments By default the 700 and 800 channel images are shown overlaid If the color scheme is the default red green color scheme areas that are yellow have intense fluorescence in both channels To look at each channel separately during scanning the Alter Image Display window can also be used to display one channel at a time If no fluorescence is visible even after sensitivity adjustments or if there is signal saturation white pixels cancel the scan described
21. Display Band Quantification C Use the band s percentage of total lane Integrated Intensity total IT Band positions can be specified in one of three units in the Image View lane profiles and reports When Display Migration in the Lane settings is set to Pixel Location band positions are reported as the number of scan lines from the top of the image When Relative Mobility is selected the top of the lane is 0 and the bottom 100 and bands are assigned a percentage based on position between the top and bottom of the lane When Size Standard is selected band positions are reported as a molecular weight value assuming size standards have been assigned The number of decimal places used when the value is displayed can be changed using the Decimal Places field Note In the Image View abbreviations for Pixel Location Relative Mobility and Size Standard are Pix Locn Rel Mob and MW respectively Band Sizing in Single Channel Mode If a scan has two images the images can be displayed individually in single channel mode or overlaid in a composite image Chapter 11 When an analysis with two images is opened for the first time the images are overlaid so lanes can be added Chapter 5 After adding lanes each image must be analyzed separately in single channel mode Switching Image Channels To display a single image choose View gt Single Channel or click on the toolbar After a single image is displayed click 7
22. Tutorial User jen Scan Description Name Scan_2 J Group jen v Modify lt Preset Membrane v L Modify 31 CHAPTER 2 Starting Scans Scanning Multiple Microplates When scanning microplates Odyssey can scan up to six microplates simultaneously During a multi plate scan a separate scan and analysis is created for each microplate and they are added to the current project For example if there are six microplates six scan files and their corresponding analysis files will be created Odyssey relies on standard size plates being placed in a known location so it is important to use the alignment guide and place the plates as described below Use the following procedure to scan multiple microplates 1 Create an empty new project or open an existing project in which to store the new scans described earlier in this chapter 2 Choose File gt Scan gt Scan Multiple Plates 3 In the Scanner Login window enter your User Name and Password case sensitive and click OK if necessary 4 In the Scan Multiple Plates Setup window enter a base name for the scan or accept the default base name that is automatically entered according to the Multiple Scan settings Settings menu Note Odyssey appends the base name with a sequential identifier for each microplate In the window below if 2005 Mar 29 4PM is the base name Odyssey will automatically use 2005 Mar 29 4PM 1 as th
23. of the pixel times its height I This is called pixel volume v So for pixel i veal Pixel volume is the appropriate measure of signal strength because it takes into account both the magnitude of the signal and the area over which it is distributed which in turn is related to the distribution of sample that is generating the signal The total signal from the entire spot is just the summation of all the pixels and is called total pixel volume V V l 1 v q 1 T ms From assumption 3 SI X l b and rearranging Dei Dh Db 2 The expression gives the summed intensity over the spot that is contributed by the sample after correcting for background The last term on the right can be rewritten as n 1 n gt b or just n b so Sl Xl n b Clearly the net pixel volume of the spot V is just adil so V a 3I nb 3 248 CHAPTER 13 Calculation Descriptions The quantity calculated by equation 3 is also called the Integrated Intensity and by assumption 2 it is proportional to the amount of sample on the membrane Note that the Integrated Intensity is the net pixel volume for the spot alone and is independent of feature size If a feature is redrawn so it includes increasing numbers of background pixels the sum of the pixels 1 will increase but since the background is assumed uniform the increase in n b will compensate appropriately In practice the background correcti
24. position where the lower boundary of the lanes should be When the mouse button is released the specified number of lanes is placed on the image Lane width is determined by the Application settings Bands are automatically found and enclosed by a band marker after the lanes are created Verifying Band Finding After lanes are created and bands are found automatically each lane should be visually checked to make sure all bands are marked with band markers and that the markers are in the correct position First switch to single channel mode by clicking Visually check and make sure there is a band marker rectangle surrounding every band and that there are no extra bands identified in the lane 74 CHAPTER 5 Creating Lanes and Finding Bands Too Many Bands If there are too many bands in a lane bands can be deleted by clicking an unwanted band marker to select it and clicking X on the toolbar Legitimate bands should have the symbol centered over a band False bands will be centered over empty image Not Enough Bands Bands are added by clicking on the toolbar centering the mouse pointer over the band on the image and clicking the left mouse button Repeat the procedure for additional missing bands Fine Tuning Band Finding If there are consistently too many or too few bands on all images the default band finding threshold can be adjusted in the Application settings as described later in this chapter Ve
25. tration The number of decimal places used when the concentration value is displayed can be changed using the Decimal Places field Values are displayed both above a feature if turned on and in a tool tip as the cursor passes over a feature When Display Values as Kilo Integrated Intensity is selected all integrated intensity values are divided by 1000 to make the values easier to read This changes the display of integrated intensity values in all windows including the Grid Sheet window ICW Analysis window Details View and any integrated intensity values displayed in the Image View window Note The Application settings control whether quantification values are displayed on the image see Chapter 11 When Substitute Trimmed Mean for Saturated Pixels is selected the calculated Trimmed Mean value Chapter 133 is substituted for any saturated pixels enclosed in a given feature before quantification Raw integrated intensity values in counts should be used cautiously when comparing shapes as detailed in Chapter 13 Raw integrated intensity values vary with resolution and the size of the feature drawn 147 148 CHAPTER 8 Quantification on the image Do not use raw integrated intensity values to compare features from images scanned at different resolutions Similarly image features drawn on the image must be exactly the same size if their raw integrated intensity values are to be compared Note For a band in a lane the quant
26. 22 CHAPTER 2 Starting Scans The Intensity fields control the detector sensitivity and affect the band intensity on the image If the intensity is set too high the detector may saturate and produce white areas in the middle of intense bands dots Saturated pixels are colored cyan if the image is being displayed as a grayscale image If the intensity is set too low the image may not show any fluorescence even though there is adequate signal from the samples LI COR Presets use an intensity value of 5 0 for membranes 8 0 for DNA gels and 5 0 for protein gels or microplates These settings may need to be optimized for your gels or membranes due to the differing background fluorescence of various materials Intensity values from 1 to 10 in increments of 0 5 can be chosen as well as low intensity values LO 5 to L2 0 L2 0 is the lowest intensity value Odyssey can use for scanning Scan Area parameters are used to specify the portion of the 25 x 25 centimeter scan surface to scan The Size and Origin cm can be set by clicking and dragging a rectangle on the scan grid as shown below Scanner Back Click and hold down the H mouse button in the A lower left corner of the area to be scanned E HH FA H E ie TN E am D E BE E a aman gE mi Drag the cursor to the CEREC fence fens
27. 24 Sample 482920 41 Sample 66418039 Sample 20224 84 Backgnd 411576 96 Sample 180728 14 448002 87 Sample l Print l Export l Copy If the data table is empty and a red status message is displayed indicating Invalid ICW calculation parameters the ICW calcu lation parameters background wells 100 response wells etc have not been set up To set up the ICW calculation parameters click Change Params to open the Change ICW Parameters window described earlier in this chapter Excluding Empty Wells Initially all wells are shown in the data table Wells that were desig nated as Not Used in the well assignment window listed as Unused in the Well Type column can be excluded from the data table by selecting Show Only Used rather than Show All a ccm Sorting Data Start by selecting Sort Ascending or Sort Descending to set the sort order to ascending or descending respectively Next click the column header of the data you want to use to sort the table Color Coded Cells for Percent Response Values The percent response values are color coded to visually distinguish cells with a given response Table cells are initially colored according to the ICW View settings To change response color codes choose Settings gt ICW View ICW View Response Color Settings Percent Response Set Points and Colors
28. Ascending check box is selected the order will be ascending otherwise the sort order is descending Use the By Field drop down list to choose the field on which to sort By default data for both image channels are included in reports Deselect either the Include 700 Channel Features or Include 800 Channel Features check box to include only one image channel in the report Saving the Template After editing the template click Save to save the finished template Field Definitions Feature Reports Field Definition ID ID numbers are automatically assigned to every feature lane circle square etc Name User supplied name entered in the properties Description User supplied description entered in the properties Lane Name User entered name for the lane entered in the Properties Channel Name of the channel 700 or 800 in which the fea ture is found Concentration Calculated concentration of the feature Conc Units Raw Int Intensity Units selected in the Concentration Standards window See description in Chapter 13 Int Intensity See description in Chapter 13 Avg Intensity Average intensity of all pixels that comprise the fea ture See description in Chapter 13 Peak Intensity Highest intensity value of all pixels that comprise the feature Minimum Inten sity Pixels Lowest intensity value of all pixels that comprise the feature Number of pixe
29. Either a new or existing project can be used Start the download by choosing File gt Scan gt Download Scan Scanner Login Scanner West Lab User User Name ron Password trate In the Scanner Login window select the scanner on which the files are stored and enter your user name and password 52 CHAPTER 4 Importing and Exporting Scans and Images After logging in the Download Scan window is used to select a scan Download Scan Scan Group jen M Scan Name 2003 Jul 01 9AM 1 Available Scans 2003 Jul 01 9AM 2 2003 Jul 30 4PM 1 2003 Jul 30 4PM 2 2003 Jul 30 4PM 3 2003 Jul 30 4PM 3_1 2003 Jun 30 4PM 1 o i aiz First choose a scan group The Scan Group drop down list shows only scan groups that the current user belongs to After choosing a scan group select a scan from the Available Scans list Chapter 12 describes the User Administration settings and how they are used to change access to scan groups if needed Select a scan and click OK To use a different scan name when the files are downloaded to avoid duplicate scan names etc enter a new name in the Scan Name field The selected scan will be downloaded into the current project The scan can be analyzed by creating a new analysis Chapter 3 after the files have finished downloading A progress bar in the Download Scan window shows how much of the download has been completed 53 Impor
30. If a scanner is turned on or added to the same network as the computer the scanner list can be updated by clicking Auto Discover to start the automatic discovery routine If an instrument is not auto discovered it has to be added manually as described below Note If the computer and Odyssey instrument are on different networks that are connected by a router the Odyssey instrument will not be found during automatic discovery Manually Adding Scanners Use the following steps to manually add a scanner to the list of scanners 1 Click the Add button in the Scanner List dialog 2 Enter a Name that identifies the scanner 244 CHAPTER 12 User Accounts and Settings 3 Enter a Host Name The Host Name must be the IP address of the Odyssey instrument or a valid host name If a host name is used it must be registered with a Domain Name Server DNS available to the network Note that leading zeros are not entered in the Host Name field For example Name East Lab Scanner you may be told that the IP address is Host Name 242 27 44 249 242 027 044 249 a fictitious address but the address should be entered as 242 27 44 249 as shown in the Add Scanner window to the left Add Scanner Description Scanner In Dr Smith s Lab 4 Enter a Description to identify the scanner optional 5 Click OK to add the scanner Editing and Deleting Scanners Scanners can be edited or deleted by openin
31. SUPPORT SERVICES EVEN IF LI COR HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES IN ANY CASE LI COR S ENTIRE LIABILITY UNDER ANY PROVISION OF THIS AGREEMENT SHALL BE LIMITED TO THE GREATER AMOUNT OF ACTUALLY PAID BY THE LICENSEE FOR THE SOFTWARE PRODUCT OR U S 5 00 PROVIDED HOWEVER IF YOU HAVE ENTERED INTO A LI COR SUPPORT SERVICES AGREEMENT LI COR S ENTIRE LIABILITY REGARDING SUPPORT SERVICES SHALL BE GOVERNED BY THE TERMS OF THAT AGREEMENT 6 4 Exclusive Remedy TO THE EXTENT THAT THE LICENSOR IS LIABLE THE EXCLUSIVE REMEDY AT LI COR S OPTION SHALL BE EITHER A RETURN OF THE PRICE PAID IF ANY OR B REPAIR OR REPLACEMENT OF THE LICENSED PROGRAM THAT DOES NOT MEET LI COR S LIMITED WARRANTY AND WHICH IS RETURNED TO LI COR WITH A COPY OF PROOF OF PURCHASE This Limited Warranty is void if failure of the Software Product has resulted from accident abuse or misapplication Any replacement Software Product will be warranted for the remainder of the original warranty period or thirty 30 days whichever is longer Outside of the United States neither of these remedies nor any product support services offered by LI COR are available without proof of purchase from an authorized international source 7 DISPUTE RESOLUTION 7 1 In the event of a dispute involving the interpretation or application of any provision of this Agreement the parties agree not to commence litigation until they have first notified each other of their intent to impl
32. Show Only Quantified Pixels icer copy Export Print close Details View shows an enlarged view of the dot band along with the shape that surrounds it The center of the feature surrounding the dot band is marked by the blue cross hairs The sides of the blue rectangle are the horizontal and vertical boundaries of the feature on the image In the case of a rectangle feature the blue line actually represents the rectangle The curves to the right and below the image plot the intensity of the pixels below the cross hairs First make sure the feature on the image is large enough to enclose all the fluorescence To get the best background calculation for quantification all pixels on the outside perimeter of the blue line should be empty background pixels no fluorescence For smeared bands or closely spaced dots this may not be possible but the background calculation method can be changed to accommodate the image data as described in the Chapter 8 After the feature is properly sized examine the cross hairs to make sure they are centered over the band dot If a feature needs to be 113 114 CHAPTER 7 Drawing Features on Images moved to center it in the cross hairs the feature must be moved on the image rather than in Details View Resizing and Deleting Features If a feature circle etc is too large or too small move the cursor toward a corner until the cursor turns to a diagonal arrow as shown
33. Standards Sets Protein Standards The table in the Size Standards Sets window shows the name number of standards in the set units and sort order for each set Click New to start a new set Enter a name for the new MW set and click OK New Size Standards Set Enter Size Standards Set Name Names cannot contain any of the following characters lt gt It is also not necessary to enter any file name extensions The Edit Size Standards Set window can be used to enter the size of all standards in the set select the units and specify the sort order for the set 97 ts basepairs Vv Sort values in descending order Save Cancel Begin by selecting the units for the set using the Units drop down list Basepairs kilobasepairs daltons and kilodaltons can be selected Standards are added by entering the value of the molecular weight standard in the Value field and clicking Add Standards can be entered as either an integer or a mixed number integer with a decimal fraction Continue entering standard values and clicking Add until all standards have been entered The order in which the standards are entered is unimportant since they are automatically sorted For typical gels with the highest MW standard at the top of the image and the lowest at the bottom the Sort Values in Descending Order check box should be selected If your image has the lowest MW standard
34. View shows the feature drawn around the fluorescence and a blue bounding rectangle The pixels used in the background calcu lation are the pixels between the blue rectangle in Details View and the edge of the image The Border Width parameter in the Background Method window determines the width in pixels of the image outside the blue border that is used for background calcu lation To illustrate the Yj location of the pixels used for Average or Median background methods the pixels have been covered with diagonal lines When Median or Average is selected background is calculated as the median value or average of the background pixels on the user selected sides of the blue rectangle respectively After selecting Median or Average choose which line segments of the perimeter rectangle to use in the calculation If All is selected pixels in all four sides of the blue rectangle are used If Top Bottom or Right Left is chosen only pixels in the two indicated sides of the rectangle are used User Defined To use the User Defined background method display both image channels and draw a feature on the image over an area of typical background The feature will be added to both 700 and 800 channel images With the channels still overlaid select the feature choose Analyze gt Background Method and change the background method to User Defined After clicking Save any new features will 157 be quantified using the new user defined b
35. a cropping template the MouseRightPosition preset in our example Available Scan Templates Oo Choose one J v MouseCenterPosition MouseLeftPosition o tightPos oteinGel 4 Click OK Crop Multiple Images Create cropped images and a new analysis for each set of images using the scan area of each Scan Preset in the list below Source images from scan SCID Mouse are T7OO TIF T800 TIF Remove Remove All 5 Repeat steps 2 4 for the center MouseCenterPosition and left MouseLeftPosition mouse positions If there are any other scan presets you would like to use add them to the list The current images can be cropped many different ways by choosing additional scan presets 6 Click OK to crop the current images and save each of the three cropped images in new analysis files 64 CHAPTER 4 Importing and Exporting Scans and Images Exporting Scans Choose File gt Scan gt Export Scan to save a copy of the current scan to any location network local etc specified in the Select Desti nation Folder window The scan folder and all of its contents will be copied including all analysis files Project Backup For archival or safety purposes the project that is currently open can be backed up to any destination by choosing File gt Backup Project Click Browse to open a standard file selection window or enter a path for the destination folder Files can be stored on any local or network drive Backu
36. a low quality assay with marginal distinction between signals for positive and negative controls and higher data variability However an acceptable Z factor target value should be determined prior to performing final validation of an assay An assay with a relatively low number of data points such as the number obtained from a 96 well plate may produce a Z factor value less than 0 5 but still be considered a good quality assay if the same value is acheived between different plates run on different days m Z lt 0 indicates unreliable data 259 A Z factor value that is close to zero or negative indicates that assay conditions are not optimized or the assay is not capable of generating meaningful data Zhang J H Chung T D and Oldenburg K R 1999 A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays J Biomol Screen 4 2 67 73 A adding scanners Odyssey instruments align overlaid images ccccccecseeeeseseeeeeees 221 analysis DUWO isie erresau e taiese sran EE EKR 6 STINT ON apsenin 6 descriptio Mase heiet 40 deleteria a a 9 naming opening multiple analyses 47 SAVING ssssusadiscasesstedssdsvsesatestentes ssodsdedavaeatsee 46 analyze menu adjust feature location 115 118 add grid ssri add multiple features background method eee concentration standards ceeeeee 149 prid sheetestedties Merete avin rename select
37. and horizontal center to center distance between objects on the image See Measuring Size and Distance on the Image below Vertical Grid Spacing Modify Grid Settings Grid Template Name 96_VVell_Plate Grid Size Rows al Columns 12 Spacing between Grid Lines mm Horizontat 90 Verticat 9 0 Location from Upper Lett Image Corner mm XOftset 58 Y Offset 37 Center in main grid when used as sub grid Well Shape and Size mm Circle O Square Quantity Vell Dia 55 Physical Well Dia 6 5 Column and Row Labels Column Labels O Letters Numbers Row Labels Letters O Numbers Main Grid Properties mal Display grid lines o Use sub grids When using sub grids Well Pite v Horizontal Grid Spacing 127 Offset From Upper Left Image Corner These X and Y coordinates determine the placement of the upper left corner of the grid The offset in millimeters is measured from the upper left corner of the image Scan TiterScan Analysis Grid Ana L X Offset Well Shape and Size Either circle or square features will be placed in the center of each cell on the grid depending on whether Circle or Square is selected for the well shape The physical size of the well spot is the actual size mm of the feature to be added to each grid cell normally just large enough to surround all the fluores cence Physical size is used to accurately place the grid The quanti
38. are possible Negative relative intensities indicate the original integrated intensity was lower than the average background when the background was subtracted Relative intensity values with low statistical significance are color coded when displayed in the View ICW Analysis window Continuing the example the next step is to use the relative intensity values from the 800 channel to normalize integrated intensity values in the 700 channel To normalize the 700 channel the integrated intensity for each well in the 700 channel is divided by the relative intensity value from the corresponding well in the 800 channel Next all wells designated 100 Standard in the 700 channel are averaged The normalized value for each well is then divided by the 100 Standard of the 700 channel and multiplied by 100 to give a value that is the percentage response to the control in the 100 Standard The calculation is slightly different if rows are linked When rows are linked all the integrated intensity values for the linked wells in a given column are averaged The average integrated intensity replaces the original integrated intensity in each of the linked wells Note If the Calculate Relative Intensity in Channel field in the ICW parameters is deselected Chapter 9 the percent response will be calculated for both channels with no normalization For each channel the relative intensity values are divided by the 100 Standard and multiplied by 100 ICW
39. below and start the scan again using new values for the Intensity scan parameter in the Scanner Console If fluorescence is too strong use lower intensity values The scan ends automatically when the entire scan area has been scanned As the images are collected image files are created both on the hard disk of the Odyssey Imager and on the computer Stopping a Scan To finish a scan before automatic completion click the Stop button in the Scanner Console window The image files will be closed and saved allowing the files to be analyzed To abandon a scan and not save the image files click Cancel rather than Stop 27 CHAPTER 2 Starting Scans Completing the Scan When the scan is complete a reduced version of the image is shown on the scan grid and the Save button is activated so the scan on the Odyssey hard drive can be saved to the computer in an Odyssey project To save the scan click Save Alternatively click Close to abandon the scan without saving any files When Save is clicked a dialog in displayed in which a new scan and analysis can be saved Save As Scan Analysis Scan Name E 007 Analysis Name 6 l OK Cancel l Advanced J The scan name and analysis name are initially determined by the naming conventions specified in the Application settings Settings gt Application then Naming Conventions but these names can be changed as needed When OK is clicked a scan folder is cre
40. below With the diagonal arrow cursor displayed the feature can be enlarged or reduced by clicking and dragging To delete a feature select the feature and press the Delete key on the keyboard or click gt on the toolbar Moving Features If the cross hairs in Details View indicate that a feature needs to be moved in order to center it move the cursor to the center of the feature on the image until the cursor has arrows in all four directions as shown below When the all arrows cursor is displayed features can be clicked and dragged to a new position Nudging Features It can be difficult to precisely place objects with the mouse Selected features can be nudged one pixel at a time using the arrow keys on the keyboard as long the all arrows cursor is displayed within the border of the feature Selecting Multiple Features Multiple features can be selected by clicking and dragging a selection rectangle around them or by control clicking additional features after selecting the first feature 115 Automatically Adjusting Feature Locations Although features can be moved manually it is often easier let Odyssey software adjust the locations of selected features As described later in this chapter choosing Analyze gt Adjust Feature Location automatically moves features over any nearby fluorescence in the image Successful use of the automatic adjustment software depends on the features being near the fluorescence that sho
41. distance when Move All Cells Together Within The Grid is selected and a consistency threshold is met Features will not be moved unless a user defined number of features are found to require approximately the same movements for correct positioning The number of features that must exhibit consistent movement is entered in the Number of Features Required field When the location of features in a grid are adjusted images are automatically aligned if Automatically Align Images is selected and the number of features requiring the same offset is equal to or greater than the number of features specified in the Number of Features Required field After alignment the fluorescent features of the two images should be in the same positions The status line at the bottom of the Odyssey window indicates which image was offset and by how much assuming the alignment was successful in some cases it may be necessary to double click the status line to open the message history window Important There is no undo operation for the image alignment routine because the TIFF image files are immediately changed 123 Restoring Default Adjust Location Settings Click Restore Defaults to change the Adjust Location settings to the values shown in the Adjust Locations window at the beginning of this section Adding Multiple Features Using Grids Odyssey s grid tool can be used to apply an array of features all at once For regularly spaced image features s
42. down the mouse rectangle surrounding button Drag downward and to the the band dot and place right Release the mouse button when the cursor in the upper the fluorescence on the image is left corner completely enclosed Drawing a feature with the Freeform tool is slightly different To draw a feature with the Freeform tool click the Freeform button in the toolbar and move the cursor to the edge of the object in the image Click and hold down the left mouse button and trace around the object on the image Release the mouse button when the freeform line completely encloses the object in the image Keyboard Shortcut The last tool selected from the toolbar is automatically selected again when F5 is pressed Using Details View to Position Features Odyssey has a Details View that is useful for verifying that a feature is correctly positioned and is large enough to enclose the fluores cence on the image To open the Details View select a feature and click on the toolbar or choose Report gt Details View Details View can also be opened by selecting a feature right clicking the image and choosing Details View from the pop up menu It is advantageous to leave Details View open while drawing features on an image because each new feature is immediately shown in Details View after it is drawn i Details View i V 24020 H 15358 T ihia 7 ID Name Integ Int Concentr Ave Inte Shape Area Pixels 963 800 E4
43. exporting an image it may be advantageous to turn on molecular weight MW values for unselected features so all MW values will be shown on the exported image Each of the Image View Feature settings is described below e Name Name is either the auto entered default object name or a user entered name in the object properties Edit gt Properties Description Description is blank by default for many objects but can be entered in the object properties Descriptions can be included in reports Quantification Quantification values are displayed as Integrated Intensity or Concentration depending on the Application settings for Display Values This annotation is generally turned off for band sizing unless bands in the lanes are to be quantified Molecular Weight The Molecular Weight annotation displays band migration in the gel as molecular weight scan line on the image or percentage of the lane according to the Application settings for Lanes 93 described below If migration is displayed as molecular weight values are displayed as n a not assigned until molecular weight standards have been identified e Boundary Each feature i e lane circle rectangle band marker etc has a boundary If boundaries are turned off only the text annotations will remain Turning off the tool tips Show Shape Tool Tips or changing the font used for annotations can also reduce screen clutter Changing the normal or selected color
44. features should be spaced equally in the horizontal or vertical dimension Features are named with a prefix base name and a numbered extension see Name Example in the window above Name extension numbers are incremented for each feature moving in the specified direction left to right right to left top to bottom or bottom to top Adding Features Along a Straight Line The next step is to draw the line Click Continue to return to the image For a straight line double click in the center of the spot band 118 CHAPTER 7 Drawing Features on Images at the opposite end of the line from the initial feature The imaginary line connecting the two points can be horizontal vertical or any angle in between Adding Features Along a Curved Line For curved lines click Continue and then single click at inflection points along the curve until reaching the spot band on the end opposite the initial feature Double click at the end point to exit line draw mode and the specified number of features will be drawn Disabling the Add Multiple Features Window For applications with repetitive sample configurations the Add Multiple Features window may not be necessary and can be disabled by selecting Don t show this dialog at the start of Add Multiple Features action For example if many blots with multiple rows from an 8 channel pipettor are scanned there is no reason to specify to add seven features for every row With the Add Multiple
45. fluorescence due to dirt fluorescence of micro plate walls and other fluorescent sources needs special consider ation For example dirt that fluoresces brightly near a selected feature can cause the feature to be moved to the wrong location Setting the threshold to a higher intensity may prevent features from being placed over spurious fluorescence but if the threshold is set too high features near low intensity fluorescent dots may be misplaced Feature locations may need to be adjusted manually on images with a lot of bright spurious fluorescence near image features Keeping the search area small is generally desirable even though it means the initial feature locations must be near the final locations If the search area is too large the search may find a bright piece of dirt some distance away from the target fluorescence The Search Area Extension slider is used to increase or decrease the image area around a selected feature that is searched for maximum fluorescent signal The area searched is defined differently for features within grids and features drawn on the image For a feature within a grid setting Search Area Extension to Small confines the search area to the boundaries of the grid cell Setting Search Area Extension to Large expands the search area to twice the height and width of one grid cell centered in the center of the original grid cell For a feature drawn on the image setting Search Area Extension to Small exten
46. for Microsoft Excel for example should use a tab as a field separator The Report Title can be entered as text or a combination of user entered text and text that is automatically entered by Odyssey software when the report is created To use auto entered text blocks click Edit Title and use the Insert button to add placeholders for auto entered text as described above for the report file name After configuring the report settings click Save to store the new report settings Note that these settings are saved only for the current Application settings file Creating Report Templates Report templates are used for printed reports and for creating the data table in the report view The current template can be selected or edited in the report view Report templates can also be created edited or deleted by choosing Reports gt Report Field Templates Select Report Template amp Template Type Feature_Data Feature Feature Lanes Lanes 189 Click New in the Select Report Template window to start a new template Create New Report Template x Name MyTemplate1 Type Feature Enter a name for the template in the Name field and select the template type Select Feature from the Type drop down list to create a template for selected features or Lane if the new template is for a report on lanes Click OK to continue Both Feature reports and Lane reports are created the same wa
47. in Single Channel mode After all the features are drawn concentration standards are identified by selecting the feature surrounding the standard and using the Concentration Standards window to enter the concentration Odyssey requires that concentration standards be added in order either lowest to highest or highest to lowest Visually identifying all the concentration standards on the image before beginning will make it easier to add the standards in the correct order As soon as the concentrations of all standards are entered the concen trations of all sample dots or bands are automatically calculated 145 146 CHAPTER 8 Quantification Quantification and Concentration Calculations When a feature is drawn over fluorescence in an image the image data within the feature are quantified immediately This applies to features like rectangles or ovals that are explicitly drawn and to band markers that are automatically assigned when lanes are created When a feature is quantified Integrated Intensity is calculated Integrated Intensity has also been referred to as Pixel Volume in other software Integrated Intensity is the sum of the intensity values for all pixels enclosed by a feature multiplied by the area of the feature counts mm Since background pixels should not be part of this calculation background is calculated and subtracted Several methods are available for calculating background The calculation method is select
48. measured in a single pixel per unit time e Sample The physical material on a membrane that generates the signal giving rise to a spot 246 CHAPTER 13 Calculation Descriptions Assumptions 1 Background is uniform though not necessarily constant across a feature whether a spot is present or not and regardless of the size of the feature or image spot 2 After correcting for background the signal in an image spot is proportional to the amount of sample generating the spot 3 The total intensity per pixel I is equal to the signal intensity arising from the sample in the pixel area l plus the signal arising from the background of the pixel area b So for pixel i f Ij b Feature Image spot Intensity Pixel 1 2 3 i n X Pixels Integrated Intensity and Integrated Pixel Volume An image can be considered a three dimensional object in which two dimensions are the x and y plane of the image and the third 247 dimension is the pixel intensity Consider an image containing spots with features drawn around them The graph above shows a slice through one such feature containing an image spot The x axis repre sents pixels and the z axis is signal intensity per pixel This graph is a two dimensional representation of a three dimensional object that also extends along a y axis into and out of the paper The total signal measured in pixel i is the area a
49. more disk space but is recommended for typical images since original copies of the scans are stored in the project 30 CHAPTER 2 Starting Scans If there is a problem with the scan and you do not want to analyze the images click Cancel in the New Analysis window instead of OK Clicking Cancel removes the new scan from the project that was open when the scan was started Creating and Editing Preset Parameters After scanning a few of your own samples you may want to create a set of Preset scan parameters or edit an existing Preset to match your scanning methods The instructions below describe creating and editing Preset parameters for Odyssey Software To create your own Presets in the Odyssey instrument consult the Odyssey Operator s Manual The Preset parameters displayed in the Scanner Console window can be created or modified by choosing Settings gt Scan Presets Scan Presets Scan Preset Name Resolution Quality Offset Channels medium 2 0 700 800 medium 700 800 medium medium i 700 800 MouseCenterPosition medium 700 800 MouseLeftPosition medium 700 800 medium 700 800 Irectivin ln s lzan eon Delete Close The Scan Presets window lists all presets To edit a preset select it from the list and click Edit To create a new preset either click New or edit an existing preset and click Save As in the Modify Scan Preset window Using the Modify Scan Preset Window T
