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ImageQuant Utilities

1. Mouse key Function Image window Mouse Pull down menus Drag Commands Click Toolbars Click Activate window Right mouse Pop up menus Magnification CTRL click Switch between Enlarge and Reduce modes Mouse keypad plus Enlarge while in Magnifier mode Mouse keypad minus Reduce while in Magnifier mode Map window SHIFT click Move Map window CTRL4click Move Map window in sync with Image window pA 2 ImageQuant Utilities User s Guide Appendix A Quick reference for Windows A 3 Toolbar functions for FluorSep Main toolbar Multichannel toolbar ENENI IS ee LE Is T Ta mig View One Channel Overlay Side by Side Channel 4 Channel 3 Channel 2 Channel 1 Image toolbar D D o tel aaan e rn aj Define Region of Interest Gray Color Adjust Map Fitto Window Magnifier Previous Frame Create Frame Pixel Distance Pixel Locator Select Pan ImageQuant Utilities User s Guide pA 3 Part five Appendixes ee A 4 Toolbar functions for ImageQuant Tools Main toolbar Multichannel toolbar EEUU i e T5 4 Coli 8 View One Channel Copy Select Region of Non Overlay Image for Excel 3D Charting Flicker Between Displayed Channels Export Individual Gel Files From Dataset pey to the Selected Folder Overlay Open Side by Side Channel 4 Channel 3 Channel 2 Channel 1 Image toolbar
2. 2 4 2 3 2 Selecting the Automatic method 000005 2 4 2 3 3 Selecting the Manual method 0 0 cece eee 2 5 2 4 Locating and placing the fluorochrome marker rectangles 2 7 2 5 Sizing and centering the fluorochrome marker rectangles 2 9 2 6 Locating and placing the background rectangles Manual method only 1 0 0 cece eee eee eee 2 10 2 6 1 Positioning the B1 through B4 rectangles 2 11 2 6 2 Determining the location for the B rectangle 2 11 2 7 Performing the separation 00 0 cece 2 13 2 8 Evaluating the results cece 2 14 ImageQuant Utilities User s Guide iii Table of contents 2 9 Choosing a background correction filter 0 cece eee eee 2 9 1 Local background correction filler 0 0 cee 2 9 2 Global background correction filter 0 ccc eee 2 10 Using the impulse filter during separation 005 Chapter 3 Using FluorSep to build a dataset file 3 1 Creating the images usuau 3 2 Building a dataset file from single images anaana 3 3 Building a two channel dataset file from separated image files Part three ImageQuant Tools utility Chapter 4 Using ImageQuant Tools 4 1 Displaying an image 0 ete 4 2 Rotating the image in 90 increments 00 0 eee 4 3 Rotating the image in 0 5 increments 0 0 ee eeue 4 4 Inverting the data values in the image
3. 0 e eee ee 4 5 Removing noise using the impulse filter 0 0 000 4 6 Removing noise using the median filter 0 0005 4 7 Cropping and uncropping a multichannel image Windows only 4 7 1 Cropping a dataset image Windows only 4 7 2 Uncropping a dataset image Windows only 4 7 3 Saving the cropped dataset image Windows only 4 8 Exporting a multichannel image as individual images Windows Only 0 isses mme 4 9 Viewing a multichannel image using a flicker effect Windows only ux PS de eee ce ees Rea eee od 4 9 1 Default settings Windows only lisse 4 9 2 Selecting the flicker settings Windows only 4 10 Copying image data to Excel for 3D charting Windows only Partfour Reference Chapter 5 File menu bil Opern r s CE E ste reet oie des t tois a EE 5 2 KESE cie et tente vie eo e aote tug S6 ete secl n iv ImageQuant Utilities User s Guide Table of contents 5 3 Build Dataset FluorSep only lisse 5 1 5 4 Save As ImageQuant Tools only 0 0 0 eee eee 5 2 5 5 Save Region of Interest As ImageQuant Tools only 5 2 5 6 Save Cropped Dataset over Original ImageQuant Tools for Windows only mi toreen neuem OE a PRG GaN Ate 5 2 5 7 Export Gel Files From Dataset to Folder ImageQuant Tools for Windows Only six caudate i brew ead neo atin a al aaa 5 3 5 8 Image Properties
4. Menus Multichannel toolbar Multichannel di Channel 1 rea 2 Channel 2 1f2 fafa nal Bg 3 Channel 3 A Channel 4 I7 Overlay T Side by Side Grayscale View One Channel Channel 1 Channel 2 Multichannel toolbar Channel 3 ImageQuant Tools for Windows only Channel 4 etsl mia 8 3 Figure 7 5 Multichannel menus and toolbar You can view multichannel images in the following ways Overlay Shows a composite image in which the separated images are displayed together Each channel appears in a different color Side by side Displays each channel in a different section of the Image window Each channel appears in gray scale View one channel Displays only the selected channel of a multichannel image The channel appears in gray scale View a combination of channels Adds or removes the channels that you select or deselect using the menu or the toolbar p7 8 ImageQuant Utilities User s Guide Chapter 7 View menu 7 11 Flicker Display Channels ImageQuant Tools for Windows only TheFlicker Display Channels allows you to view a multichannel image using a flicker effect which simulates animation You can customize the flicker effect by selecting an image to remain static the images you want to use the order in which you want the images to flicker and how fast you want the images to flicker Section 4 9 describes this feature in detail 7 12 Hide Objects