50. on the Odyssey instrument in scan groups Scan groups are like folders that require permission to access them Normally your own scan group matches your user name is listed along with any other scan groups that may have been added Modify Scan Groups Available Scan Groups Delete Manage Scans Edit Permissions Backup The following tasks can be accomplished in the Modify Scan Groups window E Add Scan Groups Click Add and enter the name of a new scan group The user who created the scan group is automatically given Control access permission All other users have no access unless their permisssion level is changed as described below E Delete Scan Groups To delete a scan group on the instrument a user with Control rights can select any of the scan groups in the Modify Scan Groups window and click Delete IMPORTANT Deleting a scan group deletes all images within the group After a group is deleted it is not possible to restore images that were in the group Images stored in other scan groups on the instrument or on the computer are not deleted E Delete Scans In A Scan Group Deleting a scan from a scan group on the instrument does not delete any scans that may be stored on the computer To delete a scan from a scan group select the scan group in the Modify Scan Groups window and click Manage Scans All scans in the scan group are listed Select a scan and click Delete to permanently delete a scan and all a
51. shall be deemed effective upon delivery in person by courier or sent by certified United States mail postage prepaid return receipt requested to the following address LI COR Inc 4421 Superior Street P O Box 4425 Lincoln Nebraska 68504 USA LI COR Odyssey and IRDye trademarks contained in the Software Product are trademarks or registered trademarks of LI COR Inc Third party trademarks trade names and product names may be trademarks or registered trademarks of their respective owners You may not remove or alter any trademark trade names product names logo copyright or other proprietary notices legends symbols or labels in the Software Product This EULA does not authorize you to use LI COR s or its licensors names or any of their respective trademarks viii EXHIBIT A JAVA ADVANCED IMAGING SAMPLE INPUT OUTPUT SOURCE CODE LICENSE Copyright Sun Microsystems Inc All Rights Reserved Redistribution and use in source and binary forms with or without modification are permitted provided that the following conditions are met Redistributions of source code must retain the above copyright notice this list of conditions and the following disclaimer Redistribution in binary form must reproduce the above copyright notice this list of conditions and the following disclaimer in the documentation and or other materials provided with distribution Neither the name of Sun Microsystems Inc or the names of contributor
52. started a project must be open so the new scan can be stored in the open project Starting Standard Scans in an Existing Project Existing projects are opened by choosing File gt Open or by clicking amp on the toolbar The four most recently opened projects are also listed toward the bottom of the File menu The number of recent projects listed can be increased to as many as 10 in the Application settings choose Settings gt Application and select General from the Settings List Once a project is open a standard scan can be started by clicking on the toolbar or choosing File gt Scan gt Scan After entering 12 CHAPTER 2 Starting Scans your user name and password the Scanner Console window is opened allowing scans to be started as described below Starting a Standard Scan in a New Project To start a new project in Odyssey Software choose File gt New New Project Path C Documents LicoriOdyssey Projects The path and project name can be entered by clicking Browse to open a standard new file window File paths and names can also be typed in the Path and Name fields After entering the project name take one of the following actions e Click Done to create an empty project e Click Import Scan to create the project and import images from a different project Chapter 3 e Click Scan to create the project and start a standard scan that will become part of the project In the Scanner Lo
53. that is too high or too low use the Edit Size Standards window to review the position of the MW line of the lane in question The line may be snapped to the wrong band or the size may be incorrectly assigned basepairs Std 1 Channel 700 154 00 659 50 1165 00 1670 50 2176 00 162 0 242 5 333 0 423 5 514 0 After reviewing the plots for lanes with MW standards review the sample lanes to make sure each standard is correctly positioned In sample lanes molecular weight standards are assigned at the points where the MW lines cross the centerline of the lane For gels with even migration straight molecular weight lines the plots of sample lanes will be very similar to standards lanes For gels with smiles or other gel artifacts where extra points are added to bend the MW lines in a particular direction close examination of each sample lane is needed Suppose you have an image with a smile and too few points are added to the molecular weight line In this case the MW line may not accurately follow the contour of the gel and may cross the centerline of a lane at a point that is too high This will be revealed in the standards plot for the lane in question since the standard in question will be misplaced on the plot Adding another point to the MW line and moving the point to the proper position will eliminate the problem When all standards have been examined click OK to close the Size Standards window Sample bands already
54. the X axis is shown in the cursor tool tip when the cursor is stopped over the graph Below the X axis is set to display relative mobility and the cursor position is 0 14 Each Y axis value in the lane profile is the average intensity of all pixels within the profile width for a given row of pixels in the lane The profile width of the lane is determined by the Application settings for lanes Profile width is the percentage of the lane width centered in the lane used to calculate the lane profile For a lane that is 40 pixels wide and a profile width setting of 75 the pixel intensity values are averaged for the 15 pixels on both sides the lane s vertical centerline 30 pixels total The average intensity is then plotted on the lane profile to represent that row of pixels This is repeated for each row of pixels in the lane until the profile is complete Displaying Band Centers With Band Centers selected in the Lane Profile window the band number is displayed on each fluorescence peak along with a that indicates where the band marker is centered Displaying band centers is an easy way to make sure each band marker is centered over the band on the image In the lane profile below band 5 is not properly centered since the center marker is not on the apex of the peak If the image for band 5 were examined the center of the band marker would not be centered in the fluorescence on the image and should be moved so the correct size wi
55. the Z factor for an assay provides a method to evaluate whether assay conditions are optimized Z factor is a dimensionless value that indicates whether the assay has sufficient dynamic range and low enough data variability to generate meaningful data When Z factor calculations start all wells designated as background in the 700 channel are averaged and the same is done for the 800 channel Assuming that background subtraction is enabled which it normally should be the average background intensity for the 700 channel is subtracted from the integrated intensity of all 700 channel wells The background is also subtracted from the 800 channel image During setup one channel is designated as the channel used to calculate relative intensity This channel is used to normalize for sample loading by detecting a housekeeping protein or protein stain In the channel used to calculate relative intensity Odyssey software starts by finding the well with the maximum integrated intensity All 257 wells in this channel are divided by the maximum integrated intensity to obtain the relative intensity of each well The relative intensity values will normally be between 0 0 and 1 0 though negative numbers are possible Relative intensity values with low statistical significance are color coded when displayed in the View ICW Analysis window The next step is to use the relative intensity values to normalize integrated intensity values in the sample ch
56. the analysis name and then click OK Save As Scan Analysis Scan Name TiterScan Analysis Name gideeelidelilsmr mene memebers To save the new analysis in a new scan enter a new Scan Name and Analysis Name optional before clicking OK An analysis cannot be saved to an existing scan other than the current scan Crop to New Analysis Choose File gt Analysis gt Crop to New Analysis to crop image files in the current analysis and save the cropped images in a new analysis A reminder is displayed that explains how to crop a region of interest Crop Region of Interest i Select the Region of Interest ROT to crop from the current image Click the mouse over the image Then drag the mouse to select the area to crop After the ROT is defined a dialog will prompt for the new analysis name 61 After clicking OK move the mouse cursor to one corner of the region of interest on the image Click and hold down the left mouse button while dragging the cursor to the opposite corner if the region of interest Release the mouse button amp Scan IkappaB scan Analysis IkappaB Analysis Images Albrecht_700 TIF and Albrecht_800 TIF DER A bm To save cropped images in the current scan click OK or edit the analysis name and then click OK To save the images in a new scan and analysis change the Scan Name and Analysis Name optional before clicking OK An analysis cannot be saved to any scan other
57. to Excel using the current ICW report template Microsoft Excel for Shape Exports all data for all features to features Excel ReagentsWebLink Command Executes a command line statement that launches Internet Explorer and sets the page to the Odyssey reagents page at www licor com 197 Launching Plug in Reports Plug ins are launched by selecting them from the Plug in menu The example below shows the procedure for using the Microsoft Excel for Grids plug in Any user created plug in reports will follow the same general procedure 1 Open the analysis that has the grid data to export 2 Choose Plug in gt Microsoft Excel for Grids 3 In the Launch Plug In window select Create New File and enter a file name The current analysis name from the Odyssey scan is entered as the default file name An existing file can be overwritten by selecting Open Existing File and selecting a file from the file list Launch Plug in Plug in Microsoft Excel for Grids Analysis Q_Analysis r Launch Options Create New File File Name MyExcelGrid Open Existing File 198 CHAPTER 10 Reports and Data Export 4 Click OK The Microsoft Excel program should start and the grid data should be displayed in a new worksheet E Microsoft Excel MyExcelGrid data 1Odyssey Aerius Data Output File 1 0 4 Grid Tutorial Titer Scan Analysis TutorialAnalysis Scan Date Unknown Channels 2 Channel N TutorialAnaly Tutorial
58. well plates are not recommended for ICW assays Modify Grid Size ICW Grid Size 96 VVell Plate colurnns 12 rows 8 The three tabbed panels in the center of the Setup Template window are identical to those describe earlier in Changing ICW Parameters for the Current Analysis In the Setup Template window the buttons at the bottom of the window are Save and Save As One or both may be active depending on whether an existing template is being edited or a new template is being created These buttons only save the 175 current template The ICW parameters in the current analysis are unchanged Creating Reports for In Cell Westerns In Cell Western reports are not available in the standard Odyssey Software Standard reports are discussed in Chapter 10 Printing and Saving Reports To print an ICW report or save it to a file choose Print ICW or Export ICW respectively from the Report menu The form of the report is determined by the ICW report template which can be changed as described below 176 CHAPTER 9 Odyssey In Cell Western Module Changing the ICW Report Template Choosing Report gt ICW Report Template opens the ICW Export Definition window that can be used change the ICW report template In Cell Western Export Definition Sorting Sort Column Cell ID v Ascending Descending Only used cells O All cells C include header information Export File Name Project_ Scan_ICvV _Export txt
59. 01 Editing Molecular Weight Lines 104 Moving Whole Lines ceeeee 105 Adding Points to a Lin eee 105 MOVING POMS recaian an 106 Plotting Size Standards wo eee 107 Setting the Interpolation Method 107 Setting Units for Standards 0 0 0 108 Reviewing the Standards Plot for Each Ee E A 108 Chapter 7 Drawing Features on Images ONT cosesisstinncass ce Get evtxesceriee ec estenvexes 111 Drawing Features on the Image 111 Using Details View to Position Features 112 Resizing and Deleting Features 114 MOVING FEAtUmnes oct cciscedsvcctctsvcticetnseceseees 114 Copying and Pasting Features 115 Adding Multiple Features 116 Setting the Drawing Mode to COMUMUOUS cscsacusnessecvastinedsacdeseasontens chs 116 Using the Multiple Features Tool 116 Automatically Adjusting Feature Locations 118 Using the Adjust Location Settings 119 Adding Multiple Features Using Grids 123 Creating Grid Templates 124 Deleting Grid Templates 0 125 Editing a Grid Template 0 0 0 125 Grid Parameterionicscscosssneses 126 Measuring Size and Distance on the MAS Sissy sa tecrs oe oeg Sov EE E siete egesticaes 128 Applying Grids to Images eee 128 Applying a Grid Automatically 129 Moving a Grid Manually eee 129 Deleting a GH ssssccunosenanesen 130 RESIZING a GHG Ns cssscssssssencss es
60. 154 Changing the Background Method 155 No Background sassinoro 156 Average Median and User Defined Background Methods ceeeeeeee 156 Using the Lane Background Method for Ban cscccssstesacssteveosnetensagnsteneasseasneay 158 Requantifying After Changing Background xi Chapter 9 In Cell Western Module OOVEIVICW Sissi cess vepinccsnieeidenhnesbaeea eit oetbbiseteds 161 Starting a New In Cell Western ANALYSIS catiisssevsectssccsoisssccboesdvechassbocsdncvsonss 162 Applying a Grid Automatically 162 Automatic Calculations eee 163 Changing ICW Parameters for the Current Analysis z csscrscrssicsecssoconnevieses 163 Applying a Different ICW Template 164 Temporarily Changing the ICW PAPA Meters kruisers 164 Well Types Tab uu eeeeeeseseeeeeeees 165 Well Links Tab wvsc sizeversabvoncsettssaevessen s 165 Calculations Tab wvsiiscsnsbsssetoshtevnoebsnves 167 Applying the Changes c eeee 168 Examining the ICW Response Data 170 Excluding Empty Wells e 170 Sorting Data essnee 171 Color Coded Cells for Percent Response Valte enean E TERS 171 Color Coded Relative Intensity Values 171 Recalculating Response Data 172 Standard Deviation of Linked Wells 172 Exporting Response Data 0c08 172 Displaying Integrated Intensity in Kilo UAS inenen nins 173 Creating Editing and Deleting ICW Templates nner 173 Creating Rep
61. 2 ew Mero ew ew Show Response Colors in ICW View Colors are changed by clicking the corresponding Modify button The response range associated with each color is changed by typing a new integer for the upper limit in the appropriate field The lower limit of the range is always the upper limit of the previous range Deselecting Show Response Colors in ICW View prevents percent response cells from being color coded Color Coded Relative Intensity Values If the relative intensity value for a given well is less than 3X the standard deviation of the background the data will be listed in the ICW Analysis window with a font that is bold and red Similarly data values are orange when relative intensity is less than 10X standard 171 172 CHAPTER 9 Odyssey In Cell Western Module deviation of the background These color coded valued could be displayed in either the 700 Relative or 800 Relative column depending on which channel is being used to calculate relative intensity These color coded values indicate the possibility of low quality data and the wells may need to be examined for errors misalignment of the grid etc Recalculating Response Data After viewing the response data changes may need to be made and the data recalculated The following changes require recalculation of the response data e Repositioning the grid on the image e Using the ICW parameters In Cell Western menu to chan
62. 44mm The measured values give a good starting point that can be refined by applying a grid to the image and adjusting the grid template as needed Applying Grids to Images Grids are always applied to both images in exactly the same location so it is best to have both images overlaid before applying the grid use in the toolbar After defining a grid template a grid can be applied to an image by clicking the grid tool Hf on the toolbar or choose Analyze gt Add Grid 129 In the Select Grid window select the grid template from the Grid Name drop down list and click OK to apply the grid to the image Select Grid To change the grid template before applying it click the Modify button in the Select Grid window and change the grid parameters as described above under Grid Parameters After clicking OK the grid is automatically placed on the image with the upper left corner at the X Y offset specified in the grid template Applying a Grid Automatically If you have the In Cell Western Module for Odyssey Software grids can be applied automatically Odyssey software finds the correct position as described in Chapter 9 Moving a Grid Manually If the grid is not placed in an optimal position start by clicking the grid to select it Next move the cursor inside the grid so the all arrows cursor is displayed With the all arrows cursor displayed click and drag the grid until the circle or rectangle features are in the be
63. 69 169 Quality medium medium medium medium Focus Offset 0 0 2 0 0 5 3 0 mm Channels 700 800 700 800 700 800 700 800 Intensity 5 0 8 0 5 0 5 0 Scan Origin 0 0 0 0 0 0 0 0 Scan Size 10 10 10 10 10 10 13 9 Note There are Presets both in the Odyssey Imager itself and in Odyssey Software The Presets in the Odyssey Imager are used when starting scans from the front panel or from an Internet browser Presets in Odyssey Software are used only in Odyssey Software Information on using modifying and saving Presets in the Odyssey Imager can be found in the Odyssey Operator s Manual 18 CHAPTER 2 Starting Scans MousePOD Presets Full Mouse Mouse Mouse Pod Center Left Right Scan Position Position Position Resolution 169 169 169 169 Quality medium medium medium medium Focus Offset 1 0 mm 1 0 mm 1 0 mm 1 0 mm Channels 700 800 700 800 700 800 700 800 Intensity L1 0 L2 0 L1 0 L2 0 L1 0 L2 0 L1 0 L2 0 Scan Origin 0 0 8 0 0 0 15 0 Scan Size 25 19 9 19 10 19 10 19 See Odyssey n vivo Imaging Guide for details on scanning with the Odyssey MousePOD Accessory 19 Editing Scan Parameters for Standard Scans All scan parameters are listed in the middle of the Scanner Console window Each parameter can be edited as described below Scanner Console BandSizing User ron Description Name 2007 05 08 154727 Group ron Preset Membrane _ Modify _ Scan Para
64. All Users Application Data Licor Logs 2 1 6 log For detals of the software update process Adding and Deleting Scanners The network address of any Odyssey instrument scanner must be added to Odyssey software before the instrument can be operated Odyssey instruments are generally added during installation but this section describes using the Scanner settings in the event of network changes or if the instrument is moved to a different network Each time Odyssey software starts any scanners on the same network as the computer are automatically detected and added to the list of available scanners If the scanner is on a different network than the computer and the networks are connected by a router Odyssey software will not automatically detect the scanner but the scanner can be added to the scanner list manually Chapter 4 of the Odyssey 243 Operator s Manual has complete details on network setup for the Odyssey instrument To add delete or edit a scanner on the scanner list choose Settings gt Scanners The Scanner List contains all Odyssey instruments that have been automatically detected or manually added to Odyssey software Scanner List Name Location LI COR Odyssey ODY 1426 ODY 1426 Next to 4300 ODY 1224 by brad and mark Auto Discover Adding Scanners via Auto Discovery When Settings gt Scanners is chosen any scanners detected when Odyssey software was started are shown in the scanner list
65. Analysis to open the New Analysis window Creating a new analysis makes copies of images from another analysis allowing the images can be analyzed separately from the original analysis A new analysis may need to be created for a variety of reasons For example if the original image had multiple membranes scanned for different users each user may want to independently analyze the portion of the image that is of interest to them If the original image is in the wrong orientation a new analysis can be created to flip or rotate the image Opening the New Analysis Window To start a new analysis open a project and click on the scan containing the images you want to copy into the new analysis Next choose File gt Analysis gt New Analysis to open the New Analysis window ii 40 CHAPTER 3 Creating A New Analysis New Analysis Name ThirdAnalysis Description Available Analyses Analysis Image My Analysis 1 fri i Flip SecondAnalysis fal A Rotate Subtract crop B Fiter ey x a Adjust Image Curve E Alter Image Display Naming the Analysis The Name field is used to name the new analysis In general it is best to use numbers letters underscore characters or dashes Do not use slashes colons or commas Entering a Description Any text entered in the Description field can be included in reports after analysis is complete Chapter 10 A list of the scan parameters and any alte
66. Analysis_800 Images C Documeni C Documents and Settings RON_WWALicor Odyssey Projects Tutorial Titer Scan Inten Undefined Undefined Scan Foct Undefined Scan Qual Undefined Scanner N lt Imported Image gt Odyssey Scan Desc BarCode Undefined Pixel Size 169 492 169 492 Well Shap C Rows 8 Colurnns 12 Feature N 1 Cell A01 Cell A02 Cel AD3 Cell AD4 Cell AD5 Cell A0G Cell AO Cell A08 Cell ADE Cell B01 Cell B02 Cell B03 Cell B04 Cell B05 Cell B06 Cell B07 Cell B08 Cell B0S v m4 4 Mh MyExcelGrid aks zi Ready See Troubleshooting Plug in Reports below if the program does not execute properly Editing Plug in Reports Though most parameters for plug in reports will not need to be edited plug in reports such as those for Microsoft Excel include a path to the application exe file that may require editing for new versions of Microsoft Office 199 To edit a plug in report choose Plug in gt Plug in Settings Plug In List Plug Ins Feature Data Report Microsoft Excel for Grids Microsoft Excel for ICV Microsoft Excel for Shapes ReagentsVVebLink In the Plug in list select the plug in report that needs to be edited and click Edit The Name field is the only field that does not directly influence the operation of the plug in Edit Plug In Name Aicrosoft Excell for Lanes Command Line 2s Microsoft Office OFFICE1 1 EXCEL EXE Type Report w Template liane pata v
67. B UH Rho VBR CLE BE RAAMEST WEES Ky S 7 A A na BA Scan Western Analysis First_Analysis_1 Images Firs C BandSizing 5 8 Western B First_Analysis i First_Analysis_1 Ox oF x Scans View Image View The Scans view is always open left and lists the projects scans and analyses for the current project Double clicking on an analysis opens an Image view right containing the images from the analysis The View menu can be used to display other file information in the main Odyssey window The Folders view shown below displays a directory tree similar to that found in Windows Explorer The purpose of the Folders view is to aid in finding and opening Projects which are displayed in the Scans view center The Thumbnails view shows a thumbnail sized image of the scan or analysis selected in the Scans view The Folders Scans and Thumbnails views are discussed below H Odyssey BandSizing File Edit View Analyze Lane In Cell Western Report Plug in Settings Window Help DSHS OF BUY ljo Vi RE CAREER EB Be ve we nA SA H e sh Folders x Scans Western Thumbnails X Scan Western Analys Desktop 4 Bandsizing y 5 My Computer 89 Western edi 3 Floppy 4 First_Analysis B S Local Disk C Analysis E Aerius a AlignIRv2 DELL 9 Documents Folders View Scans View First_Analysis Thumbnails View Image View Folders view and Thumb
68. CHAPTER 5 Creating Lanes and Finding Bands Re finding Bands If mistakes are made in band editing the Undo choice on the Edit menu can sequentially undo each edit At times however it may be easier to just start over To re find bands using Odyssey s automatic software select a lane or lanes and then choose Lane gt Refind Bands Use this function carefully All band data in the selected lanes are deleted before bands are found again Using the Lane Profile Window A fluorescence profile for a selected lane can be displayed by clicking amp on the toolbar or choosing Lane gt Lane Profile 2 Lane Profile View Lane 1 800 channel oR IKN A N F 0s Poa TIRE 0 14 at cursor position Band Finding Display Settings Profile Percent Band Boundaries Threshold F Z y Band Background C Lane Background C Lane Background Removed Fewer Bands vra 15 20 More Bands The title bar of the lane profile indicates which lane is profiled and the image on which the lane is located If lane profiles are opened with channels overlaid two profiles are opened one for the lane in the 700 channel and one for the lane in the 800 channel Similarly if more than one lane is selected profiles will be opened for all selected lanes All lane profile windows can be closed by choosing Window gt Close Lane Profiles 77 The lane profile can be used for the following tasks e To check band fi
69. ER THIS EULA IS OFFERED BY LI COR ALONE The SOFWARE PRODUCT is protected by copyright laws and international copyright treaties as well as other intellectual property laws and treaties The SOFTWARE PRODUCT is licensed not sold 1 GRANT AND SCOPE OF LICENSE This Agreement grants you the following limited rights 1 1 Applications Software You may install and use one copy of the SOFTWARE PRODUCT or any prior version for the same operating system on a single computer 1 2 Storage Network Use You may also store or install a copy of the SOFTWARE PRODUCT on a storage device such as a network server used only to install or run the SOFTWARE PRODUCT on your other computers over an internal network however you must acquire and dedicate a license for each separate computer on which the SOFTWARE PRODUCT is installed or run from the storage device A license for the SOFTWARE PRODUCT may not be shared or used concurrently on different computers 1 3 Multiple Users License If you have acquired this Agreement in accordance with a Multiple User s License from LI COR you may make the number of additional copies of the computer software portion of the SOFTWARE PRODUCT as authorized in writing by LI COR and you may use each copy in the manner specified above 2 DESCRIPTION OF OTHER RIGHTS AND LIMITATIONS Notwithstanding the license granted above Licensor retains all of its ownership and license rights in the Licensed Program and all Modifications a
70. Edit File Name Export Path ts and Settings RON LicoriOdyssey Reports Browse The arrangement of data in a report is determined by choosing the column on which to sort and whether the data should be sorted in ascending or descending order The Export section is used to output data for all cells in the table or to exclude cells that are marked as empty wells in the microplate When Include Header Information is selected information about the project scan and analysis is included in the beginning of the file If Include Header Information is selected the report will also include Z factor values if Z factor calculations are enabled The name for the export file can be entered using one of the two methods described below This file name will be the default file name listed in the Save File window when the report is exported e Enter a simple file name in the Export File Name field e Click the Edit File Name button to create a file name that includes text such as the analysis name that is added automatically when the file is created Names that are generated automatically save time when scanning multiple plates Specify ICW Export Filename Template x Type the name into the As Stored area using the Insert butto to invoke any selected special fields rset Jose IOA Data FromMy Analysis 2003 11 26 Exarnple In the file name above the text ICW Data From is followed by
71. Features window turned off all that is necessary is to add the initial feature click the Multiple Features tool and double click in the center of the last spot The latest configuration number of features names etc of the Multiple Features tool is always stored in memory for the next use Note If the Add Multiple Features window is turned off it can be turned back on by choosing Settings gt Application selecting General from the Settings List and deselecting the Don t show the Create Multi Features Dialog check box Automatically Adjusting Feature Locations When placing features circles squares polygons etc on an image it is not necessary to place them precisely because selected features can be moved to enclose nearby fluorescence by choosing Analyze gt Adjust Feature Location After selecting the features Adjust Feature Location can also be invoked by clicking on the toolbar or right clicking the image and choosing Adjust Feature Location from the pop up menu Note The Adjust Feature Location function moves features only It does not size them If the initial size is too small to enclose the fluorescence features will not be moved to optimal locations Tip If features are misplaced choose Edit gt Undo Note Even though Adjust Feature Location can be used with grids or features within grids it generally does not place features accurately due to the fluorescence of the plate walls Using the Adjust Loc
72. Features described in this chapter are disabled unless you have purchased and imported a key to unlock the In Cell Western Module It has been well documented that protein phosphorylation dephos phorylation by kinases and phosphotases is a critical process regulating almost every aspect of life Abnormal phosphorylation is a cause or consequence of major diseases like cancer diabetes and rheumatoid arthritis An In Cell Western assay has been developed to simultaneously detect both the phosphorylated protein and normalize for total protein relative number of cells in each well In Cell Westerns are highly reproducible sensitive and linear over a wide dynamic range Features distinguishing In Cell Westerns for analysis of signal transduction from other currently available technologies include 1 Near infrared probes with excitation emissions at 700nm and 800nm minimize interference from auto fluorescence of cells and plastic plates and from chemical compounds and other materials particularly potential drug candi dates 2 n situ detection to simplify sample handling and avoid the degradative effects of sample extraction 3 Simultaneous assessment of two targets enables quantitative and accurate measurement of phosphorylation of one target because of data normalization with the another target Furthermore the Odyssey In Cell Western software has been designed for the purposes of background subtraction data normalization and percentag
73. HAPTER 1 Introduction Selecting an Application Settings File at Start up When the Odyssey application starts a window is displayed that asks the user to choose the application settings file for the current session This makes it easy for users to have their own settings file that deter mines important parameters such as background calculation method Until you understand the Odyssey application it is best to choose the default application settings stored in Odyssey_Settings as shown below The active settings file can be changed at any time by choosing Settings gt Select Active Settings Chapter 12 describes adding deleting and changing the active application settings file Set Active Application Settings x Available Application Settings Odyssey Settings C Don t show this dialog at start up Setting Up Users and Scanners Before you can do anything in Odyssey software you must have your own user account The Settings menu is used to to add user accounts Chapter 12 describes this procedure The Odyssey instrument network address must also be added Settings gt Scanners to Odyssey software before the instrument can be operated Odyssey instruments scanners are generally added during installation but Chapter 12 describes this function should you need to perform it The Odyssey Help System The Odyssey help system can be invoked by choosing Help gt Contents or by pressing F1 on the keyboard amp odyss
74. Integrated Intensity has all of the properties of Integrated Intensity except it varies with resolution When pixel size is small compared to feature size the values of large pixels will be similar to that of small pixels over the same area but the number of small pixels will greatly exceed the number of large pixels so the sums will also vary Raw Integrated Intensity is not multiplied by pixel size to correct for this Therefore it makes comparisons between experi ments where resolution could differ more subject to misinterpre tation For this reason Raw Integrated Intensity should not be used to quantify image spots Integrated Intensity Integrated intensity is defined as Integrated Intensity a 3I Pixels b where area per pixel a resolution x 10 and Pixels is the number of pixels enclosed by the feature Units are counts mm integrated pixel volume 2 When background is uniform integrated intensity is independent of both the size of feature drawn on the image and the resolution Integrated intensity is proportional to the amount of dye labeled antibodies on the membrane and therefore can be accurately used for quantification 251 Average Intensity When background is zero average intensity is raw integrated intensity divided by the pixel count Raw Integrated Intensity Average Intensity Poel Count ixel Coun Since average intensity is dependent on feature area it should not be used to quantify ima
75. Name TiterScan Analysis Name Grid Analysis OK Cancel Note The Save Analysis and Save As New Analysis menu choices can also be accessed by right clicking an analysis in the project directory tree and choosing the desired menu choice from the contextual popup menu Deleting an Analysis Caution Use this function very carefully The analysis is deleted immediately when this function is performed and the deleted analysis cannot be recovered To delete an analysis click the analysis in the Scans view in the Odyssey window and choose File gt Analysis gt Delete Analysis 47 Having More Than One Analysis Open More than one analysis can be open at once allowing images to be compared When multiple image views are open the Window menu can be used to switch between windows The window name includes both the scan name and analysis name Clicking the analysis name in the Scans view will also bring that analysis window to the front Chapter 4 Importing and Exporting Scans and Images There are four methods to get scan images into a project in Odyssey software e Start a Scan Images are automatically transferred to the computer when the scan is completed Download a Scan Scans started using one button scanning on the Odyssey Imager can be retrieved from the instrument using the Download Scans function e Import a Scan Scans stored in any Odyssey project can be imported into the current project e Import Ima
76. PixelLocn 38 0 Standards Settings Lane Lane 1 Channel 700 Units basepairs Interpolation method Linear v Selecting the correct interpolation method is important because it affects the size assigned to sample bands Linear Log Reciprocal Fit 108 CHAPTER 6 Band Sizing and Exponential interpolation methods can be chosen from the Interpolation Method list In general the Reciprocal Fit interpolation method usually produces the best results There are exceptions however If the plot of the standards is very linear the Linear interpolation may yield slightly better results If the image has sample bands with a size that is higher or lower than the standards use the Exponential interpolation method Any time there is a need to extrapolate beyond the size standards the Exponential interpolation method usually produces the best results The interpolation applies to all lanes on the image Setting Units for Standards Like the interpolation method the Units apply to all lanes and should be set before examining the plot of the standards for each lane Basepairs kilobasepairs daltons and kilodaltons can be selected for units Reviewing the Standards Plot for Each Lane The Lane drop down list in the Size Standard plot window is used to select the lane to plot Start with the lanes containing MW standards Look for anomalous bands For example if the smooth curve in the plot below is broken by a standard
77. Removal filter typically removes background speckles from an image Noise removal calculates a median pixel value within the 3x3 filter region and replaces the current pixel value with the median Noise removal is most noticeable where the median replaces pixel values that are much brighter or darker For band sizing on noisy images noise removal can improve band finding accuracy because the lane profile will be smoother and have fewer noise peaks that might be identified as bands Smooth The Smooth filter changes the current pixel value to the average of the pixels within the 3x3 filter region 45 Local Maximum and Minimum The Local Maximum filter replaces the current pixel value with the maximum pixel value within the 3x3 filter region Similarly Local Minimum replaces the current pixel value with the minimum pixel value within the filter region The Local Maximum filter reduces noise on images where there are background pixels that are much darker than surrounding pixels The Local Minimum filter reduces noise on pixels where the background is fairly uniform except for some pixels that are much brighter than others Sharpen The Sharpen filter multiplies each pixel in the region by sharpening coefficients and then sums the pixel values to create a new pixel value that replaces the current pixel value The sharpening coefficients have been chosen so the edges of objects are enhanced Changing Brightness and Contrast When the New An
78. Report Application Settings Report Settings The report settings are opened by choosing Settings gt Application and selecting Report from the Settings List Application Settings Odyssey Settings Settings List Export Options General Report File Name Report txt Edit Name Image View Features Image View Display Report Path Settings My Documents Licor Odyssey Reports Display Values E m las Fi 5 H FOwSe Adjust Locations Include Field Names in Report Auto Shape Naming Conventions Fjeld Separator Tb M Print Options Report Title MyReportTitle Edit Title The Export Options are all related to creation of data files The Report File Name can be entered directly in the Report File Name field ora name can be created by clicking Edit Name Names created using Edit Names can contain placeholders for text such as project name scan name etc The file name is assembled automatically when the report is exported Specify Report File Name A Type the name into the As Stored area using the Insert button to invoke any selected special fields s stored Project Scan txt Insert Sean Example Tutorial MyScan txt In the title above two auto entered text blocks project name and scan name are followed by a file name extension txt Auto entered text blocks can be separated by commas spaces or other text as long as the resulting file name remains a l
79. Scanner Utilities Scanner Information r Scanner Diagnostics Update Scanner Software Managing Users Click Manage Users in the System Administration settings window to make changes to any user account Manage Users Available Users Change Password Edit Rights 236 CHAPTER 12 User Accounts and Settings Adding Users Click Add in the Manage Users window Add User User Name ron New Password HAART Reenter New Password aaaaee nal User Rights control Enter the name and password for the new user and repeat the password Select the access rights according to the definitions given above Click OK to add the user When a user is created a scan group in the Odyssey instrument is also created The new scan group has the same name as the user name Deleting Users To delete a user select the user from the Available User list Manage Users window and click Delete Note however that this only deletes the user account Any data must be deleted by deleting the scan group using the Manage Scan Groups button in the System Administration window Changing Passwords Passwords for any user can be changed by selecting the user from the Available User list in the Manage Users window and clicking Change Password Enter the new password and then verify the password by entering it again and click OK Editing Access Rights Access rights for any user can be ch
80. Select MV Set et Apply Std Set X Edit Mode O Add Points Modify Lines O Modify Points O Select Lanes Snap to Lane MW lines for all standards in the set are added to the image at once when Apply Std Set is clicked The next step is to identify which lanes contain standard bands and then automatically match the MW lines to the bands in the standards lanes To select the lanes containing standards click Select Lanes to set the Edit Mode to Select Lanes Edit Size Stds for S800 TIF Add MWN Line or Select MVV Set New MW Value Add MW Line Select MVV Std Set 100bp DNA Ladder he Apply Std Set m Edit Mode O Add Points O Modify Lines Omano QE sene Apply to Both l Apply Cancel Select all lanes containing size standards After selecting the first lane hold down the Ctrl key and click additional size standard lanes to select them TIP Lanes are selected by clicking the centerline but the centerline is often covered by band markers Clicking near the top or bottom is usually easier When the lane is selected the dashed centerline turns to the highlight color 100 CHAPTER 6 Band Sizing When all the size standard lanes are selected click Snap To Lanes The MW lines are snapped to the bands in the standards lanes A control point square is added to the center of each MW marker band and the MW line is drawn between the control points Note In samp
81. TER 4 Importing and Exporting Scans and Images A project must be open before importing images Images can be imported into an existing project or a new project can be created To import images choose File gt Scan gt Import Images Import Images Scan Name MyScan Analysis Name FirstAnalysis Channel Images 800 Browse Cancel The Scan Name and Analysis Name fields are used to name the new scan and analysis for the imported images Images can only be imported into a new scan The Browse buttons next to the 700 and 800 channel image fields are used to browse for the image to import After selecting one of the images the image from the other channel is automatically found as long as the two image files are in the same directory If the two image files are in different directories click the button next to the other channel to browse for the second image After clicking OK to import the images the new scan and analysis are displayed in the directory tree of the Scans view Chapter 1 in the main Odyssey window Importing Images From Other Imaging Systems The Import Images function is designed primarily for LI COR TIFF images created by the Odyssey Imager However some 16 bit grayscale TIFF images from other imaging systems can also be imported 8 bit grayscale TIFF images from common image editing programs cannot be imported 55 Exporting Images Exporting an Image Vie
82. The Odyssey User Guide and this help system contain more in depth information about each software function The Help window has two frames The left frame contains naviga tional links Click a topic to display content for the topic in the right frame Folders in the navigational frame contain additional topics and are opened by clicking their plus symbol To search for something specific click the search tab magnifying glass icon and enter the search text The help system contains most of the information found in the Tutorial Manual and this User Guide However the information is organized in a more task oriented way that should help if you forget how to do something 4 CHAPTER 1 Introduction Toolbars The Odyssey user interface makes extensive use of toolbars to provide single click access to most functions The function of each tool is given in a tool tip that can be displayed by stopping the cursor over the tool on the toolbar A description of each tool can also be found in the online help system DEH OF RB UH R i via RS YX Ee ARAM Se ele Ses Most tools on the toolbar corre sponds to a menu choice on the menu bar that does the same function The examples throughout this manual use both the toolbar and menu functions If the toolbars get in your way you can easily hide them To hide a toolbar choose View gt Toolbars and deselect the toolbar you want to hide Context Sensitive Menus T
83. Thumbnails view is opened or hidden by choosing View gt Thumb nails View A close button in the upper right corner hides the view One composite two channel thumbnail image is shown for each analysis in the scan folder Double clicking a thumbnail image opens the corresponding analysis Initially if a scan or analysis is not selected in the scan list the message No Thumbnail Defined is displayed All the thumbnails are created as soon as a scan or analysis is selected The thumbnails are real files that are saved as JPEG files in the scan folder The file name convention is ScanName_AnalysisName_tn jpg If changes are made to the image such as cropping or rotating these changes will not be updated in the thumbnail until the analysis is saved Chapter 2 Starting Scans How fo Start Scans Scans on the Odyssey Imager can be started using the Windows based Odyssey Software an Internet browser or from the front panel of the Odyssey Imager Chapter 6 of the Odyssey Operator s Manual discusses starting scans using an Internet browser Front panel operation is described in Chapter 7 of the Odyssey Tutorial Manual and Chapter 3 of the Odyssey Operator s Manual The remainder of this chapter is dedicated to starting both standard scans and multiple microplate scans with Odyssey software Scanning mice with the MousePOD Accessory is discussed in the Odyssey n vivo Imaging Guide included with the MousePOD Before a scan can be
84. ackground The background feature is labeled background to distinguish it from other features To use more than one feature as background select Ctrl click all background features before opening the Background Methods window Note For some applications such as in vivo imaging background features are added to each image individually rather than with channels overlaid The background in the User Defined method is calculated as the average intensity of the pixels enclosed by the feature that is selected To find out what the calculated background value is open the Background settings again The calculated background value is shown next to the User Defined radio button If the scan is a two channel scan a value is shown for each channel channel 700 followed by channel 800 The values are also shown in Details View Choosing Between Average Median or User Defined The blue rectangle in Details View should be observed when determining which background method to use If the pixels just outside the blue rectangle are dark background pixels then either the Average method or the Median method with All boundaries will produce good results If there are bands that are vertically close together with fluorescence in between them it is best not to use the top and bottom segments in the background calculation Median with Right Left segments will produce good results Similarly when bands or dots are horizontally close together with fluorescen
85. age Scans Select the scan from the list of scans and click Delete 237 238 CHAPTER 12 User Accounts and Settings Backup To quickly backup all the files in a given scan group from the Odyssey instrument to the computer click Backup in the Modify Scan Groups window In the Backup Scan Group window enter a destination folder or click Browse to open a standard file selection window To move the scans to the destination folder rather than copying them select the check box labeled Remove Scan Group Images After Transfer To Destination Folder To overwrite scans that are already in the destination folder select the check box labeled Overwrite Images In The Destination Folder After configuring the backup click OK to transfer the files Scanner Information Click Scanner Information in the System Administration settings to display the Scanner Information window The items displayed include the scanner name serial number and software version number of the instrument software The minimum and maximum focus depth are the smallest and largest values that will be accepted for the focus offset parameter when starting a scan Scanner Information Scanner Tech Support Serial Number ODY 0109 Software Yersion 2 0 13 Min Focus Depth 0 0 mm Max Focus Depth 4 01235 mm Close Scanner Diagnostics The Odyssey instrument has a built in procedure to diagnose hardware problems that could result in faulty operation If you are exp
86. al MW on the gel The placement and shape of the line is not important yet After the first line is added add the rest of the MW lines by entering their MW and click Add MW Line as described above After all the size lines have been added the lines need to be linked to specific MW bands in standards lanes First the lanes with 103 standards need to be selected Click the Select Lanes radio button to set the Edit Mode to Select Lanes Edit Size Stds for 800 TIF Add MW Line or Select MVV Set Select MV Std Set Select a Std Set v Apply Std Set Edit Mode O Add Points O Modify Lines O Modify Points qE Snap to Lane Apply to Both Apply Cancel With the edit mode set to Select Lanes the centerline of a lane can be selected by clicking it To select additional standards lanes hold down the Ctrl key and click the centerline of the lane All lanes with standards should be selected and there should be no sample lanes selected If a lane is difficult to select because the center line is covered with band markers click near the top or bottom of the lane a ee Se ern G 4 ee a aa o s a Caa ee a i i Sd de G 4 4 1 1 1 i f 1 1 1 1 i b 104 CHAPTER 6 Band Sizing Click the Snap To Lanes button in the Edit Size Stds window to link the MW lines to bands in the standards lanes a 5 Edit Size Stds for 800 TIF Add MVV Line or Select MVV Set
87. alysis Analyses In the project named Bandsizing above there is one scan named Western and two analyses named First_Analysis and MyAnalysis The project above has only one scan but for projects with many scans it may be useful to sort the scans Right clicking the project name opens a context menu with choices for sorting scans by name or date using either ascending or descending order The default sort order can be set by choosing Settings gt Application and selecting General from the Settings List To view all the analyses associated with a scan click the plus icon 4 to expand the scan folder To open an analysis double click it in the scans list The first analysis in a scan folder can be opened by double clicking the scan folder An analysis can also be opened using Thumbnails view as described below A scan or analysis can be deleted by selecting it in the Scans view and pressing Delete on the keyboard An analysis can also be deleted by right clicking the analysis and choosing Delete Scan from the popup menu Additionally the operating system can be used to delete files in the normal way If Odyssey software is open when files are deleted using the operating system the Scans view will not immediately show the files have been deleted The refresh button in the upper right corner of Scans view can be used to refresh the scan list and show any changes Thumbnails View
88. alysis window is opened the image thumbnail is shown using the last used brightness and contrast settings For repet itive scans the last used settings may work well For other scans brightness and contrast may need adjustment in order to see any fluorescence in the image If fluorescence cannot be seen changing brightness or contrast may be necessary to perform other image manipulations like cropping To change the brightness and contrast or to view only one of the two images click either Adjust Image Curve or Alter Image Display The operation of these two functions is described in Chapter 11 46 CHAPTER 3 Creating A New Analysis Saving an Analysis Any of the following methods can be used to save analysis files Choose File gt Save or Ctrl s is used to save a project All analyses with unsaved changes will be saved Similarly File gt Save As saves all files and any unsaved changes from the current project to a new project folder Choose File gt Analysis gt Save Analysis to save only the current analysis All other analyses with changes will remain unsaved Choose File gt Analysis gt Save As New Analysis to save the current analysis as a new analysis Rename the analysis using the Scan Name field if the new analysis will be saved in the current scan To save the analysis in a new scan change the Scan Name an analysis cannot be saved to an existing scan other than the current scan Save As Scan Analysis Scan
89. an parameters are the same as those described earlier for standard 33 34 CHAPTER 2 Starting Scans scans with a few exceptions First the scan dimensions are not editable since a standard plate size is assumed If non standard plates are used the scan size can be changed in the Multiple Scan settings Second set Focus Offset to 3 0 for standard microplates as recom mended in the Odyssey Operator s Manual The last difference is that the Scan Multiple Plates function automatically flips images to the correct orientation so the image has well A1 in the upper left corner Presets for microplates can be created as described earlier in this chapter but note that the scan size and origin are not used by the multiple scan software 8 Set the number of plates to scan If less than six plates are scanned click the plate icons corresponding to empty locations in six through one order Deselected plates appear as shown below Plate Six Not Scanned Plates should be added to the Odyssey scanning surface in the order 1 through 6 shown in the multi plate scan setup window Consult the Operator s Manual for more details on placement of the scanning guide and plates 9 Click Start to send the scan parameters to the Odyssey instrument and start the first scan The image from the first microplate is displayed in real time in the Scanner Console window The name extension in the Name field indicates which plate is being scanned 1 2
90. and the microplate should be approxi mately centered in the scan area For some images such as those with weak fluorescence grid alignment may fail in which case the grid is placed according to the X and Y offset specified in the grid template 163 On some images the grid may be placed correctly but the circle or square features are too large or too small This is possible because features are sized according to the size specified in the grid template To get a better fit delete the grid change the size parameters in the grid template and place a new grid Images with a lot of background surrounding the microplate wells and images in which the micro plate is not centered are also likely to cause problems with automatic grid finding Automatic Calculations When a grid is applied integrated intensity is calculated for each well see Chapter 13 After integrated intensity is calculated percent response is calculated for sample wells using the ICW template that was most recently used Results can be viewed by choosing In cell Western gt View ICW Analysis If the current microplate is setup differently than the most recently used ICW template background wells etc the ICW parameters can be changed and applied as described below Changing ICW Parameters for the Current Analysis Choose In Cell Western gt Change ICW Parameters to change the parameters used in the ICW Calculations The Change ICW Param eters window is used to
91. anged by selecting the user in the Manage Users window and clicking Edit Rights Click the down arrow button in the Permission column next to the appropriate user to drop down a list of access rights Choose the access rights and click Close Managing Scan Groups To add or make changes to scan groups in the Odyssey instrument click Manage Scan Groups in the System Administration window Modify Scan Groups Available Scan Groups admin public Delete ron Manage Scans Edit Permissions Backup Adding Scan Groups Click Add in the Modify Scan Groups window enter a name for the group and click OK Editing Access Permission to a Scan Group Select the scan group to edit and click Edit Permissions Click the button in the Permission column across from the user name whose access permission you want to change Select a new permission level and click Close Select None to prevent a user from accessing the scan group Select Access to give a user the ability to download and analyze scans Select Control to give a user the ability to start new scans that will be stored in the scan group as well as all the rights that come with Access permission Deleting Scan Groups Select a scan group from the Available Scan Groups list and click Delete The scan group and any scans stored in it are deleted Deleting Scans Select the scan group containing the scan to be deleted from the Available Scan Groups list and click Man
92. annel The integrated intensity for each well in the sample channel is divided by the relative intensity value from the corresponding well in the channel used to calculate relative intensity The wells designated positive and negative controls are now ready for the Z factor calculations Z factor is calculated according to the following equation 7 Vea Pel 30 30 1 30 4 30 Patel Mor Pe l Where 30 4 3 standard deviations of positive controls 30 3 standard deviations of negative controls Pe is the mean of the positive controls maximum signal Pe is the mean of the negative controls minimum signal and Vea Pe is the assay dynamic range Vet e 304 30 is the separation band between posi tive and negative control signals 258 CHAPTER 13 Calculation Descriptions These variables are represented in the illustration below Hc Data Variability Band T E Separation Band A Hc Data Variability Band v 0 96 Sample Dimensionless Z factor values are always 1 or less as described below m Z 1 isan ideal assay As standard deviations become very small or the difference between signals for positive and negative controls approaches infinity Z factor approaches 1 m 1 gt Z 0 5 indicates a high quality assay exhibiting a wide separation between signals for positive and negative controls and low data variability m 0 5 gt Z gt 0 may indicate
93. another analysis and choose Edit gt Paste to paste the grid into the second analysis IMPORTANT Any existing grid in an analysis will be replaced when a new grid is pasted Grids can also be saved for use in future analyses by selecting the grid and choosing Analyze gt Save Grid as Template The new grid template can then be applied using the grid tool like any other grid template Note however that templates saved in this fashion cannot be edited 135 Using Subgrids In the image to the right there is one main grid 2 rows x 1 column DOUODUSBUOBUU and each cell in the main grid contains a subgrid 10 rows x 10 columns A strategy for creating subgrids is outlined below ee Designing a Subgrid A subgrid cannot be resized after it is included in a main grid so the first task is to create the subgrid and adjust its size 136 CHAPTER 7 Drawing Features on Images 1 2 3 4 5 6 7 Click and drag a selection rectangle as shown below to measure the grid spacing The width and height of the rectangle will be listed in the status message at the bottom of the Odyssey window Example for measuring grid spacing X Measure the size of a well spot by dragging a selection rectangle that encloses the fluorescence as shown below k Choose Settings gt Grid Template and click New in the Grid Templates Window to create a new template for the subgrid Enter the number of rows and columns i
94. are processed for a given area on the membrane in order to make one pixel on the image For typical scans Medium is recom mended but there are five settings Choosing Highest quality will reduce noise in the image data but significantly increase scanning time due to the slower scanning speed Similarly choosing Lowest will decrease scan time but increase noise in the image data For high resolution scans where samples have very little fluorescence High or Highest may be a better choice than Medium When Quality is set too low the image may become noisy or grainy particularly in the background Focus Offset should always be zero when scanning membranes For gels set Focus Offset to half the gel thickness in millimeters For microplates recommended by LI COR Operator s Manual Chapter 3 focus offset is 3 mm The maximum possible focus offset is 4 mm Select Microplate flip image when scanning single microplates When selected images are flipped automatically after each scan so the origin well A1 of the plate is in the upper left corner Microplate images must be flipped because the plate is scanned through the bottom Deselect Microplate flip image when scanning membranes gels or mice The Channels check boxes is used to specify whether to detect fluorescence in the 700 channel the 800 channel or both When both are selected fluorescence from each dye is detected separately and stored in a separate image file 21
95. arty shall deliver to the other Confidential Information that is in its possession 2 9 Irreparable Harm The parties agree that breach of the above obligations shall be deemed to cause irreparable harm 3 UPGRADES ENHANCEMENTS If the SOFTWARE PRODUCT is labeled as an upgrade you must be properly licensed to use a product identified by LI COR as being eligible for the upgrade in order to use the SOFTWARE PRODUCT A SOFTWARE PRODUCT labeled as an upgrade replaces and or supplements the product that formed the basis for your eligibility for the upgrade You may use the resulting upgraded product only in accordance with the terms of this Agreement If the SOFTWARE PRODUCT is an upgrade component of a package of software programs that you licensed as a single product the SOFTWARE PRODUCT may be used and transferred only as part of that single product package and may not be separated for use on more than one computer 4 COPYRIGHT All title and copyrights in and to the SOFTWARE PRODUCT including but not limited to any images photographs animations video audio and text incorporated into the SOFTWARE PRODUCT are owned by LI COR or its suppliers The SOFTWARE PRODUCT is protected by copyright laws and international treaty provisions Therefore you must treat the SOFTWARE PRODUCT like any other copyrighted material except that you may install the SOFTWARE PRODUCT on a single computer provided you keep the original solely for backup or archival purp
96. at all file types are being displayed in the file selection window Click Update An information window is displayed that explains what the update procedure is going to do Update Scanner Software You have requested to update the scanner s embedded software Q from Version 2 1 1 to Version 2 1 6 You must be logged into the scanner with a user account that has admin rights to perform this operation Please note that the update will replace the current scanner software and will take a few minutes to complete Are you sure that you want to update scanner software 5 Click Yes to start the software installation 242 CHAPTER 12 User Accounts and Settings There will be several minutes of apparent inactivity while the new software is being uploaded When the update is complete a report is displayed that indicates the success of the update procedure If the update was successful the word success is displayed next to each update component as shown below Contact LI COR Technical Support if any of the update components fail Technical Support may request a copy of the update log The path to the update log file a text file is displayed at the bottom of the update report window Software Update Success Q The software update completed with the following status VERSION 2 1 6 STOPPING SUCCESS COMPONENTS SUCCESS MODULES SUCCESS SERVER SUCCESS DATABASE SUCCESS STARTING SUCCESS Refer to C Documents and Settings
97. ated in the current project and the TIFF image files are copied to the scan folder An analysis with the specified name is also created in the scan folder Both the scan and analysis are shown in the project directory in the main Odyssey window Occasionally it may be necessary to manipulate the images before creating a new analysis Click Advanced rather than OK in the Save As Scan dialog to manipulate the images before saving them 29 When Advanced is clicked the New Analysis window is opened New Analysis Name Original Analysis Description Analysis Image Any image manipulation operations Subtract performed within the New Analysis dialog are applied to the acquired image when the OK button is pressed The original image data from the scan is replaced by the altered data Adjust image Curve FE Ater image Display Using the New Analysis window a scan can be finished using one of two methods 1 Enter an analysis name and click OK to save unaltered original images in a new analysis To start analyzing the images create another new analysis Chapter 4 using copies of these original images 2 Name the analysis and prepare the images for sizing or quantifi cation using the buttons for flipping cropping rotating background subtraction or brightness and contrast adjustments See Chapter 3 Then click OK to create the new analysis and proceed with sizing or quantification The first method uses
98. ated intensity s csciese cis cnr teenies intensity scan parameter ranges and recommendations 0006 22 ACJUSUING eisein rni intensity adjustment CUIVES s s sssssssssisissseseene 213 263 264 INDEX interpolation method size standards 108 149 L lane background method cseseeeeeees 158 lane menu add lahenema 66 add multiple lanes 72 edit size standards cccccccccesseeeeseee 98 101 lane profile Multi lane profilene sasn 82 placemulti anessen 86 sample normalization cece eee 84 save lanes as template c ccceeeeeeeees 85 SIZEeStAN GALS so ces deasassansvaseuesetseutsenvenind seve 107 lane profiles lane profile WINdOW wo cess ceeeeteeeeeeees 76 multi lane profiles 82 profile widthisetting sssisssiisissii irisse 87 lanes application settings COlOE eves xsssseinvesesvestearses copying and pasting creating straight lanes ccceccceeseeeeeees 66 creating Curved lanes s sssistisiisisiisisrsssss 67 creating multiple lanes ssassn 72 deleting sssri seis seresiaieiserestassneatnendesdegd 70 displaying quantification as a percentage 89 display migration settings ccccceeee 88 editing size standards eissira 98 MOVING ss snsiassstcencedhsneasteresasenencderedssssaussvveodes 68 normalization 83 profile width 87 reshaping 09 NOSIZING iese e E EN 69 sele tNE icss carsiscisiccssssssects vane vindsedessnuysstsints 99 sele
99. ately shown on the image s in the Image View window The Show and Only Show Current Channel check boxes can be used to determine whether one or both image channels are displayed The color of the current image can be changed using the color drop down menu the colors in the Application settings for new analyses are not changed Pseudo Color is available as a color if you have purchase a key to unlock the Odyssey MousePOD module see Odyssey In vivo Imaging Guide The Invert Grayscale check box inverts the color map whites become black and blacks become white for grayscale and pseudo color images Using the Intensity Adjustment Curve The intensity adjustment curve graphically illustrates how grayscale values in the image file are mapped to the display As indicated below the X axis is the original or input intensity value from the TIFF image file The Y axis is the grayscale value that is output to the display For both axes dark pixels lowest intensity are located near the origin The intensity adjustment curve is used to calculate the output intensity for a given input intensity 215 For purposes of illustration a linear intensity adjustment curve is shown in figures A and B below A B A A F S E E 4 g g Input Intensity Input Intensity Note that as the slope of the curve increases B the output intensity of a given pixel increases The adjustment curve shown in the Adjust Image Curves windo
100. ation Settings The locations of selected features on the image are moved based on a localized search for maximum signal The Adjust Locations settings are used to refine the search criteria and improve spot finding To open the Adjust Location settings choose Settings gt Application and select Adjust Location from the Settings List 119 120 CHAPTER 7 Drawing Features on Images Application Settings Odyssey Settings Settings List Spot Finding General Minimum Finding Threshold Image View Features Image View Display 9 nl Display Values f bine Low Intensity High Intensity Adjust Locations Search Area Extension Auto Shape Naming Conventions g Report FeO A A N Serie 9 a ra E A een Small Large V Display interactive messages dialog boxes Cell Grid Automatic Image Alignment K Reset cell locations if not Found Move all cells together within the grid Number of Features Required 4 w Minimum Alignment Threshold Low Intensity High Intensity Restore Defaults Improving Spot Finding The Adjust Location settings determine the search area how far from the original location to search and threshold intensity for spot finding The Minimum Finding Threshold slider sets the threshold intensity for spot finding Since images vary in background and fluorescent intensity this setting must be determined empirically for your images Spurious
101. ation settings General to change the display units Creating Editing and Deleting ICW Templates ICW templates can be created edited or deleted by choosing Settings gt ICW Setup In Cell Western Analysis Templates x ICV Template Name Rows Columns Controls Backgrou Links 24 16 none The In Cell Western Analysis Templates window lists all current templates and a few of their distinguishing parameters To edit or delete a template select the template in the list and click the Edit or Delete button respectively To copy a template select the template 174 CHAPTER 9 Odyssey In Cell Western Module click Edit change the template as needed and click Save As to save the template under a different name To create a new template click the New button ICW Analysis Setup Template Template Name Untitled Grid Size 12 columns x 8 rows Modify Well Types Well Links Calculations 01 02 03 04 05 06 07 08 og 10 11 12 I 1 1 l The Setup Template window is nearly identical to the Change ICW Parameters window described earlier in this chapter except for some differences in the buttons at the top and bottom of the window The first thing to do in the Setup Template window is to check the grid size Click the Modify button and choose the microplate that will be scanned 96 384 and 1536 well plates are supported however 1536
102. b To change well designations begin by selecting the type of well to mark Sample 100 Standard Background or Not Used B sample C 100 standard C B Background c NotUsed Move the cursor over a well on the well assignment grid and click to assign the selected well type to that well Multiple well assignments can be made at once by clicking and dragging through a range of wells When the mouse button is released all wells within the selection rectangle change to the color assigned to the chosen well type In some cases it may be easier to mark sample wells last since there are usually more sample wells than other types of wells If Sample is selected clicking Assign Remaining designates all wells that are Not Used as sample wells To change all wells to Not Used click Clear All Rows If more than one Background well is designated all Background wells will be averaged for the ICW calculations unless individual rows are being analyzed as described below The same is true for 100 Standard wells Well Links Tab If two or more rows are exact duplicates of each other the samples can be linked in the calculations to get an average response of all the duplicate samples Suppose rows A and B are identically loaded and you want to average the response of similar samples in the two rows After designating the well types switch to the Well Links tab in the Change ICW Parameters window Next select all the wells in rows A an
103. background method 005 153 undo toolbar button or edit menu ceeeeeeeeeee 5 undo settings V view menu adjust image curves alter image display x channels overlaid csssseesesssseeseees Single channe lernis tOO Dais inene ea 4 thumbnails view folders View 000000 7 Z Z factor calculations descriptio Masinio nrinn ieii 256 enabling overview viewing Z factor values s 180 ZOOM DY drap ping nieostre teenie thats 219 zooming in out ON an IMage s s s 219 267
104. been designated If there is more than one Background or 100 Standard well in a row they are averaged before being used in the calculations but they are not averaged with any Background or 100 Standard wells outside the row When individual rows are analyzed wells between rows cannot be linked so any links designated on the Well Links tab are ignored When analyzing individual columns the same concept applies Background and 100 Standard wells must be designated in each column to be analyzed and columns cannot be linked Applying the Changes Click Apply in the Change ICW Parameters window to apply any parameter changes to the current analysis 169 170 CHAPTER 9 Odyssey In Cell Western Module Examining the ICW Response Data ICW response data can be viewed by choosing In Cell Western gt View ICW Analysis BY In Cell Western Analysis Scan4 Analysis1 Cells In Table Selected Column Headers O Show All Show Only Used O Sort Ascending Sort Descending Colored values in the Relative columns indicate that this data is less than 3X red and less than 10X orange std dev of background data 700 Integ Int 800 Integ Int Well Type 700 Relative 800 Relative 7115 25 6319 68 Backgnd 1901 25 0 0 3784 11 16865 87 Backgnd D 242 0 0 15195771 405663 19 Sample J J 0 0 178252 47 419372 98 Sample 179485 12 495595 57 Sample 628329 82 Sample 59246632 Sample 701638 65 Sample 725848 10 Sample 777615
105. calculations are performed as described above when a whole microplate is analyzed If the Individual Row Analyses or Individual Column Analyses check boxes are checked on the Calculations tab of the ICW Parameters window Response is calculated using only the wells within a given row or column For example when Individual Row Analyses is checked each row analyzed must have at least one Background well and 100 Standard well Response for sample wells in the row is calculated using the Background and 255 256 CHAPTER 13 Calculation Descriptions 100 Standard well s in the same row as the sample wells An error message will be displayed if Background and 100 Standard wells have not been designated If there is more than one Background or 100 Standard well in a row they are averaged before being used in the calculations but they are not averaged with any Background or 100 Standard wells outside the row When individual rows are analyzed wells between rows cannot be linked so any links desig nated on the Well Links tab are ignored When analyzing individual columns the same concept applies Background and 100 Standard wells must be designated in each column to be analyzed and columns cannot be linked Z Factor Calculations Before an assay can be used with various test compounds the assay should be optimized for reagents protocols instrumentation kinetics and other conditions not related to the test compounds Calculating
106. can Previewing a Scan Optional Click Preview optional in the Scanner Console window to scan a low resolution preview before starting high resolution scanning A preview scan is a low resolution scan at the lowest quality setting that takes only a few minutes to complete depending on scan area A preview scan can be used to check fluorescent signal intensity or to adjust the scan area before high resolution scanning Adjusting the scan area see Setting Scanner Parameters for Standard Scans below can shorten scan times by reducing the amount of empty background that is scanned 15 16 CHAPTER 2 Starting Scans Selecting a Scan Group A scan group is a special directory on the Odyssey instrument that has restricted access Initially users have access to the Public scan group and a scan group that matches their user name Additional scan groups can be created for special purposes For example if several people are doing scans for a particular research project it might be useful to keep all scans for that project in one scan group The Group drop down list is used to select the scan group in which the new scan will be stored Scanner Console BandSizing User ron Scan Description Name Group ron v Modify Preset Membrane a wv _ Modify Scan groups are added and deleted by clicking Modify next to Group See Chapter 12 for complete information on scan groups Setting Scanner Parameter
107. ce in between the Median method with Top Bottom will produce good results as long as the top and bottom line segments are over empty background In cases where there is a lot of fluorescence from other bands dots neither the Right Left or Top Bottom lines may be usable In this case use the User Defined method as described above 158 CHAPTER 8 Quantification Using the Lane Background Method for Bands The Use Lane Background Method For Bands check box is used when band markers within lanes are selected for quantification When Use Lane Background Method For Bands is selected the band background is calculated using the lane background profile see Chapter 6 for information on lane profiles The band background is calculated as the average intensity of the two points where the band boundary intersects the lane background profile Chapter 5 The lane background profile can be displayed by selecting lanes and choosing Lane gt Lane Profile to open the lane profiles window When Use Lane Background Method For Bands is selected and bands in lanes are being quantified all other background settings average median user defined or no background are ignored If there are both bands in lanes and other features being quantified the lane background method will be used if selected for bands in lanes and the other features will be quantified using average median user defined or no background depending on which method is selected Not
108. cting size standard lanes 1299 templates 185 Widths ea e 88 lane templates deleting renensnrnsnenaa 86 Placing ssc casniscissdsse testers desotaesrcspioderserss 86 CAVING riae aaae ana a E SEERE linear fit interpolation method z linear auto manual sensitivity controls 210 logarithmic sensitivity control 0 0 210 212 local min max pixel replace filter 0ce 45 log interpolation Method ssecsseeseeseeees 150 M median background method 000 153 155 membranes SELLING SCAN AFCA ee thae i placement on odyssey measuring distance on the image 128 136 microplates alignment GUIDE csc si ccscnetsidhcteteewiebossvtses FOCUSSEN eimina E placement on odyssey scanning MUltiplesrissssiirnise microplate flip image check box MINIMUM NENSI sissien molecular weight Calculations e 253 displaying molecular weight standards see size standards mousepod preset paraMeterS ssssiriesisisininiesisis rseeoiea 18 see in vivo imaging guide N names default scan NAMES cccccccceeesseeeeessseeeees new analysis window no background method noise removal filter normalizing lanes vu c45 sevinssicsveees aavesedeeeeseanes ines P peak intensity cciisccaisessavercctiescsnsesssieeusaeeseaes 251 percent response calculation ICW assay 254 percent saturation calculation 254 pixel LOCATON s cssssussdcscosasedcecneteosesa
109. culations start all wells designated as background see Chapter 9 are averaged for the 700 channel image Similarly the 800 channel background wells are averaged resulting in each channel having its own background intensity value Assuming that background subtraction is enabled which it normally should be the average background intensity for the 700 channel is subtracted from the integrated intensity of each well in the 700 channel The same calculation is performed on the wells of the 800 channel image using its corresponding background and integrated intensity values References to integrated intensity throughout the rest of this discussion refer to the original integrated intensity minus background intensity Odyssey software allows complete flexibility in how the image channels are used but suppose the 700 channel is used to detect phosphorylated proteins and the 800 channel is used to detect total protein In this example the 800 channel would be designated as the channel used to calculate relative intensity which indicates the relative number of cells In the channel used to calculate relative intensity Odyssey software starts by finding the well with the maximum integrated intensity All wells designated as Sample or 100 Standard in the 800 channel are divided by the maximum integrated intensity to obtain the relative intensity of each well The relative intensity values will normally be between 0 0 and 1 0 though negative numbers