5. right Low Drag to change the data values tea x Drag to change the Bend algorithm Drag to change the contrast Click to select the Curve mode Click to return to the original settings b Sigmoidal mode controls D sDemolmgs sypro gel cht m Preview Channels 4 t dmm em m m Re Histogram Channel 1 F Color m i Invert Mode E r Slope Bright zl dn Low m s ee zs Eh Drag to change the data values Drag to change the slope Drag to change the contrast Click to select the Sigmoidal mode Click to return to the original settings Figure 7 4 The Edit Lookup Table controls in the Gray Color Adjust window a The Curve mode controls and b The Sigmoidal mode controls p7 6 e ImageQuant Utilities User s Guide Chapter 7 View menu You can change the image display using the following controls Bright slider A djusts the contrast of the image M ovethe slider toward Low to decrease the contrast M ovethe slider toward H igh to increase the contrast Curvebutton and Bend slider A pply logarithmic linear and exponential lookup table definitions to the image Click Reset in the M ode area to return to the default linear lookup table definition Sigmoidal button and Slope slider Change to a sigmoidal loo
6. To determine the highest background value 1 Choose Pixel Locator ep from the Tools menu or the Image toolbar 2 Movethe pointer to the background near the top of the image and then click the mouse button A value appears in the status bar 3 Makea note of the value ImageQuant Utilities User s Guide p2 11 Parttwo FluorSep utility omms mar 4 Movethe pointer to a background location farther down the image Click the mouse button and make a note of the value 5 Repeat step 4 until you reach the bottom of the image 6 From the list of values select the highest background value 7 Click Select JB and then click the B rectangle 8 Drag the B rectangle so that it surrounds the area that contains the highest value Figure 2 9 showsthe fluorochrome marker rectangles and the background rectangles correctly positioned on the image Figure 2 9 Correctly positioned rectangles for use with the manual background correction method p2 12 ImageQuant Utilities User s Guide Chapter 2 Using FluorSep to separate images 2 7 Performing the separation After positioning the rectangles you are ready to separate the image After you separate the image you can evaluate the results and if necessary repeat the procedure using additional options sections 2 8 through 2 10 To separate the image click Perform Separation in the Fluorochrome Separation window figure 2 2 The separation process begins and the p
7. Click the Browse button for channel 2 The Open window appears 6 Locate and select the image file that contains the image data with the next shortest wavelength 7 Optional for Channel 3 only Click the Browse button J for channel 3 and select the image file with the next shortest wavelength 8 Optional for Channel 4 only Click the Browse button for channel 4 and select the image file with the longest wavelength 9 Click Build After the software creates the dataset file the Save As window appears 10 Type a name for the dataset file and click Save After FluorSep builds the dataset file a message appears stating the dataset was built successfully 11 Close FluorSep and display the multichannel image in ImageQ uant or ImageQ uant Tools p3 2 e ImageQuant Utilities User s Guide Important Chapter 3 Using FluorSep to build a dataset file 3 3 Building a two channel dataset file from separated image files The DS Builder allows you to build a two channel dataset file that uses image files created from two different datasets or from a dataset that includes more than two channels You must use FluorSep to separate the images before you build the new dataset Because the Fragment Analysis software analyzes two channel datasets only you usethe DS Builder to build datasets that can be analyzed in Fragment Analysis For example if you create a three channel dataset that includes a standard in channel 1 a
8. FluorSep only The H ide O bjects command allows you to remove the objects from the image temporarily If a check mark appears next to the H ide O bjects command the objects are hidden Choose the H ide O bjects command again to remove the check mark and display the objects ImageQuant Utilities User s Guide p7 9 Chapter 8 Tools menu The Tools menu contains Select section 8 1 Enlarge ImageQ uant Tools only section 8 2 Reduce ImageQ uant Tools only section 8 3 Zoom Area section 8 4 Magnifier section 8 5 Pan section 8 6 e Pixel Locator section 8 7 e Pixel Distance section 8 8 e Define Region of Interest ImageQ uant Tools only section 8 9 8 1 Select The Select command or button allows you to end mode commands In FluorSep the Select command allows you to select the objects on the image so that you can move them 8 2 Enlarge ImageQuant Tools only The Enlarge command doubles the magnification of theimage Choose Enlarge and then click in the area you want enlarged The pointer position is the center point for the enlargement Each click doubles the magnification Click the Select button np to end the Enlarge mode 8 3 Reduce ImageQuant Tools only TheReduce command reduces the magnification of the image by half each time you click the mouse button Choose Reduce and then click in the area you want reduced The pointer position is the center point for the reduction Cli