110. d or changed and used for the main grid Designing a Main Grid The main grid is specified in a different template than the subgrid Each cell of the main grid will have a subgrid inside it so the number of cells in the main grid should match the number of arrays on the image Start by measuring the spacing and location dimensions using the selection rectangle as described earlier in this chapter 138 CHAPTER 7 Drawing Features on Images 1 2 3 4 5 6 7 8 Measure the horizontal and vertical grid spacing as shown below Click and drag a selection rectangle starting in the upper left corner of one array Drag downward and to the right until the next arrays if any are contacted as shown in the diagram below Horizontal Spacing Vertical Spacing Choose Settings gt Grid Template and click New in the Grid Templates window to create a new template for the main grid Enter the number of rows and columns 2 x 2 in our example above Enter the measured dimensions mm for horizontal and vertical spacing Set the X and Y offsets to a small value so the grid is placed in the upper left corner of the display Select Use Sub grids to add a subgrid in every cell of the main grid and choose the subgrid template from the Sub grids list Click Save and name the template for the main grid Apply the main grid to the image using the grid tool in the Odyssey window 9 Move the grid from its initial
111. d B by clicking the first well in row A holding down the mouse button and dragging through the last cell in row B When the mouse 165 166 CHAPTER 9 Odyssey In Cell Western Module button is released all the wells in both rows turn green to indicate they are selected 01 02 03 04 05 06 07 08 og 10 11 12 Select Link Rows and click Create Link The green wells in the template turn black to indicate they are linked Each individual link A01 B01 A02 B02 etc is listed in the Existing Links list During analysis the linked wells in the Existing Links list will be averaged The average will be used in ICW calculations rather than individual integrated intensity values Only sample wells are averaged All Background and 100 Standard wells are always averaged j New Links Existing Links a add anew link OL Create a Remove All O Link Columns Ce il O Independent Link Additional links between wells with identical samples can be added by continuing to select the wells and click Create Link To link columns rather than rows click Link Columns rather than Link Rows To link a few wells that are not part of entire rows or columns select the wells select Independent Link and click Create Link To dissolve a link select the wells and click Unselect to change the well type from linked green to unlinked white L
112. d inflection points by single clicking in the center of the lane at various points in the lane Single clicking creates a multi segment lane line consisting of all the points that were clicked Finish the curved lane by double clicking the last point The more a lane curves the more inflection points will be required to make the lane conform to the image After the first lane is added additional lanes can be added using the same technique For greater efficiency however lanes can be copied and pasted or multiple lanes can be added at once Both techniques are described later in this chapter 68 CHAPTER 5 Creating Lanes and Finding Bands Moving and Resizing Lanes Moving Lanes To move a lane with image channels overlaid click the lane to select it changes color Move the cursor into the middle of the lane until the cursor shows arrows in all direc tions With the all arrows cursor displayed click and drag the lane to a new position If lanes are moved in single channel mode rather than with channels overlaid an error message will be displayed if the lanes are still linked Linked Lanes When lanes are added to overlaid images the lanes on both channels are linked In other words when the images are overlaid and the lane position or width is changed lanes on both the 700 and 800 channel images will be changed Certain operations such as trying to change lane width in single channel mode will unlink th
113. d method is displayed when an analysis is opened unless the message is turned off in the General Application Settings Stopping the cursor over the background tool in the toolbar also displays the current background method in a tool tip Changing the Background Method The background method sets the background calculation method for quantification See Chapter 9 for the background calculation method for In Cell Western assays To change the background method for the current analysis choose Analyze gt Background Method 155 Background Method for Scan TiterScan Analysis Grid A x Background Method Average Border Width 3 Median Segment for Average and Median Method All Top Bottom O Right Left O User Defined Band Specific C Use lane background method for bands When selected the lane background method is used in band quantification in place of the general shape background method defined above Save Cancel Each of the background methods is briefly described below No Background When No Background is selected zero is used for the background This is the best choice for applications such as In cell Western assays that have their own background calculation method Average Median and User Defined Background Methods Both the Average and Median methods calculate the background using pixels around the perimeter of the area being quantified The 156 CHAPTER 8 Quantification Details
114. dding points to MW lineS cee 106 applying to images changing dremesarsss control points sses creating sets a deleting cossirier sasea interpolation method ssssssssesesesesesereseseses 108 moving points on MW lines 106 plotting size standards cee 107 select lane Sheoirse eirs 103 SMAP tO LANES sssscscesssssssiezsrastoeasivsecsredsien 100 UNMIS css vsscorevesceveesn sess E EEEa a ET aE iia 108 Smoothing filtersssssonnennn araa 44 statistics display and export eee 205 subgrids designing a SUDgTIC ssseeseeeteeeeeees 135 system administration ACCOUNE TIGNES asesssisi siasio esris 234 adding and deleting users 00c 236 backing up scan groups 33 changing user passwords ceeeeee 236 changing user access rights 0 0 cee 236 managing SCAN BrOUPS srissrssersriiesesisiseses 237 system cooling MESSAGE eee eee eet tees 26 T text display area around images cee 228 tool tips Nidigere ersi rsss 93 toolbars hiding and Viewing ssrcsransirinreiaiis 4 thumbnails view OPOMINGiscscasssessncacssassyevissanssiesseveesbassssseescaates 9 double clicking thumbnail images to open 9 timmed THEAN aves cs cissdcceas consnacesearageh niae 251 U updating Odyssey instrument software 240 user administration changing password on your account 231 managing your scan groups see system administration also user defined
115. ds the search in all directions top left bottom right by one quarter of the minimum dimension of the feature Setting Search Area Extension to Large expands the search in all directions by the minimum feature dimension Enlarging the search area slows the software Note If Adjust Feature Location is used with microplate images that have fluores cence from the microplate walls grid features may not be accurately placed no matter how the threshold and search area are configured While new feature locations are being calculated a variety of warnings or error messages may be displayed depending on the image features and original location of the features To suppress these messages deselect Display Interactive Messages Messages can still be viewed by double clicking the message bar at the bottom of the Odyssey Window to display the Status Message History window 121 122 CHAPTER 7 Drawing Features on Images Grid Features and Image Alignment For grids the settings can also be used to move all the selected grid features by an equal amount and to reset features to their original location if not found If Reset Cell Locations If Not Found is selected selected features in a grid are moved to the center of their respective grid cells if a location cannot be found using the Minimum Finding Threshold Features not associated with grids are never moved if their location cannot be found All features within grids are moved the same
116. e Add MA Line Select MVV Std Set Select a Std Set x Apply Std Set Edit Mode O Add Points O Modify Points Allows the Selection and Move of existing Size Lines Apply to Both Apply Cancel Click a MW line to select it and move the cursor over the line until it becomes an up down arrow cursor as shown below i ee Click and drag the selected line up or down until it is correctly positioned then release the mouse button To move more than one line hold down the Ctrl key while clicking multiple lines Adding Points to a Line To accommodate smiles frowns or other irregular electrophoresis artifacts it is necessary to add extra points to the MW line After the points are added the next section below shows how to move the 106 CHAPTER 6 Band Sizing points into new positions that follow the contour of bands on the image To add points to MW lines change the Edit Mode in the Edit Size Standards window to Add Points as shown below Edit Size Stds for 800 TIF Add MVY Line or Select MWY Set New MW Value Add MW Line Select MW Std Set Select a Std Set Apply Std Set Edit Mode O Modify Lines Allows the Addition of new Points on Size Lines Click on a MW line at a position where an inflection point needs to be added Any line can be clicked and any number of points can be added by continuing to point and click as long as the Edit Mode is still set to Add Point
117. e The background method used for each feature can be included in reports and is displayed in the data table in Details View Requantifying After Changing Background Method When changes are made to the background method all existing features and band markers are automatically quantified again Any new features or band markers added to the image will be quantified using the new background calculation method A manual method to requantify features is also available To recalculate existing features choose Analyze gt Requantify 159 Quantification Using Grids After all the features in the grid are properly positioned Chapter 7 quantification can proceed as described earlier in this chapter Concentrations are assigned to wells containing concentration standards by selecting the individual circle or square features and choosing Analyze gt Concentration Standards Integrated intensities can be viewed immediately in the Grid Sheet choose Analyze gt Grid Sheet After the concentration standards are assigned concentration values are automatically calculated for all other features in the grid Concentration values can also be viewed by generating a report Chapter 10 that lists the concentrations for each feature Note Background calculations are set to No Background when a grid is applied to an image so a background method should be selected before quantification ina Chapter 9 In Cell Western Module Overview Notice
118. e lanes on the two images Band markers are never linked and can never be edited with channels overlaid Bands in the lanes are found independently and automatically on both images immediately after lanes are marked 69 Changing Lane Width To change lane width click the lane to select it Move the cursor over the left or right boundary of the lane until the cursor turns to a right left arrow cursor With the right left cursor displayed click and drag one side of the lane boundary the centerline and opposite boundary are not moved To symmetrically change both left and right lane boundaries click the Properties button 48 and select the Symmetric Left and Right Boundaries check box Changing Lane Height To change lane height click the lane to select it Move the cursor over the top or bottom boundary line of the lane until the cursor turns to an up down arrow cursor With the up down cursor displayed click and drag the lane boundary to a new position Changing Lane Shape If one of the end points is not centered in the lane and needs to be moved to match the shape of the lane on the image start by clicking the end point to select it When the end point changes color and is 70 CHAPTER 5 Creating Lanes and Finding Bands surrounded by the white selection box as shown below the end point can be clicked and dragged to a new location Straight lanes can be made vertical or slanted by moving one of the two e
119. e methods of mapping grayscale values to the monitor Typically either Linear Auto or Linear Manual gives the best results A manual Logarithmic mapping method is also provided Linear Auto uses computer algorithms to predict the sensitivity that will give the best image appearance When Linear Auto fails choose Linear Manual and use the sensitivity slider to interactively change the sensitivity until the image appears the way you want Linear mapping which is preferred in most applications can best be explained by considering a graph that has image grayscale values on the X axis and monitor grayscales on the Y axis 255 Linear grayscale mapping Monitor Grayscales 0 65535 Image Grayscales 211 With Linear mapping a given change in image intensity results in a proportional change in display intensity The three filled circles on the X axis above represent the grayscale values of three bands of relatively low intensity When bands have low intensity on the monitor the most common way to intensify the bands is to change the sensitivity setting This changes the slope of the linear response line as shown below The result is that low intensity bands are displayed with higher grayscale values on the monitor ae Linear grayscale mapping after increasing sensitivity Monitor Grayscales 0 65535 Image Grayscales Increasing the sensitivity will produce satisfactory results as long as there are no bands on the ima
120. e response of each sample over positive controls 162 CHAPTER 9 Odyssey In Cell Western Module Starting a New In Cell Western Analysis This section describes the operation of Odyssey software after samples have been prepared according to one of the ICW protocols in the Odyssey Application Protocols 1 Place the microplate on the Odyssey scan surface and align it using the alignment guide Only 96 or 384 well microplates can be analyzed with Odyssey In Cell Western software module See Operator s Manual for tips on scanning microplates 2 Start the scan and select the Microplate preset or a similar preset 3 When the scan is complete click Save and accept the default scan and analysis names or enter new names Applying a Grid Automatically Note Grids can also be placed manually as discussed in Chapter 7 Automatic grid alignment is only available in the In Cell Western Module For microplate scans Odyssey software can automatically place grids on images as long as the images meet certain restrictions To automatically place a grid choose In Cell Western gt Align Grid Select a grid template that matches the microplate and click OK Automatic grid placement is only available for images of 96 or 384 well microplates that have been scanned using Odyssey software 2 0 and above Trying to automatically place grids on older images will generate at error message Microplate images should be approxi mately 12 cm W x 8 cm H
121. e averaged The average integrated intensity replaces the original integrated intensity values in all the linked wells in the column resulting in them all having the same value Note If the Calculate Relative Intensity in Channel field is deselected for a two channel scan the percent response will be calculated for both channels with no normalization For each channel the relative intensity values are divided by the 100 Standard and multiplied by 100 The Calculate Response check box displays the channel s that will be used to calculate percent response It cannot be deselected and has no other purpose but to provide information Analyzing Individual Rows or Columns When the Individual Row Analyses and Individual Column Analyses check boxes are not checked the ICW calculations are performed as described above Separate ICW Analysis O Individual Row Analyses C Individual Column Analyses When Row Analysis or Column Analysis is used Well Links are ignored If either of these check boxes is checked Response is calculated using only the wells within a given row or column For example when Individual Row Analyses is checked each row analyzed must have at least one Background well and 100 Standard well Response for sample wells in the row is calculated using the Background and 100 Standard well s in the same row as the sample wells An error message will be displayed if Background and 100 Standard wells have not
122. e band The bands are used to calculate the normalization Factor for each lane Normalization Channel A The reference channel is used to calculate a normalization factor for each selected lane The band having the highest integrated intensity is assigned a normalization factor of 1 0 This reference band is then used to calculate the normalization factor for all other selected lanes by dividing the band integrated intensity by the integrated intensity of the reference band On the sample image a band s normalized integrated intensity is calculated by Band Integrated Intensity Lane Normalization Factor After the calculations are complete the normalization factors are shown in a table The table shown to the right indicates that lane 3 contained the reference band and was assigned a normalization factor of 1 0 Both the normalization factor and the normalized integrated intensity can be included in reports or exported data Sample Normalization Results The normalization results For the 700 channel View the full results in Report View using normalization fields Lane Name Normalization Factor I 1 Counts Lane 2 0 40 85330 38 Lane 3 1 00 214208 76 Lane 4 0 85 181464 96 Lane 5 0 76 163743 00 Lane 6 0 47 100801 38 Lane 7 0 61 129756 43 Lane 3 0 50 107153 79 Close 85 Creating and Using Lane Templates Lane templates record lanes from one analysis
123. e obligations of Section 2 5 shall not apply to information that is a in the possession of Licensee without obligation of confidence to Licensor before receipt thereof from Licensor b available to the public without fault of Licensor or c is disclosed to Licensee without restriction by a third party who is not under any legal obligation either by agreement with Licensor or otherwise prohibiting such disclosure 2 6 1 3 Licensee may disclose Confidential Information to governmental agencies or in litigation as required by law Licensee will give Licensor the greatest practicable notice of any such compelled disclosure 2 7 Treatment of Licensee Confidential Information by Licensor Licensor will have no confidentiality obligation with regard to confidential material or information that is a in the possession of Licensor without obligation of confidence to Licensor before receipt thereof from Licensee b available to the public without fault of Licensor or c is disclosed to Licensor without restriction by a third party who is not under any legal obligation either by agreement with Licensee or otherwise prohibiting such disclosure Licensor may disclose Licensee confidential material to governmental agencies or in litigation as required by law Licensor shall give Licensee the greatest practicable notice of any such compelled disclosure 2 8 Return of Confidential Information Upon expiration or termination of this Agreement each p
124. e scan name for the first plate followed by 2005 Mar 29 4PM 2 etc If another scan is started using the same base name a letter is also appended Thus the first scan with a duplicate base name would be 2005 Mar 29 4PM a 1 the second would be 2005 Mar 29 4PM a 2 etc The letter is incremented each time a new scan is initiated using a duplicate base name Scan Multiple Plates Setup Scanner Tech Support User jen Scans r Common Description Base Name 2005 Mar 29 4PM Group jen v Modify Preset I MicroPlate2 RA Modify Scan Parameters Scan Location Selection Resolution 169 v 50000000006 500000000006 9000000999 em SAOOODOC Quality medium 200000000000 AAAA AAAA gO Focus Offset 3 0 20000000000 r Channels 700 800 Intensity 50 50 v Image Size 672 Kbytes per channel Se AAO OOO OOS Default Values 00000000004 Plate One Scanned Plate Two Scanned Cancel 5 Choose the scan Group in which to store the scan Use of scan groups in the Odyssey instrument is described earlier in this chapter 6 Enter a common Description if any that will be used for all microplates 7 Choose a set of Preset scanner parameters designed for micro plates such as the default Microplate2 preset and skip to the next step Alternatively the individual scan parameters can be edited The sc
125. ed the last choice on the View menu will be Channel 700 Displaying a Second Image View Window For scans with both a 700 and 800 channel image a second image view can be opened by choosing View gt Display 2nd View or by clicking on the tool bar The two image view windows are displayed side by side in the Odyssey window If the first image view was in single channel mode before selecting Display 2nd Channel the second image view will be the opposite channel If the first image view shows both channels overlaid the second image view will be the 800 channel image in single channel mode Hiding Image Annotations If annotations obscure other image features all annotations can be temporarily hidden by clicking 8 on the tool bar or choosing View gt Hide Annotations To view hidden annotations click 8 on the tool bar again or choose View gt Show Annotations To reduce screen clutter the Application settings can be used to control which annotations are displayed as described below ia Using the Application Settings to Display Labels Image View Feature settings are opened by clicking on the tool bar or by choosing Settings gt Application and then selecting Image View Features from the settings list Image View Feature settings are important when analyzing membranes because they can be used to display integrated intensity molecular weight or other values over each band spot For grids labels are not displayed rega
126. ed in the Application settings as described later in this chapter Chapter 13 describes all of the calculations used in Odyssey software When the concentration of each standard is identified a concen tration value is assigned to the calculated integrated intensity for each standard After the standards are all identified concentration values of sample dots bands are calculated from their integrated intensities using the interpolation method specified by the user Displaying Quantification Values The Application settings determine how concentration and quantifi cation values are displayed for band sizing or quantification on membrane scans To change how values are displayed choose Settings gt Application and select Display Values from the Settings List Application Settings Odyssey Settings Settings List Display Values General Integrated Intensity counts mm 2 Image View Features Image View Display Display values as Kilo Integrated Intensity Display Values Raw Inti ted Intensit t PA Raw Integrated Intensity counts Adjust Locations Concentration Auto Shape 5 gt aul Naming Conventions Poem Mca 2e Report Quantifications C Substitute Trimmed Mean for Saturated Pixels Quantification values can be displayed as integrated intensity select Integrated Intensity counts mm2 raw integrated intensity select Raw Integrated Intensity counts or concentration select Concen
127. ed path is assumed to be a scan folder if scn and tif files are found in the folder Scan folders found during the search are listed in a search results window F List of Scans from Top Level of C Documents and Settings RON icor Odyssey Projects Scans in folder total found 20 ae Double click on scan to open project Folder of this scan Close or select scan and Open Project at any time including during search Scan Western Project C Documents and Settings RON Licor Odyssey Projects BandSizing Scan Date unknown a Western C Documents and Settings RON Licor Odyssey Projects BandSizing_2 unknown TkappaB scan C Documents and Settings RON Licor Odyssey Projects IkappaB Quantification unknown MouseScan C Documents and Settings RONI Licor Odyssey Projects Invivo_tutorial unknown ICW_Scan C Documents and Settings RON Licor Odyssey Projects IRDye_Brochure unknown Western C Documents and Settings RON Licor Odyssey Prajects IRDye_Brochure unknown MyScan C Documents and Settings RON Licor Odyssey Projects MyProject unknown Scan C Documents and Settings RON Licor Odyssey Projects MyProject unknown Scan_1 C Documents and Settings RON Licor Odyssey Projects MyProject unknown FirstScan C Documents and Settings RON Licor Odyssey Projects Tutorial unknown Joy 4 C Documents and Settings RON Licor Odyssey Projects Tut
128. ed shapes PEQUANTIFY sipsi save as grid template eee annotations hiding Molecular Weight ccssesscssscesssssssrtsssenases 92 N a NOt assigned e eee eect e eee 143 TOTALING 53 eccecesessursacsstenevavssivs E EAS 142 application settings adjust OCatON ssiru 119 changing the active application settings 229 deleting the active application settings 230 image View display sssseeseeeeseeees image view features LAN OS easa naming conventions PEPOME caa sates rewteveesectunteaynsves cyeveneny saving a new application settings file 229 selecting settings file at startup undo Settings seers AUTO shape TOOL aaron en average background method 0 153 155 AVEFALES INCEMSILY voscasecsssesssoncescasessessonceesdzeveouses 251 B background calculations sssrini 249 background display lane profiles e 79 background subtraction cece 43 80 background method CHOOSING ennnen weciiaieicieh 151 155 current background method 00 154 definitions of background methods 155 details Vie Wiisser 113 grids no background method 04 123 lane background method cece 158 VECiPYING usare e A a 151 backing Up projecties ninsis 64 backing up SCan gro pSu sarrende 233 band markers too Many OF tOO FEW ee ceeeeeeeseeeseeeeeeeeeees CONTEHING ss cssisececcasasnesrvonees hiding boundary lines PEFINGING vsssns
129. eects 22 setting size for membranes ceeeeee 23 SCAN grid sironnan niasa anii 22 scan group DACKUP Mlesdehacds Jovestecstadiveniecsevte taste 233 238 deleting eenma S 237 SEIECH MG eiin ia ieietees i 16 265 266 INDEX changing deleting adding 232 237 DELIMISSIONS susie eisioes sssrin 233 scan parameters OGITING seresa scanner add or delete scanner AiagnOstiCs siisi iiris s anner INLOMMATION saiae skeras Sanner NOG sessao tiniee aiaiai s anner updates eia ins scans view opening scans and analyses 6 7 refresh button esssccesseeeeeeesceeeeseeseeseeeees 9 searching for SCANS esisiini 50 sequential file NAMES eeeceeeseeeeseeteeeeeeeeeeeees 32 settings menu adjust location display values grid templates wis ccstscsv svestenc reeves ICW OXPOM bs ssisc cccsseseadeceecseasvisiev esses ICW Setup swissessscccesiessrsssesessssatesteesseasenteeses IGW VIEW is soinsscenestzsaesds voeseatevasess seca sbeetiates multiple scan a scan Presets yraa a ean ScanMan ae ne a select active settings sessirnir 229 size standard sets esseeesesiserrrrereesiseserrrreesa system administration user administration cesses shapes see features signal to noise ratio cece ese eeseee ee eeees 252 single Channel VIEW csscssecssscsssvssccssensescsctssessesesss 95 size standard display migration 78 88 94 size standards adding manually eee eseeeeeeeees 100 a
130. egal file name An example of how the file name might look is shown in the Example field Text that can be entered automatically includes the date time analysis name project name scan name organization name and report name Choose Settings gt Application and then General to enter the organization name To insert a text placeholder at the cursor position select the type of text to enter from the dropdown list and click Insert Placeholders always start with a tilde character Click OK when finished entering a title The Report Path location of the exported file can be entered directly in the Report Path field or by clicking Browse to browse for the path using a standard file selection window All exported reports will be stored in the same folder using the selected path To store exported report files in the same folder as the scan rather than a central location select Save File in Scan Folder 187 188 CHAPTER 10 Reports and Data Export Analysis programs have varying requirements for input data files Include Field Names in Report can be deselected if the analysis program does not allow field names at the top of each column A comma tab or space can be chosen as the Field Separator character The separator character is used by analysis programs to separate data values into columns The separator character is also important for plug ins since this character is automatically used as the field separator Any plug in designed
131. ement the terms of this Section after first having employed their best efforts to jointly resolve such dispute If the parties cannot resolve their differences in such fashion within thirty 30 days of either party s receipt of such notice of the intent of the other party to implement the terms of this Section the following alternative dispute resolution process the venue of which shall be Lincoln Nebraska shall be immediately implemented 7 2 Upon written request of either party the dispute will be referred for negotiation to representatives of the parties who have no direct operational responsibility for the matters involved in the dispute and who have authority to resolve the dispute 7 3 If these representatives have not agreed on a resolution of such dispute within ten 10 Business Days of its referral to them the dispute shall be promptly submitted to a neutral adviser the Adviser who shall be chosen from the list of arbitrators registered with the American Arbitration Association For purposes of this Section Business Day shall mean each weekday and the hours of such weekday in which Licensee is open for business The Adviser shall within fourteen 14 days of the submission recommend in writing a procedure for resolving the dispute and shall specify in such writing whether such procedure shall be binding non binding or involve a combination of binding and non binding procedures 7 4 If the parties do not mutually agree upon
132. eriencing difficulty with your instrument the first step is to contact LI COR Technical Support Someone in Technical Support may ask you to run the scanner diagnostic routine This routine runs 239 a series of tests on various hardware components The results can be e mailed to LI COR Technical support automatically if your network permits such operations Scanner Diagnostics Scanner Tech Support Email Options O Always send diagnostic results O Send diagnostic results only if an error occurs Send To LI COR technical support cc Reply To Start by selecting one of the three options for e mailing the results Never Send Diagnostic Results Displays results but does not e mail them e Always Send Diagnostic Results Always sends results to LI COR Technical Support and also to any e mail address included in the carbon copy CC field The Reply To field should be used to include the address of the sender e Send Diagnostic Results Only If An Error Occurs Same as above except that results are e mailed to LI COR only if one of the diagnostic tests fails 240 CHAPTER 12 User Accounts and Settings Click Run to start the diagnostic test Normally all tests will have a status of PASSED if the hardware is operating normally A status of FAILED indicates a problem and INCOMPLETE may also indicate a problem Scanner Diagnostics Results Performing Diagnostics Summary Resul
133. es Odyssey file organization starts with the project folder A project folder is a folder anywhere on a local or network drive that is used to store Odyssey scans Project folders can be used to separate scans into a logical structure that compliments your research Project folders are created when new projects are started choose File gt New Within a project folder there can be many scans Each scan is a folder containing one or two TIFF images from the Odyssey Imager depending on whether probes for one or both dyes were used Scans 6 CHAPTER 1 Introduction can be started from the New Project window or by clicking the Scan button on the toolbar if a project is already open Scan folders also contain the analysis files generated when an analysis is performed on the scan Analysis files hold all the data concentrations etc and annotations created when the scan was analyzed At the end of each new scan the first analysis on the new image files is saved A set of images can be analyzed as many times as needed A new analysis can be created by clicking the New Analysis button on the toolbar Displaying Projects Scans and Analyses in the Main Odyssey Window The main Odyssey window shown below is the default window configuration that displays the Scans view left and the Image view window right fal Odyssey BandSizing File Edit View Analyze Lane In Cell Western Report Plug in Settings Window Help OSHS OF G
134. et After clicking Reset or using Undo to reverse all changes the images selected in the Available Analysis panel can be changed 41 42 CHAPTER 3 Creating A New Analysis Flipping an Image To flip an image vertically click the Flip button and select Top to Bottom from the Flip Options To flip an image horizontally click the Flip button and select Left to Right from the Flip Options N Flip Flip Options Rotating an Image If the image is not in the desired orientation click the Rotate button and use the Rotate Options window to rotate the image If the image was scanned sideways or upside down use the Direction radio buttons to rotate the image either Clockwise or Counter Clockwise and then choose 90 180 or 270 degrees of rotation A Rotate Rotate Options Degrees s O18 O270 Note The image does Pind Ore not need to be straight or in any particular orientation to be analyzed amp Free rotations change the image data for the new analysis and may affect quantification resuits If the membrane was place on the scanner at a slight angle it can be straightened using the free rotation option First choose Clockwise or Counter Clockwise rotation Next click Free and enter the desired rotation in degrees Note however that quantification results are changed due to image interpolation when images are rotated to some angle that is not a multiple of 90 degrees 43 Performing Backgrou
135. etc The status line at the bottom of the Scanner Console window indicates the time required to finish scanning the current plate and a progress bar indicates how much of the plate area has been scanned If no fluorescence is displayed where it is expected use the Alter Image Display or Adjust Image Curves button to adjust brightness and contrast By default the 700 and 800 channel images are shown overlaid If the default red green color scheme is being used the areas that are yellow have intense fluorescence in both channels If these adjustments do not display any fluorescence you may need to start the scan again and scan with a different intensity value After the first scan is complete an analysis containing the images is automatically created for the scan using the same name as the last analysis that was created When the scan of the first microplate is complete Odyssey automatically begins to scan the second plate and repeats the scan procedure until all designated plates have been scanned Note If the microplates are poorly centered in the images try adjusting the grid template to match the grid to the wells in the image If some wells are truncated or the grid is off the image see Multiple Scan settings below for instructions on changing the scan offset to center the wells on the image for new scans Stopping a Multi plate Scan To finish the scan of the current plate before automatic completion click the Stop button The i
136. ey Infrared Scanning System Version 3 0 Change Summary H Tutorials File and Project Organization S E Projects Starting Scans Scanning Multiple Microplates Resolution vs File Size H Creating and Editing an Analysis E Importing and Exporting Scans and Image w O Lanes H Band Finding B E Band Sizing 8 Drawing Features on the Image Background Method Details View Calculation Descriptions 8 Quantification Displaying A Grid Sheet S E In Cell Western Assays S O Feature Annotation O Image Modifications B E Reports S E Plug In Reports lt m Using the Help System The left side of the Odyssey help window lists all the topics in the help system in a convenient navigation tree When you click a topic the contents of that topic are displayed in the right side of the window Help topics are grouped in folders Clicking any closed folder displays additional topics There are two tabs on the left side of the window The first tab displays the navigation tree The second tab performs a full text search of the entire help system If you enter a word in the search field and press Enter a list of help topics that contain the search word is displayed Clicking a help topic in the search results displays the selected help topic If you are new to Odyssey the Odyssey Tutorial Manual has a broad overview of the entire system and step by step tutorials for all the main functions including scanning and analysis
137. fication size specifies the size analyzed during quantification and is often the same as the physical size However if there is background fluorescence from the microplate in the well edges setting the quantification size slightly less than the physical size will eliminate quantification of background signal See Measuring Size and Distance on the Image below for details on measuring the size of image features Column and Row Labels These buttons control whether the row and column labels on the outside of the grid are numbers or letters Display Grid Lines When this check box is selected grid lines are shown on the image Use Sub grids The Use Sub grids check box and the Sub grid drop down list for specifying a sub grid are discussed below in Using Sub grids 128 CHAPTER 7 Drawing Features on Images Measuring Size and Distance on the Image Size and distance values in the grid settings must be fairly precise for optimal operation but these values are difficult to visually estimate Size and distance on the image can be determined precisely using the selection rectangle Example for measuring grid spacing X To measure grid spacing offset or feature size drag a selection rectangle over the area to be measured The X Y dimensions of the selection rectangle will be displayed in the status message at the bottom of the Odyssey window as shown below T Selection Rectangle width 51 36mm height 2 88mm diag 51
138. fs fafa CHEI 5 upper right corner of 5 10 15 20 the area to be scanned Scanner Front Specify the scan area by dragging and release the mouse your mouse over the grid display button To reposition the scan area click inside the red rectangle and drag the scan area to a new position To resize the scan area move the cursor over one of the red lines or corners until an arrow cursor is 23 displayed With the arrow cursor displayed click and drag to resize To reset the scan area click and drag a new rectangle on the scan grid starting outside the current red rectangle If necessary double click outside the current red rectangle to erase it before drawing a new one The tip of the arrow in the front left corner of the scanning surface on the Odyssey Imager corresponds to the Origin of X 0 Y 0 on the scan grid in the Scanner Console window Left border See the Odyssey of scan area Operator s Manual for lower border additional information of scan area on sample placement Origin If the size and origin are known the dimensions can be entered in the Size and Origin fields In general it is best not to place the membrane or gel at the 0 0 position The scan area drawn on the scan grid should always be larger than the membrane or gel so text annotations placed on the image during analysis will be displayed properly For low or medium resolution scans make the scan area about 1 cm larger than the membrane or gel on all fou
139. g lanes click on the tool bar until both images are overlaid in a composite image assuming the scan has two images 6 CHAPTER 5 Creating Lanes and Finding Bands Creating the First Lane To create a lane choose Lane gt Add Lane or click the amp add lane tool on the toolbar amp Scan FirstScan Analysis MyAna DER S ry Click at the top of the lane you want to add The mouse pointer should be horizon tally centered in the lane Finding Straight Lanes Scan FirstScan Analysis MyAna DER A For straight lanes vertical or slanted center the mouse under the bottom of the lane and double click The lane will be added using the lane width specified in the Appli cation Setting Bands in the lane will be found and enclosed by band markers Lanes can be added from top to bottom or bottom to top Lane 1 67 TIP If you click and hold down the left mouse button at the top of the lane and drag the mouse downward to the bottom of the lane a white dashed line extends from the point of the mouse click to the current cursor position The white line indicates where the centerline of the lane will be The white line in useful for creating straight lanes When the white line is in the correct position release the mouse button and double click where the endpoint of the line should be Finding Curved Lanes For curved lanes begin by clicking at the top center of the lane as usual Next ad
140. g the Scanner Settings selecting the name in the scanner list and clicking the Edit or Delete button Editing a scanner may be necessary if the IP address for the scanner has changed which can occur when using automatic addressing DHCP Odyssey software does not automatically delete scanners from the scanner list because some scanners may be turned off or on another networks where they cannot be automatically detected If a scanner has been removed from your network a scanner can be deleted from the scanner list by selecting it and clicking the Delete button 245 Chapter 13 Calculation Descriptions One of the most important features that Odyssey brings to protein analysis on membranes is the ability to provide linear quantitative data This chapter describes the calculations that are vital to under standing how to use the quantification tools in Odyssey software Derivation of the Mathematical Expressions Understanding the derivations and assumptions supporting the mathematical expressions will make it easier to use the computed data properly Definitions of terms e Feature any area enclosed by a user using Odyssey shape tools e Spot an area with increased intensity spot within a feature e Pixel the smallest area unit of an image that is measured with a single intensity value 2 the physical area of an image pixel in mm e Pixel area a mm e Signal intensity or just intensity signal counts
141. ge spots Average intensity can be used only when an image spot has uniform intensity for example when estimating background with background correction turned off Average intensity should not be used when there are both spot and background intensities within the defined feature Trimmed Mean Trimmed Mean is similar to Average Intensity except that five percent of pixels with the highest and lowest pixel intensities are excluded from the intensity summation and pixel count Peak Intensity Peak intensity is the highest pixel intensity within a feature The peak intensity value does not have background intensity subtracted Minimum Intensity Minimum intensity is the lowest pixel intensity within a feature The minimum intensity value does not have background intensity subtracted 252 CHAPTER 13 Calculation Descriptions Signal to Noise Ratio Signal to noise ratio is defined as follows Peak Intensity Background b Std Dev Background b SN Ratio Concentration Concentration is the amount of fluorescent material present within a given feature Concentration is calculated relative to user defined concentration standards on the same image The units of concen tration are always the same as the standards To calculate concen tration the intensity of each concentration standard is plotted and fitted with a curve using one of four interpolation methods linear log reciprocal fit or exponential Next the concentra
142. ge well desig nations linked lanes or how the calculations are performed the channel used as the relative channel etc If changes are made that require the calculations to be performed again the Recalculate button is activated Standard Deviation of Linked Wells Standard deviations are calculated and reported for the integrated intensities of any wells that were linked during ICW setup Values are reported in the 700 II Std Dev and 800 II Std Dev columns For wells that are not linked standard deviation values are listed as n a not applicable Exporting Response Data Response data can be exported to a tab delimited text file by clicking Export Use the standard file dialog to enter a file name and add any file name extension that your analysis program may require 173 Data can also be copied or printed Click Print to send the data to the default printer To copy the entire data set to the clipboard press Ctrl a to select all cells and then Ctrl c to copy the data Single columns are copied by clicking the column header to select the entire column and pressing Ctrl c to copy the data Multiple columns can be selected by holding down the Ctrl key while clicking additional columns Displaying Integrated Intensity in Kilo Units The integrated intensity columns for the 700 and 800 channels may be displayed in standard units or divided by 1000 to make the numbers more readable Chapter 8 describes how to use the Appli c
143. ge with high grayscale values Bands with high grayscale value will saturate as the sensitivity is increased When there are bands over the entire grayscale range but weak 212 CHAPTER 11 Changing The Appearance Of The Image bands need to be intensified Logarithmic mapping can help because it intensifies the display of weak bands while preventing strong bands from saturating 25 Logarithmic mapping inten Oo o ae sifies weak bands while keeping strong bands from saturating Monitor Grayscales 0 65535 Image Grayscales Note These image display controls only influence image display and do not change image data Changing Image Display Style Most analysis functions are performed with only one of the two images displayed The Show This Image check boxes in the Alter Image Display window are used to show or hide one of the two images Similar controls for selecting which image to display are discussed later in this chapter If only one image is displayed the image can be switched between color and black and white using the Show Colored Image and Show Gray Scale Image radio buttons When an image is displayed as grayscale the Show White Bands on Black check box can be used to invert the image display style to white bands on a black background Normally fluorescence shows up as dark bands on a white background 213 The Reset button changes the Brightness Contrast and Sensitivity back to the way they were when the wind
144. ges Images that no longer have a scan file scn can be imported into the current project and stored under a new scan name Starting a new scan is discussed in Chapter 2 The remaining three methods for importing images are discussed in this chapter The following methods to export scans and save images to a new analysis are also discussed i e Export Scans Saves a copy of the current scan to any specified location e Flip or Rotate to New Analysis Copies image files from the current analysis into a new analysis and flips or rotates the images in the new analysis e Crop to New Analysis Crops image files in the current analysis and saves the cropped images in a new analysis e Crop to Multiple Images Crops the current images using the scan area in a scan preset file and saves the cropped images to a new analysis 50 CHAPTER 4 Importing and Exporting Scans and Images Searching for Scans Before importing or exporting a scan you may need to search for it Choose File gt Scan gt Search for Scans to generate a list of all scans in a folder or disk drive A file dialog is displayed to choose the starting location for the search If all Odyssey projects are kept in one folder that folder may be a good starting place for the search Entire drives can be searched by starting at C D etc Note however starting at a specific folder takes less time than searching an entire drive During the search any folder in the specifi
145. gging its header left or right to a new position Click Show Only Quantified Pixels to turn all pixels black except for those that will be used for quantification the band spot should be examined to make sure it fully encloses the fluorescence on the image On the image below the circle feature fully encloses the spot and is roughly centered over it as indicated by the blue crosshairs If a feature needs to be moved it must be moved on the image rather than in Details View Features can be nudged in one pixel increments using the arrow keys as long as the mouse cursor is over the selected feature To the right and below the image are curves that plot the intensity of the pixels below the crosshairs The background method used when the integrated intensity of this feature was calcu lated is listed in the Bknd Method column of the data table V 15430 H 16790 Concentr TD Name Integ Int J Ave Inte Shape Area Bkad let ji F 0 74 3 Click Select Fields to change which fields are displayed in Details View ja A Show Only Quantified Pixels Select Fields l Clear Copy _ Export Print Close Each dot band should be examined to make sure the background method was correct In most cases one background method will be correct for most dots bands on an image but there may be a few that require recalculation using a different method As me
146. gin window select the scanner if necessary enter your User Name and Password and click OK Scanner Login Scanner West Lab If someone else is already logged in click Logout before entering your User Name and Password a ron Password titat User 13 Note User names and passwords must be added by a user with Administrator admin access privileges The system administration functions Settings gt System Administration are used to add users and set access privileges see Chapter 12 Scanner Console Window for Standard Scans Whether a scan is started in a new or existing project the Scanner Console window is used to specify the scan parameters and start the scan It can also be used for a quick preview scan During each scan the Scanner Console displays the scan in real time as it is collected and displays progress indicators for the scan Scanner Console BandSizing User ron Scan Description Name 2007 05 08 154727 Group ron Modify Preset Membrane Modify Scan Parameters Scanner Back Resolution GE v Quality medium HE Focus Offset 0 0 H H m Eee C Microplate flip image Channels E i T 700 T 800 H Intensity 5 0 5 0 Scan Area cm X Coord Coord Origin 0 0 width Height Size 10 10
147. gs to save the report template ICW Export Settings Another way to display the ICW Export Definitions window described in the previous section is to choose Settings gt ICW Export Assay Optimization With Z factor Calculations Calculating the Z factor for an assay produces a statistically derived dimensionless value that can be used to characterize the quality of an assay during assay optimization and validation The Z factor indicates whether the assay has sufficient dynamic range and low enough data variability to generate meaningful data See the calcu lation descriptions in Chapter 13 to learn how Z factor is calculated Z factor values are always 1 or less as described below m Z 1 isan ideal assay m 1 gt Z 0 5 indicates a high quality assay exhibiting a wide separation between signals for positive and negative controls and low data variability m 0 5 gt Z gt 0 may indicate a low quality assay with marginal distinction between signals for positive and negative controls and higher data variability However an acceptable Z factor target value should be determined prior to performing final validation of an assay An assay with a relatively low number of data points such as the number obtained from a 96 well plate may produce 179 a Z factor value less than 0 5 but still be considered a good quality assay if the same value is acheived between different plates run on different days mg Z lt 0 indicates u
148. gt GridSheet Main Grid o 3 27 13 44 53 61 68 70 78 30 80 84 E 3 53 40 39 32 99 27 30 29 19 3 85 12 59 26 58 28 01 31 66 Data for one channel at a time is displayed in the Grid Sheet To switch between channels use the Channel drop down menu The Show menu can be used to display integrated intensity concen 134 CHAPTER 7 Drawing Features on Images tration or raw intensity values see Chapter 13 for calculation descriptions The values shown in the Grid Sheet can easily be transferred to a spreadsheet for plotting or analysis Click Export to create a text file with tab separated values The text file will contain all data in the Grid Sheet plus a header that describes the scan Click Copy to copy all data to the clipboard Individual rows can be selected by Control clicking the row s and pressing Ctrl c to copy the data to the clipboard Reports and the Microsoft Excel for Grids plug in report can also be used to export grid data The Print button sends the entire grid sheet to the specified printer in the page orientation of your choosing Changing Font Size in the Grid Sheet The font size used in the Grid Sheet can be changed by choosing Settings gt Application and changing the Grid Sheet Font Size setting in the General settings Copying Grids Between Analyses The easiest way to copy a grid from one analysis to the other is to select the grid choose Edit gt Copy switch to
149. hannel 800 to 700 Next switch to the Well Types tab in the Change ICW Parameters window For Z factor calculations each well should be designated as a positive control negative control background well or not used To change well designations begin by selecting the type of well to mark from the Well Type Selector Well Type Selector Pos Control O El Neg Control O packaround O E Not Used Move the cursor over a well on the well assignment grid and click to assign the selected well type to that well Multiple well assignments can be made at once by clicking and dragging through a range of wells When the mouse button is released all wells within the selection rectangle change to the color assigned to the chosen well type If Assign Remaining is clicked any wells that are Not Used will be assigned the currently selected well type Clicking Clear All Rows changes all wells to unused Viewing Z Factor Values Choose In Cell Western gt View ICW Analysis to see the calculated Z factor value The Z factor is listed in red above the data table in the View ICW Analysis window Standard deviation and mean of the positive and negative controls are also listed Percent response fields 181 in the data table contain n a since ICW values are not calculated during Z factor calculations my In Cell Western Analysis Assay_Optimization_2 Z Factor3 i Cells In Table Selected Column Headers e O Show All Show Only y 4
150. hat cannot be entered in the Grid Template settings For example if you rotate a grid right click the image and choose Save Grid As Template the amount of rotation will be stored in the template even though rotation is not a parameter that can be entered in the Grid Template settings Any changes become part of the template when Save Grid As Template is clicked Note Templates saved using Save Grid As Template are not editable nor can they be used for creating subgrids Deleting Grid Templates To delete a template select the template in the Grid Templates window and click Delete Editing a Grid Template 1 Select a template from the template list in the Grid Templates window and click Edit 2 Change the grid parameters as needed 3 Click OK to save the changed grid template 126 CHAPTER 7 Drawing Features on Images Grid Parameters When you edit or create a grid template the grid parameters are listed in the Modify Grid settings window Each of the parameters are discussed below Grid Size Enter the number of rows and columns in the array of image features For example to create a template for a 96 well microplate you would enter 8 rows and 12 columns horizontal orien tation Grid Spacing The vertical and horizontal spacing in mm of the grid lines can be changed in order to match the spacing between objects on the image The vertical and horizontal spacing should be the same as the vertical
151. have a MW assigned so after any adjustments are make to the MW lines sizing is complete and the MW assigned to each sample band is final 109 Chapter 7 Drawing Features on Overview Images For images that do not have bands in lanes analysis begins by drawing individual features circles squares etc that surround all the fluorescent dots or bands in the image Features can be drawn on the image using a shape tool rectangle circle oval free form shape etc A grid tool is also available to quickly apply features to an image in a regularly spaced grid pattern The grid tool is ideal for scans of microplates Each grid can also have a subgrid in each grid cell making the grid tool useful for protein arrays Drawing Features on the Image For quantification each image should be analyzed separately When adding individual features to an image only one image should be displayed Begin by clicking on the toolbar until only one image is displayed in Single Channel mode assuming the scan has two images Click the shape tool in the toolbar that most closely matches the dot or band on the image Rectangle O Circle Oval and Freeform tools are available ha 112 CHAPTER 7 Drawing Features on Images Draw a feature that encloses the dot band Features are drawn the same way they are in most drawing programs Rectangles circles and ovals are drawn as shown below Imagine a bounding Click and hold
152. he Modify Scan Preset window is used to modify each of the scan parameters displayed in the Scanner Console Window Scan param eters were defined earlier in this chapter After you are done editing the scan parameters check the image size to make sure it is under 14 MB and click OK if you just want to edit the Preset If you want to create a new Preset with a different name click Save As instead of OK If you make a mistake and want to set all values to stored factory default values click the Default Values button To abandon changes without saving them click Cancel Note The status of the Microplate flip image parameter is ignored when a preset is used for a multi plate scan The images are always flipped for multi plate scans Preset Name Membra j Modify Scan Preset Scan Parameters 63 medium Resolution Quality Focus Offset 0 0 C Microplate flip image r Channels 700 bo m 800 50 Intensity r Scan Area cm X Coord o Width jio Y Coord Height 10 Origin Size 700 Kbytes per channel Default Values Image Size Tip The Modify Scan Preset window can also be opened by clicking Modify in the Scanner Console window as shown below Note however that the Scan Area fields are not editable unless the Modify Scan Preset Window is opened from the Settings menu Scanner Console
153. he Report menu format data according to the current report template which is selected in the Report View 185 When Report gt Export or Report gt Print is chosen the name of the current report template is shown in the title bar of the window that is opened Feature_Data is the current template shown below Report Export Feature Data Save in Reports v m E Report11792 txt E Report11793 txt My Recent Documents Printing Reports Choose Report gt Print to open the print window Print Feature Data Print Content Title My Report VJ Print Scan Parameters Printer Setup Printer Ad2 MARCOMM_HP8100 Orientation Portrait Page Setup Change Printer The default report title in the Title field is controlled by the Report Application Settings but the title can be changed as needed The scan parameters resolution etc can be appended to a report by selecting Print Scan Parameters Page Setup page orientation and Change Printer are standard printer functions and Preview shows a facsimile of the page that will be printed 186 CHAPTER 10 Reports and Data Export Exporting Report Files When Report gt Export is chosen a standard save file window is opened and the default path is the Report File Name and Path in the Application Settings Settings gt Application gt Report The field separation character tab etc used in the file is also determined by the
154. hen the X axis is expanded the Min Pt Cntr and Max Pt buttons can be used to quickly scroll to the minimum point midpoint center and maximum point respec tively The bar over the top of the histogram indicates the maximum intensity If the cursor is stopped over the bar the maximum intensity is listed in the tool tip If maximum intensity its very low the image may have been scanned with the intensity parameter set too low to get adequate signal strength Conversely if intensity is set too high during scanning saturated pixels may result A red bar all the way across the intensity indicator as shown below indicates saturation mN lt m gt 219 Cropping Rotating and Flipping Images In Odyssey most image manipulations like cropping flipping and rotating are done in the New Analysis window as described in Chapter 3 When a new analysis is added the image can be flipped rotated cropped or filtered as needed The background fluorescence can also be subtracted These functions are accessible only when a new analysis is created Magnifying the Image All of the zoom functions described in this section can also be accessed via the View menu Zoom Functions on the Toolbar Click on the toolbar to magnify the image Click the down arrow next to the magnify icon to access other functions bo a amp vho v Ey zh E zoom m amp Zoom Out 100 fe Zoom by Dragging Zoom Me
155. i Display 2nd View EB Alter Image Display ra Adjust Image Curves Q Zoom In amp Zoom Out 100 Zoom by Dragging m QA Hide Annotations A Channels Overlaid 5 Channel 700 Grayscale GP Color E AEEA Alt F10 Alt F9 F11 F12 N v File v Edit Ctrl F y view v Analyze Ctrl F5 v Report v Help Odyssey has context sensitive menus that change depending on what is selected when the menu is opened To open a context sensitive menu select a feature on the image such as a band marker and right click the image Using context sensitive menus you EP Properties can do things like open the Properties Mo Te Rotation f b Rotate Text CCW 45 degrees window for an object rotate text Rotte Text CCW adidegroos annotations and plot a histogram of Z Details View quantification values Ut chart view Rename Selected Shapes Sj Bands Equal Height Crop To New Analysis Flip or Rotate To New Analysis Correcting Mistakes Odyssey software has extensive Undo capabilities that are accessed on the Edit menu By continuing to choose Edit gt Undo or clicking the tool you can undo the last 100 operations since the Odyssey program was opened with a few exceptions The number of undo s can be changed in the Application Settings choose Settings gt Appli cation and select General from the settings list Odyssey Project and File Organization Projects Scans and Analys
156. ich is used to detect total protein should be selected in the Calculate Relative Intensity in Channel field The next calculation compares integrated intensity values in the 800 channel total protein in order to find the well with maximum integrated intensity All wells designated as Sample or 100 Standard in the 800 channel are divided by the maximum integrated intensity to obtain the relative intensity of each well The relative 167 168 CHAPTER 9 Odyssey In Cell Western Module intensity values will be between 0 0 and 1 0 which also indicates the relative number of cells in each well The relative intensity values from the 800 channel can now be used to normalize the integrated intensity values in the 700 channel which is used to detect phosphorylated proteins To normalize the 700 channel the integrated intensity for each well in the 700 channel is divided by the relative intensity values from the 800 channel This normalized value for each well is divided by the 100 Standard of the 700 channel and multiplied by 100 to give a value that is the percentage response to the control in the 100 Standard If more than one well is designated 100 Standard in the 700 channel they are averaged before being used to calculate percentage response The calculation is slightly different if rows are linked in the well assignment window When rows are linked all the integrated intensity values for the linked wells in a given column ar
157. ification value of the band can also be expressed as the band s percentage of the total integrated intensity of all bands in the lane see Application settings at the end of Chapter 5 Entering the Concentration of Standards Important For scans with both 700 and 800 channel images concentration standards in one channel cannot be used to quantify bands in the other channel Concentration standards for both dyes must be loaded and each image must be analyzed separately in Single Channel mode Concentration standards are identified using a shape tool rectangle circle oval or freeform shape to draw a feature that encloses the standard Circles or rectangles in a grid can also be used as standards On images where lanes have been added the band markers surrounding the concentration standard bands can be selected and used for quantification Note Image background can be added as a standard by drawing a feature around an area of uniform background and adding it as a standard as described below Important Concentration standards must be added in either ascending or descending order To help add the standards in the correct order visually identify all concentration standards on the image before beginning After drawing a feature around a concentration standard or selecting an existing feature like a band marker encompassing a concentration standard open the Concentration Standards window by choosing Analyze gt Concentration Sta
158. iii Chapter 1 Introduction How to Learn Odyssey If you are upgrading from a previous version of Odyssey software a list of changes for version 3 0 can be found in the help system In addition movies that illustrate new software features can be viewed by choosing Help gt What s New The best way for new users to learn Odyssey is to work through the tutorials in the Tutorial Manual The Tutorial Manual is a step by step guide that introduces you to scanning with the Odyssey Imager as well as analysis with Odyssey software The overview of Odyssey in the Tutorial Manual will familiarize you with basic operation of the entire system When you are ready for more information this User Guide is a reference manual with complete descriptions of sizing and quantifi cation as well as features of the Odyssey In Cell Western Module The Odyssey In vivo Imaging Guide describes optional software module and operational details for scanning mice using the Odyssey MousePOD Imaging Accessory Sample preparation is described in the Odyssey Application Protocols Manual and in the pack inserts enclosed with reagents Operation and maintenance of the Odyssey instrument can be found in the Odyssey Operator s Manual Documentation of the server software inside the Odyssey instrument is also included in the Operator s manual User account management networking trouble shooting scan control and software updates are all discussed 2 C
159. ile Save Close The image can be saved in three file formats TIFF files will have the largest file size and the highest quality because there is no compression The High Quality JPEG File setting uses a slight amount of JPEG compression to reduce file size but still maintains very high image quality Medium Quality JPEG File moderately compresses the image which produces some noticeable compression artifacts in the image compared to an image that is not compressed Medium Quality JPEG images are useful for e mails due to the small file size and may be suitable for web or slide presentations if the image artifacts are acceptable Medium Quality JPEG files are not recom mended for print publication When the file is saved all annotations currently displayed on the image will be saved in the JPEG file Use the Application settings to select which annotations are displayed Annotations can also be turned off or on by clicking Sal on the toolbar or by choosing View gt Hide Annotations Note If the Application Settings Image View Display have been used to enlarge the text display area in order to prevent annotations from being truncated at the edge of the image this extra text display area is included when an image view is exported assuming the extra text area is visible in the Image View window Exporting the TIFF Images Choose File gt Export Image gt Copy TIFF Files to save copies of the 16 bit grayscale TIFF images to any
160. ile Displaying the band background line can be useful in determining whether to use the band background or lane background as the background calculation method during quantification see Chapter 8 Displaying Lane Profiles With Background Fluorescence Removed When Lane Background Removed is selected in the Lane Profile window the lane profile is displayed with the lane background subtracted 81 Controlling Band Finding Using the Lane Profile Window The Threshold slider in the Lane Profile window is used to control the band finding software in order to more accurately find the correct number of bands in a lane 3 Lane Profile View Lane 1 800 channel Band Finding s Profile Percent Band Boundaries Threshold Band Background Fi Lane Background C Lane Background Removed Fewer Bands More Bands As the Threshold slider is moved band finding is changed in real time and bands are added or deleted on the lane profile as the slider moves If the threshold is set too high too many bands will be found when new lanes are pasted or created If the threshold is set too low too few bands will be found The correct threshold setting for a given lane is the threshold setting at which the number of bands matches the number of fluorescence peaks After adjusting the threshold click Apply to apply the changes to the lane on the image To optimize the default threshold setting observe typical thre
161. images the default sensitivity can be changed by picking a value from the Sensi tivity list Changing Image Colors From Red Green Using the 700 Channel and 800 Channel drop down lists the color of the 700 and 800 channel images for any new analysis can be changed to red green or blue Each channel must have a different color to distinguish fluorescence from each channel when channels are overlaid Choose View gt Adjust Image Curves to change colors for the current analysis Extending the Text Display Area Around Images When features or annotations are drawn near the edge of an image some of the text data values etc associated with the feature may be truncated at the edge of the image This is most likely to happen when Extended Text Area Around Image Edges is set to None the default If some of the text on an image is truncated increase the text display area by setting Extended Text Area Around Image Edges to Small or Large This does not add pixels to the image data it only allows truncated text to be displayed Note This additional text display area is included when an image view is printed File gt Print Image View or exported to a file File gt Export Image gt Export Image View 229 Chapter 12 User Accounts and Settings Application Settings When the Odyssey application starts the window shown below is displayed which can be used to choose the application settings for the current session This make
162. inks can also be dissolved by clicking Remove All to remove all current row links or selecting links in the link list and clicking Remove Selected Shift click and Ctrl click are available for multiple selections Calculations Tab After assigning any well links click the Calculations tab A complete description of the calculations can be found in Chapter 13 Well Types Well Links Calculations Select Calculations Used Subtract Background on All Channels Calculate Relative Intensity in Channel 800 v Normalize Channel 700 to 800 Calculate Response in 700 Channel When the ICW calculations start background subtraction is performed on all wells for each channel if Subtract Background on All Channels is enabled Background subtraction is normally enabled During background subtraction the integrated intensities of all wells designated as background in a given channel are averaged and subtracted from the integrated intensity of every well References to integrated intensity throughout the rest of this discussion refer to the original integrated intensity minus background In a typical ICW analysis the 700 channel might be used to detect phosphorylated proteins and the 800 channel used to detect total protein Phosphorylated proteins in this case are probed with IRDye 680 labeled secondary antibodies and total protein is detected using IRDye 800CW labeled secondary antibodies The 800 channel wh
163. ion draw a selection rectangle from the upper left corner of the image to the upper left corner of the main grid and get the offsets from the status message at the bottom of the Odyssey window Alternatively the properties can be opened and another template created thereby storing the current X and Y offsets and all other values in the new template The Save Grid As Template command on the Analyze menu is also useful for saving grid templates in certain cases The utility of this command is based on the fact that it saves everything about a grid 139 140 CHAPTER 7 Drawing Features on Images i e much more information than is in the grid template It saves everything about the grid including any adjustments to individual features grid rotation etc Using the Auto Shape Tool The purpose of the Auto Shape tool Y is to automatically create a feature that encloses fluorescence from irregularly shaped tumors or organs of small animals The Auto Shape tool is disabled unless you have purchased a key to unlock the software for the Odyssey MousePOD Module Naming Features and Adding Annotations Each feature drawn on the image is automatically assigned an ID number by Odyssey software To make a dot or band easier to identify on reports aname and description can be assigned To name a dot band select the feature that encloses it and click Properties 3 on the toolbar The name and description are retained with the feature
164. ion settings for lanes can increase band finding accuracy and make the initial lane boundaries more closely match the lanes in the image Profile Width The Profile Width field is the percentage of the lane width starting from the center that is used to generate the lane profile The default value of 75 seldom needs to be changed but unusual band shapes could require a higher or lower percentage in order to generate an accurate lane profile 88 CHAPTER 5 Creating Lanes and Finding Bands Total Width Each new lane is created with a width in millimeters equal to the Total Width parameter Setting Total Width to match the typical lane width on your images minimizes the lane width adjustments that need to be made when creating new lanes Band Finding Threshold The Band Finding Threshold slider is used to control the band finding software in order to more accurately find the correct number of bands in a lane If the threshold is set too high too many bands will be found when new lanes are pasted or created If the threshold is set too low too few bands will be found The proper threshold setting for a given lane can be determined using the Lane Profile window described earlier in this chapter After using the profile window to observe typical thresholds for a variety of scans the default value can be set more accurately Display Migration Band positions on images with bands in lanes can be specified in one of three units p
165. is and to change the sort order of the features on the X axis The Axis Label field can be used to switch between the feature name and 204 CHAPTER 10 Reports and Data Export ID When Sort is selected features on the X Axis will be sorted in descending order using the selected Field Features are sorted in ascending order when Ascending is selected X Axis Properties x Axis Label me RY r Sort Options M Sort Ascending Field Name v The font size on the X axis is set automatically and decreases as the number of data points increases If more than 45 data points need to be plotted on the X Axis the labels become unreadable and are removed automatically Information about a specific data point is displayed in a tool tip when the cursor is moved over the data point and stopped Using Templates Chart View templates store the Chart Style X Axis Properties and Display Values so they can quickly be loaded using the Load From Template list in the Chart View window After a template is loaded it can be changed by clicking Modify The Chart Style X Axis Properties and Display Values in the Edit Template window operate as described above for the Cart View window After editing the template click Save to change the current template or Save As to create a new template New templates can also be created by choosing Settings gt Chart View Templates Displaying and Exporting Statistics 205 Statistics can be disp
166. is or between images in different analyses Deleting Lanes Lanes can be selected and then deleted by clicking the X button If image channels are overlaid when a lane is deleted lanes in both image channels are deleted 72 CHAPTER 5 Creating Lanes and Finding Bands Creating Multiple Lanes The Add Multiple Lanes tool can be used any time there are evenly spaced lanes on the image This tool does not find lanes but instead creates the specified number of lanes and spreads them evenly over the area marked by the user Only vertical straight lanes can be created Before starting check the default lane width using the Appli cation settings as described at the end of this chapter Start by clicking the multi lane tool 8 or choosing Lane gt Add Multiple Lanes In the Add Multiple Lanes window enter the number of lanes to create and click OK Add Multiple Lanes Number of Lanes to be added Press OK Then drag a rectangle over the image area where the lanes should be added The first and last lanes will be centered on the left and right edges of the rectangle After clicking OK click and drag a rectangle over the image area where the lanes should be added Start in the center horizontally of the first lane at a height that will correspond to the upper lane 73 boundaries Release the mouse button when the right side of the rectangle is centered in the right most lane and at the vertical
167. ith the rotation cursor displayed click and drag the cursor upward or downward to rotate the grid Moving Features To move individual features first click the feature to select it and move the cursor inside the feature until the all arrows cursor is displayed With the all arrows cursor displayed click and drag the feature until it encloses all the fluorescence on the image Multiple features can be selected by holding down the Ctrl key while clicking additional features or by dragging a selection rectangle around multiple features 132 CHAPTER 7 Drawing Features on Images Note The circle or rectangle features in the grid do not have to stay within the bound aries of the grid cells The only thing that is important is that the features fully enclose the fluorescence in the image The grid lines are only a visual placement aid Changing the Feature Size or Type The size of a feature or type of feature can be changed after a grid is applied using the grid properties To open the grid properties select the grid and click the properties button Right clicking the image and choosing Properties from the popup menu also opens the grid properties Edit Grid Properties Grid Name 96_Vell_Plate Grid Size Rows 8 Columns 12 Spacing between Grid Lines mrn Vertical 9 00 Horizontal 9 00 Well Shape and Size mm O Square Well Diameter 6 50 Display VJ Show grid lines Desc
168. ixel location percent of lane or as a molecular weight value assuming size standards are assigned The number of decimal places used when the value is displayed can be changed using the Decimal Places field Pixel Location is the number of scan lines rows of pixels from the top of the image When Relative Mobility is chosen the top of the lane is 0 and the bottom 100 and bands are assigned a percentage based on position between the top and bottom of the lane Note In the Image View the band migration value is labeled Pix Locn when Pixel Location is selected Rel Mob when Relative Mobility is selected and MW when Size Standard is selected 89 Lane Color The lane border color and font color can be changed for lanes that are selected or unselected using the two sets of font color controls Controls are provided to pick colors for both color and black and white image display styles some colors do not work well for both display styles Changing the lane font color can make it easier to distinguish lanes from band markers Choose Image View Features in the Application settings list to set band marker color Displaying Band Quantification as a Percentage When Use Band s Percentage of Total Lane Integrated Intensity is selected the data value for each band is not integrated intensity but rather the band s percentage of the total integrated intensity of all bands in the lane The percentage is displayed in the tool tip as the cur
169. known values of the concentration standards to find the concentration of the unknown dot band In general the Reciprocal Fit interpolation method usually produces the best results There are exceptions however If the plot of the standards is very linear the Linear interpolation may give slightly better results If the image has sample dots bands that are a higher or lower concentration than any of the standards use the Log interpo lation method Whenever there is a need to extrapolate beyond the known concentration standards the Log interpolation method usually produces the best results Reviewing the Standards Plot The standards plot should also be used to look for anomalous standards Any standard that is out of position on the plot may need editing For example if a set of standards has a linear plot but the straight line is broken by a standard that is too high or too low the standard should be reviewed Standard 2 in the standards plot below appears to have a concentration value that is too high micromoles 800 300 00 237 50 175 00 112 50 50 00 p 0 11 21 62 43 13 64 64 86 15 When reviewing the anomalous standards make sure the standards are fully enclosed and that the feature drawn on the image is 151 centered over the fluorescence Also make sure that the correct concentration has been assigned to the correct feature After examining the standards for one channel use the Channel drop down list to switch t
170. layed for a set of features by selecting the features and choosing Report gt Stat Table View The Statistics Table displays the median average standard deviation minimum and maximum for the following variables for the set of selected features e Raw Integrated Intensity Integrated Intensity e Peak Intensity e Average Intensity e Trimmed Mean e Concentration e Background Each of these variables is described in the Chapter 13 Statistics for Selection for Scan TiterScan Analysis Grid Analysis Channel i 700 Quantification Raw Inten Median 19019 09 Average 18693 17 Std Dev 151575 Minimum 15772 63 Maximum 21190 88 i 800 Raw Inten 3075 52 4377 39 2925 27 1559 76 11002 91 i 700 Integ Inten 441 43 426 92 39 91 342 32 494 67 i 800 Integ Inten 76 53 113 99 80 61 35 47 291 56 i 700 Peak Inten 65 54 65 54 0 0 65 54 65 54 i 800 Peak Inten 7 24 14 48 18 34 3 27 65 54 Ave Inten 18 92 18 60 1 51 15 69 21 09 Ave Inten 3 06 4 36 2 91 1 55 10 95 Trimmed Mean 1776 17 54 0 94 15 49 19 53 Trimmed Mean 3 02 431 2 89 1 53 10 87 Concentration nia nia nia nia nia Concentration nia nia nia nia nia Background 3 65 3 81 0 55 2 88 4 68
171. le automatic image alignment when the Adjust Locations function is used to move features see Chapter 7 Changing to Grayscale Image Display Style If a single image is displayed rather than two images overlaid the image display style can be switched to grayscale by clicking in the tool bar or choosing View gt Grayscale The Alter Image Display window can also be used to switch between grayscale and color display as discussed earlier in this chapter Changing to Color Image Display Style While a single image is displayed the image display style can be switched to color by clicking in the tool bar or choosing View gt Color The Alter Image Display window can also be used to switch to the color display style as discussed earlier in this chapter Changing to Pseudo Color Image Display Style The pseudo color display tool on the tool bar is grayed out unless you have purchased a key to unlock the Odyssey MousePOD Module See the Odyssey In vivo Imaging Guide for details on pseudo image display Switching Between Image Channels If a single image is displayed rather than two images overlaid clicking 7 in the tool bar or choosing the channel on the View menu switches between the 700 channel image and the 800 channel image If the 700 channel image is displayed Channel 800 223 224 CHAPTER 11 Changing The Appearance Of The Image can be chosen from the View menu as shown below If the 800 channel image is display
172. le lanes molecular weight standards are assigned at the points where the molecular weight lines cross the centerline of the lane Control points are inflection points on the line that can be moved For gels with even band migration no other editing may be necessary since the straight lines between control points accurately represent the location of that molecular weight across the gel For gels with smiles the MW lines need to be edited and reshaped so the MW line curves and follows the contour of the gel Editing MW lines is described below Applying Standards to the Image The MW lines are now shown on the image but have not been permanently applied MW lines are applied to only the current image or to both images using the Apply or Apply to Both buttons respectively Apply is used when analyzing one image or when each image has its own MW markers When Apply to Both is clicked the MW lines on the current image are used to size bands on both images MW bands don t need to be present on the second image When Apply to Both or Apply is clicked the size standards are identified and all sample bands are automatically sized Adding MW Lines One at a Time The Edit Size Standards window described above can also be used for adding MW lines individually rather than in sets For gels with a few size standards that will be run just a few times it is better to add size standards individually for each gel rather than to save a set that w
173. lected Shapes C Name C Description Quantification Molecular Weight lt Boundary Font Color for Selected Shapes Color images white Grayscale images white x Grayscale images white x Shape Annotation Font for all Shapes Name Dialog v Size 14 v Rotation Rotate 45 COW Fonts Example Annotation Text Shape Tool Tips V Show Shape Tool Tips 92 CHAPTER 6 Band Sizing The settings for image view features can also be opened by choosing Settings gt Application and then selecting Image View Features in the Settings List The settings for image view features are an important part of any analysis because they can be used to reduce the screen clutter that can occur when annotations are displayed on closely spaced screen objects There are two groups of Image View settings those for selected screen features and those for unselected features One strategy to reduce screen clutter is to treat selected and unselected features i e band markers etc differently If all annotations are turned on for selected features and all annotations except boundaries are turned off for unselected features the display will stay unclut tered and each individual feature can be clicked to display its annotation molecular weight etc In other situations like exporting annotated images it is useful to have annotations turned on for unselected features For example before
174. ll be assigned Boundary Line Displaying Band Boundaries When Band Boundaries is selected in the Lane Profile window vertical magenta lines are placed on the profile at the beginning and end of each band as shown above These lines correspond to the upper and lower sides of the band marker on the image If lane finding identifies too many bands band boundaries are useful for identifying false bands False bands are usually centered on some small noise peak in the fluorescence curve rather than on a fluores cence peak If a band marker is too small it will be apparent in the lane profile because the band boundaries will be on the sides of a peak rather than down in the image noise between peaks Displaying Band Background Fluorescence With Band Background selected the calculated band background is displayed as a horizontal line between the band boundary lines Band Background Line 79 80 CHAPTER 5 Creating Lanes and Finding Bands Displaying the band background line can be useful in determining whether to use the band background or lane background as the background calculation method during quantification see Chapter 8 Displaying Lane Background Fluorescence When Lane Background is selected a profile for the lane background is displayed beneath the lane profile Lane Background Line The lane background line is calculated by selecting appropriate minima points from the lane prof
175. location 16 bit TIFF images can be used in Odyssey software and other analysis software that accepts 16 bit grayscale images Commercial image editing programs Adobe Photoshop etc generally require 8 bit TIFF images see Exporting 8 bit Grayscale Images below Copy Tiff Files Scan Name FirstScan Analysis Name Original Analysis New Images 700 ments and Settings RONULicor Odyssey Reports CopyofS700 TIF 800 ments and Settings RONILicoriOdyssey Reports CopyotS800 TIF Browse Remember this location next time Automatically fill all channel locations The Browse button opens a standard file selection window If Automatically fill all Channel Locations is selected the path for the second channel is automatically updated when the path for the first 57 58 CHAPTER 4 Importing and Exporting Scans and Images channel is chosen using the Browse button To always save the copied images in the same location select Remember this Location Next Time Exporting 8 bit Grayscale Images Choose File gt Export Image gt 8 bit Grayscale TIFF to export 8 bit grayscale TIFF images rather than the 16 bit grayscale images scanned by the Odyssey System 8 bit grayscale images can be used with commercial image editing software and presentation software such as Microsoft PowerPoint The Create Grayscale Tiff window works as described above for copying TIFF files Note Quantitative analysis should no
176. location and place it so the features in the subgrid are aligned as well as possible with the fluores cence on the image Tip When a subgrid is selected right clicking the image opens a pop up menu that has several useful choices for selecting all or part of a subgrid single rows columns etc 10 Select Analyze gt Adjust Feature Location to move the features to their final location If spot finding is not very accurate select Edit gt Undo Adjust Feature Locations change the spot finding param eters in the Application settings discussed earlier in this chapter and select Analyze gt Adjust Feature Location again On images with a lot of extraneous fluorescence e g microplates with fluorescing side walls it may be necessary to move the features manually to their final locations Tips If the main grid cannot be moved far enough because it contacts the side of the image move the subgrids instead An easy way to select all the subgrids is to select the main grid right click on the image and select Select All Subgrids On Grid from the context menu All the subgrids can then be moved by clicking and dragging them The subgrids can be moved outside of the cells of the main grid without causing any problems If the main grid template is to be used for multiple similar images the template can be fine tuned by measuring the X and Y offsets and entering the offsets in the main grid template After the grid is in its final locat
177. ls Overlaid which is used to overlay the images again In addition menus are activated to switch between channels and to switch between various image display styles Icons on the tool bar are activated to change the image display style from color to grayscale 8 or back to color as well as pseudo color M if you have the optional Odyssey MousePOD Module To switch between channels click 7 Aligning Images When scanning at high resolution 84 42 and 21 um it is possible for the 700 and 800 channel images to be misaligned The image below shows a microplate scan where the 700 channel image red is horizontally offset from the 800 channel green image Images can be aligned by choosing File gt Align Images Important There is no undo operation for image alignment because the TIFF image files are immediately changed 222 CHAPTER 11 Changing The Appearance Of The Image Align Images of Scan Alignment Direction Reference Channel 800 s Up Left l Cancel Reset To correct an offset start by using the zoom buttons to make the offset clearly visible Next select the reference channel that stays stationary while the other channel is moved Last move the channel not designated as the reference using the Alignment Direction buttons The Up Down Left and Right buttons move the image one pixel at a time To start over click Reset Note The Application settings can be used to enab
178. ls in the feature Shape Area Area of a feature in mm Area is the number of pix els multiplied by the area per pixel Area per pixel a resolution x 10 191 192 CHAPTER 10 Reports and Data Export Field Definition Width Width of an imaginary bounding rectangle around the feature Height Height of an imaginary bounding rectangle around the feature Shape Shape of a feature circle rectangle etc Ref Point X X coordinate value of the upper left corner of the feature Ref Point Y Y coordinate value of the upper left corner of the feature Center X X coordinate value of the center of the feature Center Y Y coordinate value of the center of the feature Background Background value calculated for the feature See Bkgd Method description in Chapter 13 Background calculation method used to calculate background See Chapter 8 Project Name of project containing the feature Scan Name of scan containing the feature Analysis Name of analysis containing the feature MW Molecular weight of a band MW w units Same as MW with units label Sat Number of saturated pixels divided by the total number of pixels multiplied by 100 to give a per centage Probability See description in Chapter 13 Trimmed Mean SN Ratio See description in Chapter 13 Signal to Noise Ratio See Chapter 13 Field Definition Std Dev Standa
179. lways 249 equal to the number of pixels included in the feature itself If you want to remove background correction as a variable set background equal to zero In that case it is best to draw your features so they fit the spots as closely as possible and use Integrated Intensity as the measure Integrated Intensity should be used as the basis for quantitative measurements Odyssey Calculations Number of Pixels Pixel Area and Shape Area The number of image pixels enclosed by a feature is the variable Pixels which can be listed in reports The area enclosed by a feature mm is reported in the variable Shape Area Shape Area Pixels x a where area per pixel a resolution x 10 Background Background b is the average intensity of pixels selected as the background region b Eb np where bj is the intensity of the i background pixel and ng is the number of pixels in the region selected as background The pixels used to calculate background are assigned by choosing Analyze gt Background Method The background can be set to No Background Average Median User Defined or Lane Background Method For Bands Chapter 8 describes each of the background methods in detail 250 CHAPTER 13 Calculation Descriptions Raw Integrated Intensity Raw Integrated Intensity is defined as Raw Integrated Intensity Xl where is the total intensity of the i pixel enclosed by the feature Units are counts Raw
180. ly the same position as the original analysis and molecular weight standards are applied in the same way If Refind Bands on Lanes is selected lanes are placed in the same position but the stored band markers are discarded and the automatic band finding software is used to find bands within the lanes Deleting a Lane Template Click Eg to open the template selection window Select a template and click Delete Using the Application Settings To open the Application settings for lanes choose Settings gt Appli cation and then select Lane from the Settings List 87 Application Settings Odyssey Settings Settings List New Lanes General Profile Width 75 Image View Features 4 Image view Display Total Width left right mm 4 Display Values Band Finding Threshold Lane Adjust Locations y Auto Shape T Naming Conventions o 5 10 15 20 Report Fewer Bands More Bands Display Migration Pixel Location Relative Mobility RF O Size Standard Decimal Places 2 ry Font Color for Unselected Lanes Font Color for Selected Lanes Color images D white Color images magenta Grayscale images I white v Grayscale images E yelow Display Band Quantification C Use the band s percentage of total lane Integrated Intensity total II Application settings for lanes are used to control the characteristics of a lane when it is first created Adjusting the Applicat
181. mage proceed with analysis and adjust the grid template to match the location of the wells If the wells are truncated or if the grid is off the image adjust the Scan X Offset and Scan Y Offset in the Multiple Scan settings to center the microplates in the images Only integer numbers should be entered and units are 0 1 mm For example a Scan X Offset of 15 is an offset of 1 5 mm For non standard plates scan sizes and offsets have to be determined experimentally Start with the default values above and increase or decrease them as needed Set the scan width and height inside the plate edges but outside of the rectangle defining the well bound aries Set the Scan X Offset to the distance along the X axis from the right boundary of one scan to the left boundary of the next scan Similarly set the Scan Y Offset to the distance along the Y axis from the upper boundary of one scan to the lower boundary of the next scan If the scan size and offset are changed you will also need to edit a grid template so the template matches the non standard plate size 37 Chapter 3 Creating a New Analysis Overview An analysis holds all the sizing or quantification data created when ascan is analyzed For each scan there can be many analyses At the end of each new scan a new analysis is named and saved along with the new scan as described in Chapter 2 For existing scans a new analysis can be created by choosing File gt Analysis gt New
182. mage files will be closed and saved and Odyssey will start the scan of the next plate if any To abandon a scan and not save the image files click Cancel rather than Stop Clicking Cancel also cancels all other plates from being scanned Multiple Scan Settings In the setup window for multi plate scanning a default base name is presented that is used to name all plate scans for a given series of scans The default base name can be specified on Base Name tab in 5 36 CHAPTER 2 Starting Scans the Multiple Scan Settings window To open the Multiple Scan settings choose Settings gt Multiple Scan Multiple Scan Settings x i Locations O Date Stamp YYYY_MMM_DD Date Stamp YYYY_MMM_DD_HH O Text Scan The default name can be set to the current date the current date plus current hour or user designated text In the setup window for multi plate scanning the user is not given the option of setting the scan size or XY offset This is because the scan size is based on a standard microplate size The scan size and XY offset for microplates recommended by LI COR are shown below Multiple Scan Settings ed Base Name Scan Width Scan Height Scan X Offset Scan Y Offset All values in tenths of mm Example 1278 is 127 8 mm These settings are only a guide and every instrument may not produce images with the microplate exactly centered If the micro plate is not centered in the i
183. meters Resolution v Quality medium Focus Offset 0 0 Scanner Back C Microplate Flip image Channels 700 800 Intensity 5 0 v 5 0 i Scan Area cm X Coord Coord Origin o o Width Height Size 10 10 Image Size 700 Kbytes per channel Scanner Front Specify the scan area by pressing the left mouse button and dragging the cursor estimated scan time 6 minutes 49 seconds Resolution can be set to 21 42 84 169 or 337 um For typical scans of membranes or gels 169 um scans should suffice As resolution increases file sizes get very large The table below shows the resolution and scan size limits for starting scans with Odyssey Software File sizes under 7 MB per image scan well and can be analyzed in Odyssey Software without cropping the image into smaller pieces File sizes from 7 14 MB are marginal and Odyssey Software may 20 CHAPTER 2 Starting Scans run out of memory during a scan Scans with file sizes larger than 14 MB per image can be performed using the browser interface as described in the Odyssey Operator s Manual but should not be attempted with Odyssey Software The table below shows typical combinations of resolution and scan size with shading to indicate file sizes that are too large for Odyssey Software Resolution
184. microplate alignment guide is used to position the microplate at a known location on the scan surface Push the guide into the lower left corner until it contacts the bezel surrounding the scan surface on both the front and left sides Place the microplate on the scanning surface and slide it into position until it contacts both the front and left side of the alignment guide The first well in the first row A1 should be toward the back and left side of the alignment 25 guide as shown below When the microplate is placed against the alignment guide the scan size and origin parameters in the default microplate scan preset should work well Alignment Guide After placing the membrane gel or microplate on the scanning surface close the lid on the Odyssey Imager For scanning mice with the MousePOD Accessory consult the Odyssey n vivo Imaging Guide included with the MousePOD 26 CHAPTER 2 Starting Scans Starting a Standard Scan Status line To start a standard scan click the Start Scan button in the Scanner Console to send the scan parameters to the Odyssey Imager and start the scan The images are displayed in real time in the area of the Scanner Console window where the scan grid was located Scanner Console BandSizing User jen Description Name Group Preset Scan Parameters The images are displayed as they are scanned Resolution Quality Focus Offset 00 mm e lip image
185. n the subgrid Enter the horizontal and vertical spacing measured in Step 1 Select the type of feature circle square and enter the value measured in Step 2 for the physical and quantify size vary the quantify size if you need to quantify an area that is less than the entire well spot Enter a small number such as 0 5 for X and Y offsets When the subgrid is applied it will be in the upper left corner of the image which is fine for this design phase 137 8 Select the Center in Main Grid When Used as Sub grid check box so the subgrid will be centered in the cells of the main grid when the main grid is applied 9 Click Save and name the template for the subgrid 10 In the Odyssey window click the grid tool and apply the subgrid template to the image 11 Move the subgrid into position and size the entire subgrid to fit the image as needed 12 Check to make sure the feature size in the subgrid is correct If not click on the toolbar to open the properties and change the feature size to fit Repeat as necessary 13 After the grid and grid features are correctly sized click 4 on the toolbar to open the properties and click Create Template 14 Check to make sure the Center in Main Grid When Used as Subgrid check box is selected and enter small numbers e g 0 5 in the X and Y offset fields Click Save As enter a name for the new template and click OK The original subgrid template can either be delete
186. nails view can be opened using the View menu Folders View Folders view is opened or hidden by choosing View gt Folders View A close button in the upper right corner hides the view Odyssey project folders which contain scans have a unique icon as do the scan folders within project folders Projects that are open and have been edited are shown with a pencil icon W All folders can be expanded by clicking the plus icon next to the folder 9 Folders can also be expanded by double clicking them When an Odyssey project folder is double clicked the project is opened and shown in the Scans view The context menu that opens 8 CHAPTER 1 Introduction by right clicking on a folder can also be used to open and close a folder Note Starting with Odyssey software version 3 0 project folders can be stored in any location Local drives and mapped network drives generally offer the best perfor mance In addition to browsing for projects and scans in Folders view a search function File gt Scan gt Search for Scans is available to find scans if the file name is known or partially known The list of recently open projects on the File menu is also a fast way to open projects Scans View The Scans view shows all scans and analyses for the current project The name of the current project is shown in Scans view and highlighted in Folders view Project Scans andsizing EE Scan F First_Analysis i MyAn
187. nd Enhancements 2 1 Not for Resale Software The SOFTWARE PRODUCT is not available for resale and therefore notwithstanding other sections of the Agreement you may not resell or otherwise transfer for value the SOFTWARE PRODUCT 2 2 Limitations on Reverse Engineering Decompilation and Disassembly You may not reverse engineer decompile adapt translate disassemble or create derivative works based up any portion of the SOFTWARE PRODUCT except and only to the extent that such activity is expressly permitted under a Third Party Software license or by applicable law 2 3 Separation of Components The SOFTWARE PRODUCT is licensed as a single product Its component parts may not be separated for use on more than one computer 2 4 Rental You may not rent lease or lend the SOFTWARE PRODUCT 2 5 Support Services LI COR may provide you with support services related to the SOFTWARE PRODUCT Support Services Use of Support Services is governed by LI COR polices and programs described in the user manual in online documentation and or in other LI COR provided materials Any supplemental software code provided to you as part of the Support Services shall be considered part of the SOFTWARE PRODUCT and is subject to the terms and conditions of this Agreement With respect to technical information you provide to LI COR as part of the Support Services LI COR may use such infor mation for its business purposes including for product support and devel
188. nd IRDye reagents are covered by U S patents foreign equivalents and patents pending Table Of Contents Chapter 1 Introduction How to Learn Odyssey s 1 Selecting an Application Settings File at E E N T E adeeened 2 Setting Up Users and Scanners s s s 2 The Odyssey Help System eee 3 TOO A E N 4 Context Sensitive Menus ssssssssserssesees 4 Correcting Mistak soncnencccimsenicesis 5 Odyssey File and Project Organization 5 Projects Scans and Analyses s 6 Displaying Projects Scans and Analyses in the Main Odyssey Window 008 6 Folders ViW ccsccsscesseecseeceneceneeeneenees 7 SCANS Vie Wesen en en er en T R 8 Thumbnails View seee 9 Chapter 2 Starting Scans How to Start SCANS ccsvasdsierteesssetendesates 11 Starting Standard Scans in an Existing Projetista nii aE E 11 Starting a Standard Scan in a New Project 12 Scanner Console Window for Standard SCANS recocnerre eenn 13 Naming a Scan and Entering a Description sucses 14 Changing the Default Scan Name 14 Previewing a Scan Optional n s 15 Selecting a Scan Group seeeeeeeeeeeeee 16 Setting Scanner Paramters for Standard SCAMS io E r E RE E Ni 16 Loading Preset Parameters 0 0008 16 Editing Scan Parameters For Standard SCAMS eoar re REE EEEE E SEESE ES EEES 19 Placing Samples on the Scan Surface 24 Starting a Standard Scan 27 Stopping a SCAN ssse
189. nd Subtraction Background subtraction finds the minimum intensity value on the image and subtracts that intensity from all pixels in the image Subtract Before subtraction After subtraction Cropping Images To crop an image draw a selection rectangle around the desired area of the image and click the Crop button The selection rectangle is drawn by moving the cursor to one of the four corners of the crop area Click and drag the cursor while holding down the left mouse button Don t release the mouse button until the border of the selection area encloses the entire crop area Analysis Image 44 CHAPTER 3 Creating A New Analysis IMPORTANT Be careful not to crop too close to bands or other objects that will be analyzed Leave some empty image around the edges If the image is cropped too close to bands it may be difficult to add text lanes or features near the edge of the window Using Image Filters IMPORTANT When quantifying bands dots none of the filters should be used since they change the image data thereby invali dating the quantification results Filter Options Fitter Options O Smooth O Local Minimum O Sharpen O Local Maximum ay Filtering operations change the image data for the new analysis and may affect quantification results Each filter operation uses a 3 x 3 pixel convolution filter A brief operational description of each filter is given below Noise Removal The Noise
190. nd points Curved lanes can be reshaped using the same click and drag method on any point in the lane Copying and Pasting Lanes After creating the first lane additional lanes can be added by copying and pasting lanes This method however is useful only if the other lanes on the image have a similar shape Copy a lane by selecting it and pressing Ctrl c on the keyboard Lanes are pasted at the cursor position unless the Paste Special command is used Move the mouse cursor to a point on the image that is centered and at the top of an unmarked lane shown below 71 Press Ctrl v on the keyboard to paste a new lane at the position of the mouse pointer Bands in the new lane are found automatically Note Cut Copy Paste and Undo are also available as tools on the left side toolbar Copying Multiple Lanes Multiple lanes can be selected by holding down the Control key and clicking each lane or by clicking and dragging a selection box around the desired lanes Ctrl c and Ctrl v can then be used to copy and paste the lanes Using the Paste Special Command The Paste Special choice on the Edit menu can also be used to paste lanes that have been copied but the result is slightly different than the Paste choice Ctrl v Paste Special pastes lanes in the exact same coordinate position as the lanes that were copied Paste Special is generally used to copy lanes between images Lanes can be copied between images in the same analys
191. ndards or click 4 on the toolbar Concentration Standards Standard Plot micromoles 0 of 2 required standards currently assigned 00 integ Inten 0 0 Standards Settings Channel s00 w Clear Interpolation method Linear he Enter New Standards Values o fucronces Select Enea then add a standard First make sure the Channel 700 or 800 matches the image being analyzed Next enter the concentration for the selected standard in the Enter New Standards Values field select the units and click Add New Note Changing units changes the units for all standards on both images The units need only be set once for each analysis The first standard is now added Continue adding the other standards by selecting a feature entering the concentration in the Concen tration Standards window and clicking Add New the Concentration Standards window can be left open The standards must be added in ascending or descending order Setting the Interpolation Method After the standards are entered the interpolation method should be set and the standards plot reviewed for anomalous standards 149 150 CHAPTER 8 Quantification The concentration standards plot shows integrated intensity on the X axis plotted against concentration on the Y axis For each dot or band of unknown concentration Odyssey uses the integrated intensity of the dot band and interpolates between the
192. nding e To verify that band markers are centered on the fluorescence peaks e To change band finding threshold and re find bands e To view a graphical display of background fluorescence in the lane e To view the fluorescence peaks with background fluorescence removed Understanding the Lane Profile The X axis of the lane profile represents the vertical position in the lane The scale for the X axis is shown above the graph and units are user selectable in the Application settings for lanes Band centers Y Axis Average intensity of all pixels within the lane profile width for a given row of pixels Lane X Axis Lane Top Molecular weight or band Bottom migration value Migration in pixels is shown Lane profile width percentage from settings pin 1 eet Lane rotated 90 counterclockwise for illustration Units for the X axis can be e Pixel Location shown distance in pixels from the image to the band center in pixels 78 CHAPTER 5 Creating Lanes and Finding Bands e Relative Mobility Relative distance from the top of the lane The top of the lane left side of lane profile is assigned a value of zero and the bottom of the lane right side of lane profile is assigned a value of one Size Standard Molecular weight calculated using sized standards currently applied to the image Units are those previously assigned to the size standard when it was created The cursor position on
193. nitial graph To open a chart view click on the toolbar or choose Report gt Chart View Chart View can also be chosen on the contextual menu accessed by right clicking the image The controls in the bottom half of the Chart View window allow the chart to be changed interactively The Chart Style can be used to switch between a line chart and a bar chart The Display Values list shows the data value average intensity integrated intensity etc graphed for each feature The list also shows the Channel the data belong to and the Color used for identification Existing values can 203 be changed using the drop down menus in the Field and Channel columns Color can be changed by clicking the color button in the appropriate row and choosing a new color from the color palette Chart View TiterScan Grid Analysis Shapes pe o cont cot cos cath gs at Name W 700 Avg Intensity W 800 Avg Intensity r Chart Template Load from template veintensityChartTemplate v Mouity Chart Style X Anis Properties r Display Values Fiela Channel Color lal Avg Intensity zl 2j Avg Intensity l L To remove a set of data values click the row number first column to select the row and click Remove To add a set of data values click Add and then set the Field Channel and Color as desired The X Axis Properties button can be used to change the label on the X ax
194. notations You May See Either integrated intensity or concentration will be displayed after a feature is created depending on the Application settings If concen tration values are displayed the concentration is n a not assigned until concentration standards are assigned and the concentration of unknown dots bands can be calculated If the Application settings are set to display integrated intensity values integrated intensity will be displayed immediately after the feature is drawn Hiding Annotations If annotations obscure other image features annotations can be temporarily hidden by clicking a on the toolbar or by choosing View gt Hide Annotations To view hidden annotations click 8 on the toolbar again or choose View gt Show Annotations Note To reduce screen clutter use the Application settings to control which annota tions are displayed for both selected and unselected features Chapter 8 Quantification Overview Quantification can begin after a new analysis is started or an existing analysis is opened Chapter 3 Before quantification features must be drawn on the image Chapter 7 or lanes and bands must be found Chapter 5 for images with bands in lanes For scans with both 700 and 800 channel images concentration standards in one channel cannot be used to quantify dots or bands in the other channel Concentration standards for both dyes must be loaded and each image channel must be analyzed separately
195. nreliable data A Z factor value that is close to zero or negative indicates assay conditions have not been optimized or the assay is not capable of generating meaningful data Enabling Z Factor Calculations Z factor calculations are enabled by choosing In Cell Western gt Change ICW Parameters and switching to the Calculations tab At the bottom of the Calculations tab select Calculations are for Z Factor 2 Factor Calculations are for 2 Factor When Z Factor Analysis is used Well Links are ignored Calculate Relative Intensity in Channel must be selected for Z Factor analysis and the appropriate Normalize Channel selected When Calculations are for Z Factor is selected In Cell Western calculations are not performed and any well links are ignored if links are defined on the Links tab of the Change ICW Parameters window If background wells are loaded make sure Subtract Background on All Channels is selected When using Z factor calculations one image channel should always be used for normalization in order to compensate for variations in cell number between wells Make sure 180 CHAPTER 9 Odyssey In Cell Western Module Calculate Relative Intensity in Channel is selected and the correct channel for normalization is chosen Change ICW Parameters for Analysis Z Factor Well Types Well Links Calculations Select ICW Calculations Used K Calculate Relative Intensity in 4 1700 v Normalize C
196. ntioned earlier the sides of the blue rectangle in Details View are the horizontal and vertical boundaries of the feature on the image The pixels on the outside perimeter of the blue rectangle are the pixels used for background calculations In the Details View shown above the pixels just outside the blue rectangle on all four sides are empty background pixels For this reason either the Average or Median All method defined below are appropriate Both of these background calculation methods use the pixels surrounding all four sides of the blue rectangle Suppose however that there was another dot very close to the dot shown in Details View and that fluorescence from the second dot contacted the left side of the blue rectangle In that case the left side should not be used in the background calculation because the added fluorescence would increase the calculated background The Average and Median All methods would not be appropriate because they use all four sides In this case the Median method that uses only the top and bottom lines would be the appropriate background method The User Defined background method could also be used Comparing Data Using Details View Details View lists a variety of useful values including concentration With the Details View open each time a feature is selected the infor mation for that feature is added to the table To compare the concen tration of two features just select them and examine the value
197. nu Choices Zoom In Same as described above e Zoom Out Decreases image magnification 220 CHAPTER 11 Changing The Appearance Of The Image e 100 Returns the zoom level to 100 Zoom by Dragging Click and drag a rectangle around an area to zoom in on and release the mouse button when the area is enclosed R The rectangular region is enlarged to fit in the current Image View window To get a lot of magnification enlarge the Image View window before using the Zoom by Dragging function Restore Zoom Restore Zoom works in conjunction with Zoom by Dragging and resets to the zoom level prior to using Zoom by Dragging 10 Use the drop down magnification list to select a specific magnification percent of original size Keyboard Shortcuts Function key F11 increases image magnification Unlike Zoom In on the View menu however the zoom is centered at the cursor position as long as the cursor is over some part of the image Function key F12 zooms out centered at the cursor position 221 Overlaid Images For scans that have both 700 and 800 channel images the images are automatically overlaid when an analysis is opened To display a single image choose View gt Single Channel or click the overlay button on the tool bar The overlay button toggles between Channels Overlaid and Single Channel display The View menu changes when a single channel is displayed The Single Channel menu changes to Channe
198. nu is used to change your own password and scan groups User accounts passwords and scan groups are stored on the Odyssey instrument See System Administration below to make changes to user accounts or scan groups other than your own Changing Your Own Password 1 Choose Settings gt User Administration 1 Scanner Login If someone else is already logged in click Logout before entering your User Name and Password West Lab User Name ron Password tee 231 2 Select a scanner from the Scanner list and enter the User Name and Password of your account Click OK to continue See Scanner Settings if no scanners are listed User Administration User ron Scanner West Lab Location User and Scan Group Management Change Password Manage Scan Groups 3 Click Change Password and enter your current password in the Old Password field 4 Enter a new password in the New Password field and repeat it in the Re enter Password field Change Password Old Password tirrr New Password EEEE EE Reenter New Password seeee 5 Click OK to change your password Managing Your Own Scan Groups 1 Choose Settings gt User Administration 2 Select a scanner from the Scanner list and enter the user name and password of your account Click OK to continue 3 Click Manage Scan Groups 232 CHAPTER 12 User Accounts and Settings Scans are stored
199. o the other channel and examine the plot of the concentration standards on the other image Changing and Deleting Concentration Standards After a concentration standard is defined in the Concentration Standards window it cannot be changed To assign a different concentration value to a standard all standards must be cleared and reassigned using the new values Standards are cleared by clicking the Clear button in the Concentration Standards window Note Deleting a feature on the image that encloses a concentration standard does not delete the standard Using the Details View for Background Verification Having the Details View open when new features are drawn on the image gives immediate access to a variety of useful information about the newly quantified feature Details View also provides an easy way to compare data values for various features on the image The Details View is opened by choosing Report gt Details View or by clicking on the toolbar Details view can also be opened by selecting a feature right clicking the image and choosing Details View from the pop up menu With Details View open an enlarged view of the band spot is displayed each time a feature is added to the image Details View is useful for verifying the location of the feature over the band spot and the background calculation method The feature shown surrounding 152 CHAPTER 8 Quantification Any column in the Details View can be moved by dra
200. of the font reducing the size of the font or even changing to a different font can improve the readability of notations on the image A sample of the currently selected font and font colors is shown in the Fonts Example area Separate controls are provided to pick font colors for both color and black and white image display styles some colors do not work well for both display styles Annotations can be rotated 45 or 90 degrees counter clockwise using the Rotation list Checking the Display Migration Settings Odyssey can display the migration of a band through the gel as a molecular weight value scan line pixel row on the image or a percentage of the lane To change the way band migration is displayed choose Settings gt Application Settings and then select Lane from the Settings List 94 CHAPTER 6 Band Sizing Application Settings Odyssey Settings Settings List New Lanes General Profile Width 75 Image View Features gt A a Image View Display Total Width left right mm 4 Display values Band Finding Threshold Lane Adjust Locations 5 Auto Shape l va ib l a l Naming Conventions 0 5 10 15 20 Report Fewer Bands More Bands Display Migration O Pixel Location Relative Mobility RF O Size Standard Decimal Places 28 Font Color for Unselected Lanes Font Color for Selected Lanes Color images JE white v Color images IE magenta Y Grayscale images JE white v Grayscale images oy yellow
201. on is not exact both because a uniform background is not necessarily constant and statistical fluctu ations can and do occur but also because the background is not always uniform Nevertheless you can confirm that Integrated Intensity is substantially independent of feature size by drawing features of varying sizes around a spot with a uniform background and observing in the Details View that the Integrated Intensity changes very little Note also that Integrated Intensity is substantially independent of resolution Lower resolution means fewer and larger pixels For features that are large compared to pixel size the intensities recorded for pixels in similar locations over the feature will be about the same regardless of pixel size because the intensities are deter mined by the amount of sample and or background at each location but as the pixels get larger the number of terms in the summation gets smaller so the sum gets smaller This is compensated by the increase in pixel area a Normally background pixels are taken from three or so rows of pixels adjacent to the perimeter of a rectangle that encloses the feature drawn on the image If one of the sides of the rectangle includes an anomaly then either the two horizontal sides or the two vertical sides can be selected If spots are too close to reliably isolate uniform background then another region on the gel can be selected from which to compute b however the number of pixels n is a
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203. orial unknown QuantScan lt C Documents and Settings RON Licor Odyssey Projects Tutorial rm unknown Any scan in the search list can be opened by double clicking the appropriate row or by clicking a row and clicking Open Project If a search is still in progress opening a scan stops the search and opens the project containing the scan 51 Initially the table is sorted in ascending order by Scan name Click the Scan header to switch to descending order To sort by a different column click the column header and click again to switch from ascending to descending order as needed To close the search window click Close Close can also be clicked during a search to stop the search and close the window Downloading Scans The Download Scan function is most commonly used to retrieve scans that were started from the Odyssey front panel keypad and stored in Odyssey s public scan group see Odyssey Operator s Manual By comparison a scan started from within the Odyssey application software is downloaded to the computer automatically when the Save button is clicked at the end of the scan The Download Scan function can also be used to download another copy of a completed scan that is still stored on Odyssey Note Odyssey should not be used as a long term storage device for scans It is best to download scans immediately after they are finished A project must be open before scans can be downloaded
204. orts for In Cell Westerns 175 Printing and Saving Reports 4 175 Changing the ICW Report Template 176 ICW Export SettingS cccceeeeeeeeee 178 Assay Optimization With Z factor Cal CUlALOMSixcavsvcsesweitestiussdessdat davianeretstaes 178 Enabling Z Factor Calculations 179 Viewing Z Factor Values s s s 180 Chapter 10 Reports and Data Export Report Table View 0 0 cece renee 183 Printing and Exporting Reports 184 Printing REPOMS ccs cezccsscsdedecsgenssectgeesd 185 Exporting Report Files 0 eee 186 Report Seting iscesssecsueeesetdeesuectgenseectgeesd 186 Creating Report Templates 0 0 00 188 Choosing Fields to Include in the REPOM sesssicesSedaviecu sess sudsseetadhansst siais 190 Saving the Template eee 190 Field Definitions eee 191 Plug in REPOMS enra aa aS 196 Launching Plug in Reports 197 Editing Plug in Reports see 198 Adding and Deleting Plug ins 201 Troubleshooting Plug in Reports 201 Graphing Datars sesssssssesscezsacevessssssezestestonts 202 Using Templates ieren 204 Displaying and Exporting Statistics 205 Printing an Image VieW cceceeeeeeeee 206 Viewing and Printing the Scanner Log 207 Chapter 11 Changing the Appearance of Scanned Images Image Display Adjustments 006 209 Changing How Image Data Are Mapped to the Monitor
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206. ould become useless after the gels are run Adding MW lines individually is very similar to using size standard sets except that each MW line is created individually by entering its MW in the Edit Size Standards window As with size standard sets the number of MW lines added must exactly match the number of bands in the lanes containing standards After the MW lines are added the lines are snapped to their respective bands and the MW of each sample band is found as soon as the MW lines are applied to the image In order to add MW lines lanes should be found and only one image displayed Adding MW Lines MW lines are added by choosing Lane gt Edit Size Standards Individual MW lines are added by entering the size in the New MW Value field and clicking Add MW Line Later in the chapter you will see how to change the units in the Size Standards window 101 102 CHAPTER 6 Band Sizing Edit Size Stds for 800 TIF Add MWN Line or Select MVV Set Add MWY Line Appl Std Set New MW Value 75 0 Select MWY Std Set Select a Std Set v Edit Mode O Add Points Modify Lines O Select Lanes O Modify Points Apply to Both Apply Cancel 4 i nba t After clicking Add MW Line a MW line is displayed on the image that will connect all the standard bands of a particular weight 75 bp in the example above The MW line itself represents all the points of equ
207. ow was opened The Default button changes the image display settings to the default programmed settings Adjusting Image Curves Adjust Image Curves window can be opened by clicking on the tool bar or choosing View gt Adjust Image Curves The Adjust Image Curves window can also be opened from the Scanner Console window or the New Analysis window The channel area is used Adjust Image Curves to select the channel to a view and change its 800_53 TIF show appearance C only show current channel Inverse Grayscale ST iit Min 0 Max 1939 Gamma 1 35 At x 3385 mapped y 255 Contract Expand v Histogram Olmer Oog OK Cancel 214 CHAPTER 11 Changing The Appearance Of The Image Note If the images were previously adjusted using the Alter Image Display function a message may be displayed indicating the image curve settings are not stored for the image There is not always a direct conversion between the two image manipulation methods used in Alter Image Display and Adjust Image Curves so defaults are used when switching back and forth between the two methods Start by selecting the channel 700 or 800 to adjust The Channel drop down menu or the Next and Prev buttons can be used to switch channels A histogram of intensity values and an adjustment curve for the selected channel are displayed in the middle of the window Any adjustments made to the curves are immedi
208. p Project Project Tutorial Destination Folder D Ody_Images OdyProjBackup If you intend to frequently back up projects to the same directory after making changes click Overwrite Files in the Destination Folder so the older copy is overwritten by the new version 65 Chapter 5 Creating Lanes and Finding Bands Lanes can be created by several methods Typically lanes are created one at a time by selecting the lane tool clicking at the top center of a lane and then double clicking at the bottom center Lane bound aries are drawn according to the lane width specified in the Appli cation settings Immediately after a lane is created all bands are identified and marked with a band marker Before You Begin Creating or Opening an Analysis An analysis must be open in order to add lanes to scanned images For new scans a new analysis is created as described in Chapter 2 For an existing analysis double click the analysis in the navigation tree to open it Single Channel Display vs Overlaid Image Channels Scans with two images can be displayed in single channel mode or with both image channels overlaid as a composite image Lanes are usually added with images overlaid because lanes are added to both images simultaneously Lanes can also be added to each image channel separately if desired After adding lanes each image must be analyzed separately in single channel mode Chapters 6 and 8 Before creatin
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211. r sides For example if the membrane size is 5 x 5 cm set the scan Width and Height to 7 cm and set the Origin to 0 0 The membrane would then be placed at the 1 x 1 cm position on the scan surface Note After setting the scan area check the file size at the bottom of the Scanner Console window to make sure the size is acceptable 24 CHAPTER 2 Starting Scans Placing Samples on the Scan Surface In general it is easier to place the membrane or gel on the scan surface before drawing the scan area on the scan grid If the sample is placed first the 1 cm grid lines on the scan surface can be used to determine where to draw the scan area on the scan grid in the Scanner Console window Membranes should be placed face down with the top of the membrane toward the front of the Odyssey Imager Orientation can be changed by flipping or rotating the image as needed Tip Rectangular membranes or gels will scan faster if the long dimension of the membrane is oriented horizontally along the front border of the scan area Placement in a vertical orientation requires the laser microscope to travel further and increases scan time Consult the Operator s Manual and Odyssey Protocol pack inserts for tips on handling membranes and remember to touch the membrane only with a clean forceps Orienting a single microplate for a standard scan is somewhat different scanning multiple microplates is described later in this chapter A plastic
212. rations made to the image cropping etc is appended to the end of the description When the analysis is open the description can be edited at any time by choosing Edit gt Analysis Description Copying Images From Another Analysis The Available Analysis list in the New Analysis window displays all images in the current scan that can be copied into the new analysis When an analysis is selected from the list a thumbnail view of the images that will be copied is shown in the Analysis Image section of the window If there are two images in the selected analysis both the 700 and 800 check boxes will be selected indicating that both images will be imported To copy only one of the images to the new analysis deselect the image that is not wanted See descriptions of the Undo and Reset buttons below for information about changing images after image manipulations have been performed Manipulating Images The Flip Rotate Subtract Crop Filter Adjust Image Curve and Alter Image Display buttons in the New Analysis window are used to alter images before they are added to a new analysis The Undo button cancels the last change made using the Flip Rotate Subtract Crop Filter Adjust Image Curves and Alter Image Display buttons Odyssey has multiple undo capability so clicking Undo multiple times steps backward through each change made since the New Analysis window was opened To undo all changes in the New Analysis window click Res
213. rawing Features on Images After copying and pasting features they can be automatically moved into the correct position using the Adjust Feature Location function described later in this chapter Adding Multiple Features Setting the Drawing Mode to Continuous The drawing mode tool B can be used to toggle the drawing mode for features shapes between drawing single features and drawing a new feature each time you click and drag across the image To determine which mode is currently selected move the cursor over the drawing mode tool and read the tool tip A menu choice on the Analyze menu also toggles between Single Shapes indicating continuous mode is selected and Continuous Shapes Suppose you want to draw five circles and the mode is set to Single Shapes Begin by clicking the drawing mode tool B to change to Continuous Shapes click the circle tool and then click and drag on the image to create the first circle After releasing the mouse button click and drag again for the second shape and repeat until all five shapes are drawn Click to resume drawing single shapes Note The drawing mode tool works with only the circle ellipse rectangle freeform and text tools Using the Multiple Features Tool When there are equidistant image features bands spots etc along an imaginary straight or curved line the multiple features tool il can be used to add a user specified number of equally spaced features circles etc
214. rawing mode cceeeees copying and pasting 0 deleting assisi drawing single features F5 repeat SHOrtCUt ssseeesseesestsseeees hiding boundary liN ESsssssssirersssssisissriosisrer MOVING wsssdasscscrecesnsecsets moving automatically moving features in grids MAIN Bc sssssecestssdsstsvescrdysaastaxcpesseeaeeaas NOSIZIN Gs seren eS aE T E ces selecting multiple features single shape drawing mode TOG Sisivs E E E i verifying feature boundaries 112 file menu Aligia pE Seese ect ieren cis analysis gt new analysis crop to new analysis crop multiple images delete anallySisvesssccvissoscacseecessscecs tassevtessonns download Scans cccsicsssscniesperdeisecsescenscetenes export image VICW ceccesceseeseeseeteetseeeeesees export TIFF images sossccisercrisis export scan flip or rotate to new analysis ceeeee 59 IMPOM SCAN visvscessteacavectesssasveveeedacgeantevesinss import Mages 2 ssesscrssessiverssessvestesseetsesesinss NEWAPIOJECO driom na OPE asna iar print image view recent projects SAVE ANALYSIS sasise save as new analysis eee scan multiple microplates search OF SCANS es sccis sos cisiancvsssesessaeccedeses files deleting vices secu aenrike ieis storage location search for scans size based on resolution cccceeeeeee 20 starting SCANS sis csesvansecesosarasoveedeaseanatyvesives 11 seq
215. rd Deviation of background pixels Background SD See Odyssey In vivo Imaging Guide Chapter 3 Multiplier Values in this field are irrelevant unless the feature was created with the Auto Shape tool Normalized I I Normalized integrated intensity See Chapter 13 193 Reports and Data Export Lane Reports Field Definition ID ID numbers are automatically assigned to every feature lane band etc Name User entered name for a band entered in Properties Number Band number automatically assigned by Odyssey Lane Name Name for the lane entered in the Properties Description Description entered in the object properties by the user Bands Total number of bands in the lane Channel Name of channel 700 or 800 where lane is found Concentration Calculated concentration of a band Conc units Units selected in the Conc Standards window MW Molecular weight of a band MW w units Same as MW with units label Background Background value calculated for the feature See description in Chapter 13 Bkgd Method Background calculation method used to calculate background See Chapter 8 Rf Relative mobility Rf is a measure of the distance a band has migrated as a percentage of the total lane length Rf values are therefore a decimal value between 0 and 1 Pixels Number of pixels in the feature Shape Area Area of a band in mm Area is the number of pixels
216. rdless of how the Image View Features are set Manually added text annotations are displayed on grids Application Settings Odyssey_Settings Settings List Show for Unselected Shapes Show for Selected Shapes General Name Name Image View Features 0 0 Image View Display Description Description ipa Values Quantification Quantification ane Adjust Locations O Molecular Weight O Molecular Weight Auto Shape V Boundar V Boundar Naming Conventions H Report Font Color for Unselected Shapes Font Color for Selected Shapes Color images i yelow v Color images E white v Grayscale images white v Grayscale images white Shape Annotation Font for all Shapes lt Name Dialog Size 14 x Rotation Rotate 45 COW v Fonts Example Annotation Text Shape Tool Tips Show Shape Tool Tips The Image View Feature settings can be used to reduce the screen clutter that can occur when labels are displayed on closely spaced features Notice that there are two groups of settings those for 226 CHAPTER 11 Changing The Appearance Of The Image selected features and those for unselected features One strategy to reduce screen clutter is to treat selected and unselected features differently If all desired labels for selected features are turned on and all labels except boundaries are turned off for unselected features the display will stay uncluttered and indi
217. re automati cally modified to use the new template Shape exports a complete data set for each selected feature shape The complete data set is all fields available in reports ICW exports the complete In Cell Western data set that is displayed in table form when In Cell Western gt View ICW Analysis is chosen Parameters should always be set to data for ICW exports Command Only is the same as typing the contents of the Command field and the Windows command prompt followed by the param eters in the Parameters field To understand this plug in type examine the default ReagentsWebLink plug in The Command field contains the path to Internet Explorer and the Parameters field is the URL for the page that Internet Explorer is supposed to open when it launches The Parameters field should normally be one of the following data for plug ins that export data template for report plug ins where the text that follows the equal sign is the name of a valid report template Parameters that must follow the command for a Command Only plug in 201 Important Select the features or grid for which to export data before running the plug in Only data for selected features are exported to plug ins Adding and Deleting Plug ins Plug ins are created by choosing Plug in gt Manage Plug ins and clicking the Add button in the Plug in List window The Name Command Line Parameters Type and Template can be entered as described above
218. rifying Band Markers Are Centered For both molecular weight sizing and quantification it is important that band markers are centered in the bands Visually check each band to make sure the symbol in the band marker is centered in the band All bands in a lane can be checked at once using the Lane Profile window as described later in this chapter If a band marker is not centered it should be moved by selecting the band marker and moving the cursor to the center of the band marker When the cursor changes to an all arrows cursor click and drag vertically to move the band marker until the symbol is centered Verifying Bands Are Fully Enclosed If the bands are going to be quantified make sure each band is fully enclosed by the band marker and that each band marker is properly placed for the background calculation method in use see Chapter 8 In general the band marker lines should be over empty background image if possible and not touch any pixels that represent band fluorescence For MW sizing placement of the band marker boundaries is not as important as long as only one band is enclosed in each band marker If bands are not fully enclosed sizing will be unaffected To change the size of a band marker select the band marker and move the cursor over the top or bottom boundary line When the cursor changes to an up down arrow cursor click and drag to move the boundary to the desired position 4 men ri 76
219. ription Cell Grid OK Create Template Cancel The grid size and spacing are listed in the Properties for information only To change the number of rows or columns in a grid delete the grid change the Grid Template settings and apply the grid again Grid spacing can be changed by reshaping the grid as described above in Resizing A Grid If the initial features are too large or small when the grid is applied the Well Shape and Size fields can be used to change the size or even 133 the type of feature To change from circles to squares or vice versa select Circle or Square To change the size of the feature enter the Well Diameter in millimeters for circles or the Well Width and Height for squares The grid description can be changed in the Description field IMPORTANT When OK is clicked to apply the properties a new grid is applied that uses the new features and well sizes Any changes to the existing grid will be lost Displaying Grid Data in the Grid Sheet The numbering or lettering of rows and columns in the grid sheet corresponds to rows and columns in the grid Grid objects are often closely spaced making it difficult to display data values next to the features on the image For this reason data are not displayed on the image Instead a grid sheet is used to display data in table format for each feature in the grid A grid sheet can be opened by selecting the grid and then choosing Analyze
220. s To stop adding points switch to one of the other edit modes like Modify Points Moving Points When the Edit Mode in the Edit Size Standards window is changed to Modify Points any individual points can be clicked and dragged to a new position On images with large smiles or frowns it may be necessary to add many additional points to make the molecular weight line conform to the curve Remember that the point where the MW line crosses the centerline of a lane is where that molecular weight standard is located in that particular lane For images with smiles or other curves plotting the size standards described below 107 for each lane will identify any MW standards that are out of position due to misplaced points or lack of points Plotting Size Standards Band sizing is not complete until the Size Standards window has been used to set the size interpolation method and to check the standards plot for each lane The rest of this chapter discusses how to accomplish these important goals Setting the Interpolation Method When molecular weight standards are applied to an image sample bands of unknown MW weight are automatically sized using a mathematical method to interpolate the size of sample bands that lie between standard bands The interpolation method should always be verified by choosing Lane gt Size Standards Size Standards Standard plot basepairs Std 1 Channel 700 75 00 137 50 200 00 262 50 325 00
221. s at the top of the image deselect the Sort Values in Descending Order check box to reverse the order After entering all the standards click OK to save the new set 98 CHAPTER 6 Band Sizing Deleting a Standard From a Set To delete a standard select the standard in the list and click the Delete button Editing Size Standard Sets Any MW standard set can be edited by double clicking a set in the Size Standard Sets window or by selecting the set and clicking the Edit button a Size Standards Sets Size Standards Set Entries Unit 100bp DNA Ladder 3 xe asl 1h basepairs Lambda DNA Hind Ill Markers basepairs Descending Protein Standards kilodattons Descending In the Edit Size Standards Set window standards can be added or deleted as described above under Creating MW Sets Deleting Size Standard Sets To delete a size standard set select it in the Size Standard Sets window and click the Delete button If only one standard within a set needs to be deleted edit the set as described above Using Size Standard Sets Start by choosing Lane gt Edit Size Standards or by clicking on the toolbar Remember that lanes must be found and only one image displayed before molecular weight bands can be identified 99 Choose a MW set from the Select MW Std Set drop down list in the Edit Size Standards window and click the Apply Std Set button Edit Size Stds for 800 TIF yp Add MV Line or
222. s communications for the purpose of settlement and as such shall be deemed to be confidential and inadmissible in any subsequent litigation by virtue of Rule 408 of the Federal Rules of Evidence as the same may be amended from time to time 8 TERMINATION 8 1 Termination 8 1 1 By Licensor Licensor may terminate this Agreement a immediately upon Licensee s copying or modification of the Licensed Program transfer of possession of any copy of the Licensed Program to any third party other than as contemplated under this Agreement or otherwise authorized in writing by Licensor or other failure to comply with the terms and conditions of this Agreement or b upon thirty 30 days prior written notice for non payment results from a good faith dispute between the parties In such event Licensee must destroy all copies of the Software Product and all of its component parts 8 1 2 By Licensee Licensee may terminate this Agreement a immediately upon Licensor s breach of the obligations in Article 7 or b upon thirty 30 days prior written notice thereof to Licensor 8 2 Bankruptcy Termination In the event Licensor enters bankruptcy the laws and rules of the Bankruptcy Code will govern the enforceability of this agreement 9 MISCELLANEOUS 9 1 Headings Unless otherwise stated all references to Articles and Sections refer to the articles and sections of this Agreement The headings of the Articles and Sections of this Agreement are fo
223. s for Standard Scans Scan parameters such as resolution and scan area can all be entered individually in the Scanner Console window or loaded from stored sets called Presets For most scans it is easiest to load Preset param eters and then edit individual parameters such as scan area to match the current scan Loading Preset Parameters Sets of scan parameters can be chosen from the Preset drop down list When a Preset is chosen all existing scan parameters in the Scanner Console window are replaced by scan parameters stored in the Preset file Scanner Console BandSizing User ron Scan Description Name Group ron B _ Modify _ Preset Membrane a Modify Scan Parameters Scanner Back a FRE Odyssey software initially has four Preset files for general use one for membranes two for gels and one for microplates In addition there are four Preset files for the MousePod In Vivo Imaging Accessory one for each of the three mouse positions in the MousePod and one for all three positions at once Note For older instruments Odyssey Server Software version 2 0 or above is required in order to have enough focus offset to scan a microplate See Odyssey Operator s Manual to determine software version number Membrane Gel and Microplate Presets 17 Membrane DNA Gel Protein Gel Microplate2 Resolution 169 169 1
224. s in the Concentration column To clear the table click the Clear button A description of each column in Details View can be found in the field definitions for reports in Chapter 10 The calculation descriptions in Chapter 13 provide additional information The values shown in the table in Details View can easily be trans ferred to a spreadsheet for plotting or other operations Click Export to create a text file tab separated values containing a header that describes the scan and all the data in the Details View data table 153 154 CHAPTER 8 Quantification Click Copy to copy all the data in the Details View data table to the clipboard Individual rows can be copied by Control clicking the row s and pressing Ctrl c to copy the data to the clipboard To print the Details View table to the default printer click the Print button Note Data can also be viewed exported or printed using the menu choices on the Report menu Chapter 10 Choosing the Background Calculation Method Determining the Current Background Method The background method is stored with each analysis and is listed in the tool tip that opens when the cursor is stopped over an analysis in the navigation pane If an analysis is open the current background method is always shown in the message line in the lower right corner of the main Odyssey window as shown below Ave Backgnd Ave Backgnd 710 461 1963 NA A message containing the current backgroun
225. s it easy for users to have their own settings file that determines important parameters such as background calculation method In labs that do not have multiple users however the settings selection window is not needed When Don t Show This Dialog At Startup is selected the settings selection window will no longer be displayed when the Odyssey application starts The active settings file can be changed at any time by choosing Settings gt Select Active Settings Set Active Application Settings x Available Application Settings sey _ Settings C Dont show this dialog at start up To add a new set of settings click Add and name the settings file A new settings file will be created containing default settings 230 CHAPTER 12 User Accounts and Settings A settings file can also be created by copying the active settings file To copy a settings file choose Settings gt Select Active Settings select an application settings file and click OK Next choose Settings gt Application change any of the application settings and save a new settings file by clicking the Save As button and naming the new file Note Only Application settings are stored Other settings such as ICW Setup are not stored To delete a settings file select it in the Set Active Application Settings window and click Delete Note The last settings file cannot be deleted User Administration The User Administration menu choice on the Settings me
226. s may be used to endorse or promote products derived from this software without specific prior written permission This software is provided AS IS without a warranty of any kind ALL EXPRESS OR IMPLIED CONDITIONS REPRESENTATIONS AND WARRANTIES INCLUDING ANY IMPLIED WARRANTY OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT ARE HEREBY EXCLUDED SUN AND ITS LICENSORS SHALL NOT BE LIABLE FOR ANY DAMAGES SUFFERED BY LICENSEE AS A RESULT OF USING MODIFYING OR DISTRIBUTING THE SOFTWARE OR ITS DERIVATIVES IN NO EVENT WILL SUN OR ITS LICENSORS BE LIABLE FOR ANY LOST REVENUE PROFIT OR DATA OR FOR DIRECT INDIRECT SPECIAL CONSEQUENTIAL INCIDENTAL OR PUNITIVE DAMAGES HOWEVER CAUSED AND REGARDLESS OF THE THEORY OF LIABILITY ARISING OUT OF THE USE OF OR INABILITY TO USE SOFTWARE EVEN IF SUN HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES You acknowledge that Software is not designed licensed or intended for use in the design construction operation or maintenance of any nuclear facility Publication Number 984 09386 Printed January 2008 LI COR is an ISO9001 registered company 2001 2008 LI COR Inc All rights reserved Specifications subject to change LI COR Odyssey and IRDye are trademarks or registered trademarks of LI COR inc Adobe and Acrobat are registered trademarks of Adobe Systems Inc Windows and Microsoft are registered trademarks of Microsoft Corpo ration The Odyssey Infrared Imager a
227. savecesssenebes 175 report field templates 0 cee 188 EDOM VIEW cssscceids arsane Er E ETS 183 stat table VieW sisiss stscscsrsisscscseesnenttecicieneses 205 FEPOrt Seting Ses neer isra e 186 report templates choosing fields serninnrsinainsnii 190 CEAN wis iessssnncetcscncnes soesesadestessesiess dovseivere 188 field AetimitiOnS ss scsseeseosecscvsceesssvecveocsets ds 191 resolution scan parameter recommendations eesesesesseeesseseeeseees 19 related file SIZES niissrssiissssersisiseiisssssueisss 20 rotate ANNOLALIONS sis cccssisassccetesdessecedeseatzavinae s 142 rotate image new analysis WiNdOW cee eee rotate and save image to new analysis POLE gride iesea iori S scan Can eling sanii 27 35 channels scan parameter cece 21 Cefault scan NAME ssseeseceessseeeseeees 14 OX DO libsezesavezes cxitzsbencyevssctacbentassece focus offset scan parameter s IMPO eieaa n E DS intensity scan parameter s s s microplates multiple cece ORIBINANG SIZE esras parameters and presets cceeeeeseeeees PIOVIOW SCAN igien e quality scan parametel cccceeeeseeees scan area parameter scan group selecting Scanner CONSOLE c cceeeseeeeeeeeeees searching for scans ceceeees starting SCANS evecsssssssseseecadaseeises STOPPING oi s2 conseasccscevsnctsecostadensnvercesinetesss scan area parameters MOVIN and resizing 0 eeeeee
228. scanner log when diagnosing problems inl Chapter 11 Changing the Appearance of Scanned Images Image Display Adjustments Choose View gt Alter Image Display to open the Alter Image Display window The Alter Image Display window can also be opened by clicking F on the tool bar The name of the image corresponding to each 1700 11F group of controls is Brightness shown in the upper left Corirast corner Sensitivity i Alter Image Display Linear Auto O Linear Manual A O Log Show Colored Image T800 TIF Show This Image Brightness a ral Fa 3 Contrast Sensitivity Linear Auto O Linear Manual A g O Log a Fy Show Colored Image ale Image k ic hite Bands on Blac Lox Reset Default Cancel 210 CHAPTER 11 Changing The Appearance Of The Image Each image has its own set of Brightness Contrast and Sensitivity sliders The Brightness and Contrast sliders change the appearance of the background and bands in the image Changing How Image Data Are Mapped to the Monitor If a scan appears blank the cause may be due to failed reactions but it may also be because the sensitivity needs adjustment Odyssey image files contain over 65000 grayscale values but the typical computer monitor can display only 256 grayscale values This requires a scheme to map grayscale values in the image to the monitor Three Sensitivity controls are provided that give alternativ
229. scesses 77 88 94 plug in reports default reports suiesiiisascsiisi ariete 196 CHING wand conan ae REE 198 PAWN CDM Ess ses cocsersisvess cteseast xa css cretegzeatesees 197 preview scan SCAMELMG 2555s pene ar aeae E E ES E 15 preset creating parameter sets presets 0064 30 default presets iii 17 CCIE sssrini sns 30 31 DOA seins as 16 odyssey instrument presets 0 0 0 0 cee 18 probability calculation ccccceeeeeeeeeees 252 project backups isperite 64 GEANE oprek a 5 12 OPRMING eisioes e EEE SEE aaRS 11 GSTINIION sserrep aE 5 Q quality scan parameter sesesereesesrseerereesisrerreer 21 quantification calculati ON nanea 146 displaying ssicesesiscssisesesescdecasseosssscesines 92 146 displaying a band as a percentage of lane 89 OVERVIEW 9 oa nanen terer aTe EEE sracanteass 145 MUI IES sens EE E E E EEEE 147 USING BRS cisssssascsscsitoxensscscssssevevessesiscsesanid 159 USING lane Skersio ernier 158 R raw integrated iMtenSity usnis 250 reciprocal fit interpolation method 0 150 relative MODIY soseri 78 88 94 report menu details VieW ccecccccesseceessceesseeeeses 112 151 ehari Vie We ics estas sacs daesteshersedenstenknctaree 202 EXD OU cipere paa ae ERr ETEN see tedunceess 184 EXPO GW oo ceccssncessevsacserececsssenesssesetedseaas ss 175 ICW report template 176 Pii cisissteccsseesisesccsccisaacescesnenst rateeedenensiayes 184 Print ICW oc ccsccantvsececessenesssesssvesea
230. ses caventest 130 Rotating Grid eensame 131 Moving Shap sssrscssnesosansins 131 Changing the Shape Size or Type 132 Displaying Grid Data in the Grid Sheet 133 Changing Font Size in the Grid Sheet 134 Copying Grids Between Analyses 134 Using SUDgrids c cissccsscosasseoscssscsosuenecsontes 135 Designing a Subgrid 0 0 0 cee 135 Designing a Main Grid s s s 137 Using the Auto Shape Tool 140 Naming Features and Adding ANNOLA ONS csssiecsesssncdessen eteveanetesedine te sehes 140 Renaming Multiple Features 0 141 Adding Text Annotations s s s 141 Changing an Annotation s s s 142 Copying and Pasting Annotations 142 Rotating Annotations cccceceeeeeees 142 Other Annotations You May See 143 Hiding Annotations sssrinin 143 Chapter 8 Quantification OVET EW seese ee errr EE E seacadess 145 Quantification and Concentration Calculat onse naan a e 146 Displaying Quantification Values 146 Entering the Concentration of Standards 148 Setting the Interpolation Method 149 Reviewing the Standards Plot 150 Changing and Deleting Concentration Standard Saision en e ea Eaa 151 Using the Details View for Background Verificato Niega en n a ES 151 Comparing Data Using Details View 153 Choosing the Background Calculation Method aacecssiittcistaesgeds nnan 154 Determining the Current Background M th od sssaaa nnn
231. sesavecesocssosessesnacesocesvaaaoes 28 Completing the Scans sniseii itise 29 Creating and Editing Preset Parameters 31 Using The Modify Scan Preset Window 32 Scanning Multiple Microplates 0 0 0 00 33 Stopping a Multi plate Scan uu ee 36 Multiple Scan Settings eee 36 Chapter 3 Creating a New Analysis OVEIVIOW ese icseccsedcietciesciersiescieteieted ones avse 39 Opening the New Analysis Window 39 Naming the AnalySis eceeeeeeee 40 Entering a DeSCTiptiOn soseroioaesesi 40 Copying Images From Another Analysis 41 Manipulating Images 0 0 cece 41 Flipping an Image cece 42 Rotating an Mages seess 42 Performing Background Subtraction 43 Cropping IMageSic assessed dsseteecdesctenieen 43 Using Image Filters cee eee 44 Changing Brightness and Contrast 45 Saving an Analysis cseeeeseeeeeeeeees 46 Deleting an Analysis 0 cece 46 Having More Than One Analysis Open 47 Chapter 4 Importing and Exporting Scans and Images Searching for Scans mesnin ienesis 50 Downloading Scans 0 0 eeeeeseeeeeeeeeees 51 IMPOFUME E EREE 53 Importing IMageES sasessiisresesinersssssaseerssineess 53 Importing Images From Other Imaging E E E ET 5 Exporting Mage Susena 55 Exporting an Image View cseeeeee 53 Exporting the TIFF Images 57 Exporting 8 bit Grayscale Images 58 Exporting Colorized TIFF Files
232. shold settings on a variety of scans and then change the Application settings described below to match the typical threshold On images with a lot of background noise the profile may be very jagged with noise peaks on the sides of larger fluorescence peaks These noise peaks can cause inaccuracy in band finding even though 82 CHAPTER 5 Creating Lanes and Finding Bands the band finding threshold is set correctly If bands are not going to be quantified the Noise Removal or Smoothing filters can be used to reduce noise peaks and improve band finding accuracy These filters are available when starting a new analysis Chapter 3 Comparing Lane Profiles To compare lane profiles for two or more lanes select the lanes Ctrl click and choose Lane gt Multi Lane Profile The window below shows a molecular weight standard lane lane 1 and a sample lane lane 5 The lane profiles show that band 1 in lane 5 is at the same pixel location as band 4 in the molecular weight standards lane A composite image of all the selected lanes is shown to the right of the profiles Bands are colored with their respective lane colors Band labels to the left of the image are also colored with the lane color Lane colors can be changed by clicking the Modify button in the Color column in the lane table at the bottom of the window Multiple Lane Profile View Analysis First_Analysis Pixel Location a 6 8 1500 2250 Average Inten
233. sity i v 800 Description T ShowLane Band Symbol ijlane 1 He Circle Silane 1 Diamond 4Lane5 Triangle Clicking in the appropriate cell in the Band Symbol column of the lane table and selecting a symbol from the list of symbols changes the band symbol used in the lane profile To simplify the lane profiles all lanes for a given channel can be turned off by deselecting either the 700 or 800 check box Individual lanes can be turned off using the check boxes in the Show Lane column Normalizing Bands in Lanes The sample normalization function allows one image channel to be used as a reference to normalize bands in lanes in the second image channel Normalization Procedure 1 With image channels overlaid add lanes 2 Click and then as needed until the channel is displayed that will be used as the reference channel 3 Examine each lane in the reference channel image and make sure there is only one band per lane Delete any surplus band markers 4 Click to display both images again and select Ctrl click the lanes to normalize 83 84 CHAPTER 5 Creating Lanes and Finding Bands 5 Choose Lane gt Sample Normalization 6 Choose the reference Select Normalization Channel channel and click OK Select the channel to use For calculating the p v7 normalization Factor The selected lanes in the channel must have only on
234. so they can be applied to another analysis with similar lane patterns Lane templates work well in repetitive experiments such as those in which the LI COR MPX Blotter is used to produce images with consistent lane and band patterns Lane templates record all sample lanes band markers molecular weight standard lanes and the applied set of molecular weights Saving a Lane Template Start by doing all of the tasks below m Create all lanes using the single or multi lane creation tools The multi lane tool works well for sample lanes from the LI COR MPX Blotter or similar blotting systems since the lanes are uniform m Edit the band markers Add or delete band markers as needed Adjust band marker size as needed m Apply molecular weight standards to the standard lane After completing the tasks above click FR or choose Lane gt Save Lanes as Template to save the current lanes and molecular weight standards to a template Enter aname and click OK Multi Lanes Template Name My Lane Template 86 CHAPTER 5 Creating Lanes and Finding Bands Placing Lanes Using a Template To place lanes from a template click Eg or choose Lane gt Place Multi Lanes Place Selected Multi Lane Set E3 Multi Lanes Set Name Refind Bands on Lanes Cancel In the template selection window select a template from the list and click OK Unless Refind Bands on Lanes is selected lanes and band markers are placed in exact
235. sor passes over a band and is substituted for the Integrated Intensity quantification value if the display of quantification values Settings gt Application gt Image View Features is enabled Image View Display Settings for Lanes In addition to Lane settings a useful setting for lanes can be found in the Image View Display settings of the Application settings If lane names or annotations are truncated at the edge of the image choose Settings gt Application and select Image View Display from the Settings List Select Small or Large extended text area around the image see Chapter 11 Chapter 6 Band Sizing 91 Before bands can be sized an analysis with lanes defined must be open All bands within the lanes are automatically found when the lanes are created Creating lanes and finding bands is described in Chapter 5 Checking the Application Settings Before sizing bands on an image it is a good idea to check the Appli cation settings to see how annotations will be displayed Click on the toolbar to open the Application settings for image view features Application Settings Odyssey Settings Settings List Image View Display Display Values Lane Adjust Locations Auto Shape Naming Conventions Report Imai eatures Show For Unselected Shapes C Name O Description Quantification C Molecular Weight Boundary Font Color for Unselected Shapes Color images F yellow v Show for Se
236. ssey window lt Preset Membrane Entering a description in the Description field is optional however descriptions can be included in reports Changing the Default Scan Name To change the default scan name choose Settings gt Application and select Naming Conventions from the Settings List Since these are Application settings naming conventions can be saved in the settings files for individual users Chapter 12 allowing each user to have scan names automatically entered as desired Application Settings Odyssey Settings Settings List Default Scan Name General Image View Features Image View Display Display Values Lane Empty Adjust Locations Time stamp YY MM DD HHMMSS Last used name sequence LastUsedName_ Default Scan Names e Time Stamp When the Scanner Control window is opened the current year month day hour minutes and seconds is entered in the Name field automatically This time stamp can be edited or appended with other text Last Used Name Sequence When the Scanner Control window is opened the name of the last scan is entered in the Name field and appended with a sequential number For example if MyScan was the last scan name Odyssey will present MyScan_1 as the default name for the next scan followed by MyScan_2 etc e Empty When the Scanner Control window is opened the Name field is left blank so the user can enter a name at the end of a s
237. ssociated files image files scan logs etc To delete multiple scans use Shift Click or Ctrl Click to select additional files before clicking Delete Edit Permissions To Your Scan Group Any user with Control permission can allow other users to access their scan group to start new scans analyze scans or delete scans that in the scan group To change access for a given user select the scan group in the Modify Scan Groups window and click Edit Permissions All users are listed along with their current permission levels Select a user and click the down arrow button in the Permission column to drop down a list of permission levels Select None to prevent a user from accessing your scan group Select Access to give a user the ability to download and analyze scans in your scan group Select Change to give a user the the ability to start new scans that are stored in your scan group as well as all the rights that come with Access permission Backup To quickly backup all files in a given scan group on the Odyssey instrument users with Control permission can click Backup in the Modify Scan Groups window Backup Scan Group Scan Group admin Destination Folder C Remove scan group images after transfer to the destination folder FJ Overwrite images in the destination Folder dj l OK Cancel In the Backup Scan Group window enter a destination folder or click Browse to open a standard file selection window To mo
238. sssssiscsscessesssssuavescessaseazzeosess RESIZING oaeaeei aaa E O band finding controlling refinding bands threshold esitasid asiana C channels scan parameters c cccceeeseeeeeeees 21 comparing lane profiles sisessisissniseississss 82 concentration calculation 252 concentration standards add in OFder nc ceceeeeseeessceesseeeseeeees 145 148 adding changing and deleting cccceeeee 151 for two Channel scans eceeeeeeeeeeeees 145 interpolation Method sisssississsissrisisosrssse 149 plottiNg eisenii 149 context sensitive menus right clicking an image n s 3 cropping images in the new analysis window 0 cee 43 261 INDEX crop image and save to new analysis 60 crop multiple images 00 0 cece 61 cropping flipping rotating images 219 D details view background verification eee 151 blue rectangles sssi ieres possi bitni changing column order comparing data copying printing exporting data 153 TOSS MANS ease a etees 113 OPENING cs siasssssecescasssnes rvectsaceqneivocesndect vetoes 112 verifying background calculation method 113 verifying feature boundaries 0 113 E edit menu analysis description WMO Aa E ausesieeee export data from details View 0 cee 153 ICW TESPONSE Cata ssecsesesesseesesseeerees 172 F features add multiple features tool eee continuous d
239. st possible alignment with the objects in the image 130 CHAPTER 7 Drawing Features on Images Tip While the all arrows Scan Scan4 Analysis MyAnalysis Image 800 TIF cursor is displayed the grid can be nudged one pixel at a time using the arrow keys on the keyboard All arrows cursor Note Itis not necessary for features on the image to be centered in grid cells The grid is provided merely as a visual aid and is not used in analysis The only thing that is important is that the circles or squares in the grid surround the fluorescence in the image Deleting a Grid To delete a grid select it and click the delete button X on the tool bar Pressing the Delete key on the keyboard also deletes a selected grid Resizing a Grid To reshape a grid first select the grid by clicking one of the grid lines Move the cursor over one of the border lines of the grid until the double arrow cursor is displayed Double arrow cursor 131 With the double arrow cursor displayed click and drag the left or right border line to resize the grid horizontally Click and drag the upper or lower border line to resize the grid vertically Note A grid can also be resized by deleting it modifying the grid template and applying the grid again Rotating a Grid To Rotate a grid select the grid and move the cursor over the lower right corner of the grid until the curved rotation cursor is displayed i Rotation cursor W
240. switch to a different template or just make temporary parameter changes that will be applied to the current analysis Applying a Different ICW Template Use the following procedure to load a different ICW template and apply it to a grid 164 CHAPTER 9 Odyssey In Cell Western Module 1 Choose In cell Western gt Change ICW Parameters 2 Click the Load From Template button at the bottom of the Change ICW Parameters window ee Apply Load From Template Cancel 3 Select an ICW template from the list and click OK Select ICW Analysis Template X Modify the template Select ICW Template as needed before ICW Analysis Template Ete airs V app ly ing t Thi SIS a L permanent template modification Note The new template must have he same number of wells as the grid on the image 4 Click Apply in the Change ICW Parameters window to apply the new template and recalculate the response data for each well using the well designations and calculation setup in the template Temporarily Changing the ICW Parameters The Change ICW Parameters window is also used to change the parameters for the current ICW calculations without permanently changing a template The Change ICW Parameters window is divided in to three tabbed panels that are used to specify how the microplate is loaded which rows columns to average and which calculations to perform Each of the tabbed panels is discussed below Well Types Ta
241. t be performed on exported 8 bit images Exporting Colorized TIFF Files Choose File gt Export Image gt Colorized TIFF File to save a single TIFF image file that contains both the 700 and 800 channel images The images are colorized and overlaid resulting in the same appearance as the images in the Odyssey window Colorized TIFF File export can also be used for single images in pseudo color format Annotations and other markers are not included only the image data are saved 59 Flip or Rotate to New Analysis Choose File gt Analysis gt Flip or Rotate to New Analysis to copy image files from the current analysis into a new analysis and flip or rotate the images in the new analysis To flip the images select Flip and select the direction to flip the images top to bottom or right to left fl Flip or Rotate Options Fig Top to bottom Right to left To rotate the images select Rotate and select the direction and degrees to rotate the images Direction Clockwise Counter Clockwise Degrees Ois O270 Note To rotate the images by an increment other than 90 degrees create a new analysis and rotate the images in the New Analysis window Chapter 3 60 CHAPTER 4 Importing and Exporting Scans and Images After clicking OK the Save As window opens with the current scan name and a proposed analysis name already filled in To save the new analysis to the current scan click OK or edit
242. t colors is shown in the Fonts Example area at the bottom of the window Separate controls are provided to 227 pick colors for both color and black and white image display styles some colors do not work well for both display styles Annotations can be rotated 45 or 90 degrees counter clockwise using the Rotation drop down list Displaying Data in Tool Tips When the Show Shape Tool Tips check box is selected data values are displayed in a tool tip when the cursor is stopped over a feature Using the Image View Display Settings The Image View Display settings are opened by choosing Settings gt Application and selecting Image View Display from the Settings List Application Settings Odyssey Settings Settings List Default Sensitivity for New Images eneral Sensitivity 5 v Image View Features Image View Display Image View Colors Display Values Lane 700 Channel red v Adjust Locations 800 Channel Auto Shape pones green he Naming Conventions Report Extended Text Area Around Image Edges None O Small O Large Setting the Default Sensitivity for New Images When a new scan is opened for the first time the image is displayed using the sensitivity value shown in the Image View Display settings Generally the Auto setting provides good results However if the 228 CHAPTER 11 Changing The Appearance Of The Image sensitivity always needs adjustment after opening new
243. than the current scan or a new scan Save As Scan Analysis Scan Name IkappaB scan Analysis Name Cropped_IkappaB Analysis Crop to Multiple Images Choose File gt Scan gt Crop to Multiple Images to crop the current images using the scan area in a scan preset file Chapter 2 and save the cropped images to a new analysis If necessary create and save scan preset s with the scan area equal to the cropped size of the images 62 CHAPTER 4 Importing and Exporting Scans and Images Example Suppose you are using the Odyssey MousePod which has three mouse positions The original scan was collected with the FullPodScan preset which creates one image that includes all three mouse positions Now however it would be more convenient to analyze each mouse separately The Crop to Multiple Images function can be used to create three new analyses using the scan presets for the left middle and right mouse positions Here is how the three new analyses can be created 1 Begin by choosing File gt Scan gt Crop to Multiple Images Crop Multiple Images Create cropped images and a new analysis for each set of images using the scan area of each Scan Presetin the list below Source images from scan SCID Mouse are T OO TIF T800 TIF Remove Remove All 2 Click Add to add a scan preset to the list of scan presets 63 3 Choose a scan preset that has a scan area in the correct position to use as
244. the process recommended b the Adviser within ten 10 Business Days of their receipt of the Adviser s written recommendation they shall promptly convene a non binding hearing the Mediation The rules for Mediation will be established by the Adviser after consultation with the parties 7 5 If the dispute cannot be resolved either through the procedure recommended by the Adviser or through the Mediation within such period as the Adviser shall deem reasonable the Adviser shall at the request of either party certify to the parties that the matter is incapable of resolution 7 6 No litigation may be commenced concerning the dispute until the Adviser has certified in writing that the dispute is incapable of resolution provided that any party may commence litigation a on any date after which such litigation could be barred by an applicable statute of limitations or b if litigation is otherwise necessary to prevent irreparable harm to the moving party 7 7 Each party shall bear its own expenses in connection with the alternative dispute resolution procedures set forth in this Section except that the parties shall split equally the fees and expenses of the Adviser including the costs associated with any Mediation and the fees and expenses of any other person designated by the Adviser to assist the parties 7 8 All communications made in connection with the alternative dispute resolution procedure set forth in this Section shall be treated a
245. ting Scans Occasionally it may be necessary to copy a scan from one project to another to reorganize the projects Choosing File gt Scan gt Import Scan imports scans from other projects into the current project All analyses associated with the imported scan are also copied along with the scan A project must be open before scans can be imported After choosing File gt Scan gt Import Scan use the standard file selection window to find the scan and open it Only scan files scn are listed in the file selection window Change the Files of Type field to All Files if you want to see all files If a scan is not listed in a directory where it is expected to be the scan file may have been deleted In this case choose File gt Scan gt Import Images instead of Import Scan or download the scan from the Odyssey instrument Importing Images During each scan a scan file scn and one or two TIFF images TIF are created If the original scan file has been lost or deleted choosing File gt Scan gt Import Images can be used to import just the TIFF image files from a scan Import Images can only import images from a local or network storage device not from the Odyssey Imager Note When only images are imported analysis files are not imported The image resolution and scan remarks are also not imported since these data are in the scan file Always use Import Sean if you want to import analysis files with the images 54 CHAP
246. tion of unknown image spots are calculated by comparing the intensity of the area within the surrounding feature to the curve Additional infor mation on choosing the interpolation method is given in Chapter 8 Probability Probability is one of the values that can be output in a report Proba bility represents the probability that the area enclosed by the feature boundary contains a statistically significant area of higher signal Odyssey software uses a Student s t Test to determine whether the mean of a set of pixels inside a given feature is significantly different from the mean of another data set of background pixels The t Test is defined as follows Students t Test tTest ng x ny x dof ng ny ag ay Msns 1 vpn 1 253 Where ns is the number of pixels in the signal area np is the number of pixels in the background area dof np n 2 is the degrees of freedom a is the average of signal pixels ap is the average of background pixels Vs is the variance of signal pixals Vp is the variance of background pixels The value of the t Test can be positive or negative If the t Test is negative then the mean of pixels within the feature is less than the mean of background pixels Since this is meaningless for Odyssey applications Odyssey software reports n a if the t Test is negative Odyssey software versions prior to 1 1 however did report negative values The reported probability us
247. tions cee eee averaging linking wells Cal CULATIONS naei changing ICW templates 0 0 0 cee 163 COLON COMES sisiseisdcusstevipasssestinegsysssascees ones 171 designating well types ccceeceeees 165 exporting response data 00 cece 172 OVEIVIEW soi csnscscvsasedsssocvissacticosncessaucsnveceeyes 161 percent response calculation 254 FQDOMS ssccessisdsasseces ssaasseitessdacdaasasscessascunanas 175 standard deviation of linked wells 172 templates creating editing deleting 173 viewing analysis data cccceeceeeees 170 image adjust curves annotations alter image display adjust image CUIVES eee eects background subtraction brightness and contrast changing channels local min max replace filter MAGMY essri measuring size noise removal filter OVEH AIG scciesisisccstssssstsinoweestassonesecessdssivndesat sensitivity controls single Channel i isscsisescensscsssconsseeasesseisetes smoothing fIte hia vecesasessssesssrsssesssgesvaseee textarea nuen a ikes image view features settings IMPOM IMAGE S sescssnescrdesacesecsindesvsssrectesensssvvesdes import scan from new project WindoW cceeeeeee 12 froni fle MON Use iicssceverecsccsvooensvecaeoceeves sess 53 in cell western menu align grid iccsscsessstctessssasecetstevssevas setter estseacs change ICW parameters view ICW analysis i integr
248. to the left all pixels to the right of the adjustment point are mapped to maximum intensity and become much brighter As the maximum adjustment point is moved to the left the slope of the adjustment line increases making many of the other pixels brighter as well lt The midpoint can be dragged in only the vertical dimension As the midpoint is moved the shape of the adjustment curve is changed which results in dark pixels and bright pixels being adjusted by different amounts It general changing the midpoint will have the greatest impact on the mid tone pixels but the bright and dark pixels will also be changed Using the Histogram Linear vs Log The histogram shows the number of pixels Y axis that have a given grayscale intensity value X axis When Linear is selected in the Adjust Image Curves window the histogram is plotted with a linear Y axis For images that have many dark pixels and comparatively few pixels with high intensity values a linear Y axis can make it difficult to see high intensity values Clicking Log to switch to a log scale for the Y axis can make it easier to see intensity values with fewer pixels 218 CHAPTER 11 Changing The Appearance Of The Image lt ii 2 lt ii B Linear Log The X axis of the histogram can be expanded or contracted using the Contract Expand slider The scroll bar underneath the histogram can be used to scroll along the X axis W
249. ts Test Instrument Information Motor Control Card DSP Card Signals Board System Resources Focus Info Home Info Cal Data Active Diagnostics X Motor Y Motor Z Motor Active Signals Board Scanner Update Updates to the Odyssey instrument software will be posted on LI COR s FTP site and made available on CD ROM Update notifica tions will be sent that explain how to get the update on CD and will also include the FTP address name and password to download the update file IMPORTANT The update software temporarily uses space on the Odyssey hard disk while the update is running If the hard disk is too full the oldest scans will be deleted as needed Make sure all scans have been downloaded from the Odyssey before proceeding 241 Whether you have an update CD or have downloaded the update files via FTP the update procedure is as follows 1 2 3 4 If you have an update CD insert the CD into the drive If you downloaded the update file make sure the file is accessible on your computer or network drive Click Update Scanner Software in the System Administration window to open the Scanner Update window Update Scanner Software Scanner West Lab Current ersion 2 1 1 Click Browse and use the standard file selection window to select the update file from CD or disk drive If the update file name is not displayed make sure th
250. two auto entered text blocks analysis name and date Auto entered text blocks can be separated by entering underscore characters spaces or other text as needed An example of how a file name might look is shown in the Example field Text that can be automatically inserted in the file name includes the Date Time Project name Scan name Analysis name and organization name choose Settings gt Application and select General to enter an organization name To insert a placeholder for the auto entered text select the type of text to enter from the drop down list and click Insert Place holders always start with a tilde character Click OK when finished entering a title The directory path sets the default directory where the report file will be saved To always store the report in the same directory as the scan click Save File in Scan Folder and the path will be set automatically A path can also be entered by typing it in the Export Path field or by clicking Browse and selecting a path from a file selection window The path entered will be the default directory shown in the Save File window that is opened when exporting a report Make sure to enter 178 CHAPTER 9 Odyssey In Cell Western Module the Export File Name and Path in such as way as to create a unique file for each scan unless there is a reason to overwrite the export file with every new scan When finished making changes to the ICW report template click Save Settin
251. ually n a 0 99 or 10000 will also be meaningless if there is no background defined or if Lane is selected as the background method When the value of the t Test is positive and not zero it is further refined using a published iterative statistical algorithm that calcu lates a probability in percent This algorithm was translated from a perl translation of the Pascal function on p 81 of Statistical Computing in Pascal by D Cooke A H Craven and G M Clark 1985 Edward Arnold Pubs Ltd London The Pascal algorithm is itself a translation of the Fortran algorithm AS 3 by B E Cooper of the Atlas Computer Laboratory as reported in among other places Applied Statistical Algorithms edited by P Griffiths and I D Hill 1985 Ellis Horwood Ltd W Sussex England Molecular Weight The molecular weights of bands in a lane are determined by comparison to the positions of molecular weight standard bands in lanes containing standards Chapter 6 discusses the use of molecular 254 CHAPTER 13 Calculation Descriptions weight standards to calibrate the molecular weight of unknown bands Percent Saturation Percent saturation is one of the values that can be output on reports Chapter 10 Percent saturation is defined as the number of saturated pixels divided by the total number of pixels enclosed in a feature multiplied by 100 to give a percentage Percent Response for ICW Assays When ICW cal
252. uch as those created by microplates or manually placed spots in dot blots the grid tool can save considerable time compared to manually placing each feature using the shape tools For protein arrays the grid tool can also be used to apply subgrids which are grids within each cell of a larger grid Grids are often applied to images for In Cell Western assays or other analyses that have independent background calculation methods To accommodate this the background calculation method Analyze gt Background Method is changed to No Background when a grid is added either manually or automatically as described below If you want to use one of the other background calculation methods apply the grid select the background shape and set the background method by choosing Analyze gt Background Method Before a grid can be applied a grid template must be defined that specifies the size and placement of the grid as well as the size of the features and whether the features are circles or rectangles Grid templates for typical 96 and 384 well plates are supplied with Odyssey software and can be modified as needed to create new templates 124 CHAPTER 7 Drawing Features on Images Creating Grid Templates The Grid Template settings are used to save grid templates The Grid Template settings are opened by choosing Settings gt Grid Templates Grid Templates X Grid Template Name Rows Columns Quantify Size Physical Size ate 3 2 6 5
253. uential NAMING cc cece eee 32 flip image new analysis WINdOW cceeceseeeeeeeeees flip and save in new analysis focus offset scan parameter cece eee folders view OPCMING scssvivsesstcacsueesessnsiagssesccccedbaasiossesteiees 7 fonts COlOF SIZE FOtATION s nsss essas 93 G grainy IMAS cers so gre oE EE EAE hes 21 graphing atta rss icesesveves cents dhrcvtecosdonseacessosbes 202 grayscale image display eects 212 grids adjust feature locations automatically 122 applying a grid to an image applying grids automatically changing feature size or type COPYING PAS s5 cciccsssseciscsscscsssspenterssiiovess creating grid templates 0 eee default background method deleting se ccs sttalss de des Bastacee cae moving the entire grid moving individual features eee parameters size spacing etC 126 QUANTI CATION es eesssscceecreti tetas centers 159 WOSIZING ss c3 crsncossseies orvesneses RES 130 rotating a grid 131 SUD SHOSI 2s EE AREE 135 grid sheet change TOM armare aei 134 export copy print data eee 134 OPENING enan reat a eE i 133 grid templates copying templates seeiis 124 Creating templates sssessessseeseee 124 editing and deleting sussriserieiiosssinisss 125 H help system OPENING essaye oE ER S 3 ICW In Cell Western assays applying grids automatically 0 162 automatic calcula
254. uld be enclosed and large enough to enclose the fluorescence without being so large that extraneous fluorescence gets enclosed Copying and Pasting Features With one exception selected features can be copied and pasted or cut and pasted in the normal way The exception is the location at which the feature is pasted Features are pasted with the upper left corner of the feature at the current position of the mouse cursor The normal paste procedure is to move the mouse cursor to the desired position and press Ctrl v on the keyboard A variation of the Paste command is the Paste Special command Paste Special pastes features in exactly the same pixel locations as the original features Paste Special is useful for pasting features between images For example if one image has an array of dots with circles drawn circles around them the circles can be copied to the second image by doing a Select All copying the circles Ctrl c switching images and doing a Paste Special All the circle features will be pasted in precisely the same location as the first image elimi nating the need to draw all the circle features on the second image Multiple Feature Selection When multiple features are selected they can be copied and pasted as a group To select multiple features e Click the first feature Hold down the Ctrl key and click additional features e Click and drag a selection box around all the features to be selected 116 CHAPTER 7 D
255. ve scans to the destination folder rather than copying them select the check box labeled Remove Scan Group Images After Transfer To 233 234 CHAPTER 12 User Accounts and Settings Destination Folder To overwrite scans that are already in the desti nation folder select the check box labeled Overwrite Images In The Destination Folder After configuring the backup click OK to transfer the files System Administration Account Rights To use the System Administration functions on the Settings menu you must be logged in with an account that has Administrator access rights There are three levels of access rights m None Accounts with access rights set to None are inactive and the user Cannot access data or start runs E Control Most users are given Control access rights allowing them to start new runs and access data in scan groups to which the user has been given access permission E Administrator Includes all the Control rights plus the ability to change any user account or scan group update system software and other setup and diagnostic functions For greatest security accounts with Administrator access rights should not be used for daily operation 235 To use the system administration functions choose Settings gt System Administration and log in using an account with Administrator rights System Administration Software Yersion 2 0 13 User and Scan Group Management L Manage Scan Groups
256. vidual features can be clicked to display their labels In other situations like exporting annotated images it is useful to have labels for unselected features turned on Turning on quantifi cation values for unselected features for example displays all quantification values on the image Each type of label is described below Name Name is either the auto entered default name or a name entered in the properties Description Description is blank by default for many features but can be entered in the properties Quantification Quantification values are displayed as Integrated Intensity or Concentration depending on the Application settings Molecular Weight The Molecular Weight annotation displays band migration in the gel as molecular weight scan line on the image or percentage of the lane according to the Application settings described below Molecular weight values are displayed as n a not assigned until molecular weight standards have been identified Boundary Each feature circle etc has a boundary If boundaries are turned off only the text annotations if any will remain Changing Font Specifications The font used to annotate features can also be changed to reduce screen clutter Changing the normal or selected color of the font reducing the size of the font or even changing to a different font can improve the readability of annotations on the image A sample of the currently selected font and fon
257. w Choose File gt Export Image gt Export Image View to save the image to a JPEG or TIFF file The portion of the image saved is the portion visible in the Image View In the case of a pseudo color image the pseudo color legend is appended to the right side of the image when it is exported Before the image is saved the image is shown in the Export View window and the file size is listed The image can be saved at a resolution of 72 dpi or 300 dpi 72 dpi is generally preferred for electronic slide presentations and web applications Some journals however require that images have a 300 dpi resolution or greater With either resolution there is the same number of pixels in the file The pixels stored in the file are exactly the same as the pixels shown on the computer monitor To get the highest quality image for print applications make the image as large as possible on screen before exporting it to a file This will build an image file with as many image pixels as possible To get even more pixels in the file use the Windows Control Panels to set the display resolution to a high level 56 CHAPTER 4 Importing and Exporting Scans and Images Export View Press Save to create an image file of the captured view Pixel Size Width 584 x Height 536 Print Size inches Width 1 9466667 Height 1 7866666 Resolution O 300 dpi Saved image format Medium Quality JPEG file O High Quality JPEG file TIF f
258. w has three adjustable points that appear as dots on the curve These three points can be used to change the slope and shape of the curve The initial shape of the adjustment curve and location of the three adjustment points are calculated by Odyssey software To revert to the initial calculated curve click Auto End Pts Above is an unedited curve and the corresponding image data from the 800 channel Compare these data to the curves and images below the illustrations are shown in color in the Odyssey help system v ajm 216 CHAPTER 11 Changing The Appearance Of The Image Maximum adjustment point Midpoint Minimum adjustment point The adjustment curve and image above illustrate several effects of moving the minimum adjustment point lower left toward the center it only moves horizontally First many of the darker pixels in the input file are now mapped to black on the monitor All pixels to the left of the adjustment point are mapped to black The result is that areas composed primarily of dark pixels are now nearly black The second effect is a slight increase in the slope of the adjustment curve which will make some of the brighter pixels even brighter The adjustment points have additional functions with pseudo color images See Odyssey In vivo Imaging Guide for details 217 The maximum adjustment point upper right can also be moved horizontally As the adjustment point is moved
259. xt area around the image as described at the end of Chapter 11 The Annotation Properties window can be used to enter a single line annotation set the font properties of the text or add a border to the text using the Include Border Around Text check box The text properties can be changed at a later time by selecting the annotation and clicking the properties tool 4 Changing an Annotation To change an annotation click the annotation to select it and click the properties button to open the Annotation Text Properties window Make changes and click OK Copying and Pasting an Annotation To copy an annotation click the annotation to select it choose Edit gt Copy move the mouse cursor to the location where the annotation should be pasted and choose Edit gt Paste A copy of the annotation will be pasted at the cursor position Rotating Annotations After adding text annotations annotations can be rotated using one of two methods To change the rotation for all annotations on the image choose Settings gt Application and use the Rotation field in Image View Features to change the rotation to none 45 degrees counterclockwise or 90 degrees counterclockwise Annotations can also be rotated by selecting them right clicking the image and choosing the rotation from the popup menu The popup menu allows each annotation to be rotated independently rather than applying the same rotation to all annotations 143 Other An
260. y The only difference is the fields that can be included in the report The report type is always listed in the Type field at the top of the Edit Template window Edit Template MyTemplate1 Report Type Sorting Ascending Up Int Intensity Shape Area Remove gt Channel cea _ Down Concentration Include 700 Channel Features Include 800 Channel Features 190 CHAPTER 10 Reports and Data Export Choosing Fields to Include in the Report Use the Fields area at the bottom of the Edit Template window to select the fields to include in the report Definitions for each of the fields are given later in this chapter The In Use list shows fields that will be included in the report By default several fields are automatically added to the In Use list when the Edit Template window is opened These fields can all be removed and added back to the Available list by clicking the Clear button Fields in the In Use list can also be removed one at a time by selecting the field in the list and clicking Remove To add a field select the field in the Available list and click Add To change the order in the In Use list and the report select a field and click Up or Down to move the selected field Use the Sort Output check box to choose whether to sort the report data If Sort Output is selected you can choose the field by which records will be sorted and whether the order is ascending or descending If the

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