9. TheH elp menu contains Index FluorSep and ImageQ uant Tools for M acintosh only section 11 1 UsingHelp FluorSep and ImageQ uant Tools for M acintosh only section 11 2 e User s Guide ImageQ uant Tools for Windows only figure 11 3 Molecular Dynamics H ome Page section 11 4 About FluorSep section 11 5 About ImageQuant Tools section 11 6 11 1 Index FluorSep and ImageQuant Tools for Macintosh only The Index command opens a Help window containing a list of topics Reference material is provided for each topic Click the desired topic to access the reference material 11 2 Using Help FluorSep and ImageQuant Tools for Macintosh only The Using Help command provides tips on how to use H elp 11 3 User s Guide ImageQuant Tools for Windows only The User s Guide command allows you to view an electronic pdf format version of the mageQ uant Utilities User s Guide To view the guide you need the Adobe Acrobat Reader software which is included on the IQ Solutions CD 11 4 Molecular Dynamics Home Page If your ImageQ uant workstation contains an Internet connection you can use the M olecular Dynamics H ome Page command to go directly to the M olecular Dynamics H ome Page www mdyn com ImageQuant Utilities User s Guide p11 1 Partfour Reference 11 5 About FluorSep The About FluorSep command displays the version number of the FluorSep software you are using
10. 1 copy data for 3D charting 4 14 Copy Image command 6 1 Copy Image Data for Excel 3D Chart command 6 1 Crop Dataset command 6 2 crop multichannel images 4 8 D data values invert using ImageQuant Tools 4 4 Define Region of Interest command 8 4 DS Builder creating images for use with 3 1 separated image files using 3 3 single images using 3 1 window 3 2 ImageQuant Utilities User s Guide Index 1 Index Edit menu Copy Image command 6 1 Copy Image Data for Excel 3D Chart command 6 1 Crop Dataset command 6 2 Undo Crop Dataset command 6 2 Enlarge command 8 1 Exit command 5 5 Export Gel Files From Dataset to Folder command 5 3 export multichannel image as individual images 4 10 F File Info tab in Image Properties window 5 3 File menu Build Dataset command 5 1 Close command 5 1 Exit command 5 5 Export Gel Files From Dataset to Folder command 5 3 Image Properties command 5 3 most recently used list 5 5 Open command 5 1 Quit command 5 5 Save As command 5 2 Save Cropped Dataset over Original command 5 2 Save Region of Interest As command 5 2 Find Fluors using in FluorSep 2 7 Fitto Window command 7 2 Flicker Display Channel command 7 9 flicker effect 4 11 Fluorlmager 1 1 Fluorochrome Separation window 2 3 Index2 ImageQuant Utilities User s Guide FluorSep automatic background correction method 2 4 background correction methods 2 3 description of 2 1 determine background uniformi
11. 860 instruments The fluorochrome separation process removes the cross contamination that can occur when the emission spectra from multiple images overlap Because the emission spectra can overlap between the images the emissions from one fluorochrome can contaminate the collection for the other fluorochromes The fluorochrome separation process removes the cross contamination by performing a mathematical transformation on the images produced during scanning T he end result includes two or more images that under ideal conditions represent the light emitted from the fluorochromes used in the sample TheFluorSep utility also provides a method to create multichannel images by combining single images gel See part 2 for a description of the FluorSep utility ImageQuant Utilities User s Guide p1 1 Partone Introduction 1 2 Overview of the ImageQuant Tools utility ThelmageQ uant Tools utility provides several image processing tools You use these tools to modify a copy of the original image The original image remains unaltered Both images can be used for quantitation ThelmageQ uant Tools utility allows you to Rotate You can rotate or flip images Filter You can reduce the noise in an image using a median filter or an impulse filter nvert You can invert the pixel values of the image so that images scanned with a dark background high values and light spots low values are displayed as dark spots high va
12. Header Size 8 File Size 3044515 Width 1408 Height 1081 Bits Pix 16 Samples Pix 1 Min Yalue 0 012 Max Value 98333 886 Photometric 1 Background is Black Figure 5 1 TheFile Info tab of the ImageProperties window ImageQuant Utilities User s Guide p5 3 Partfour Reference Scan Info tab The Scan Info tab displays the information shown in figure 5 2 E ImageProperties x File Info Scan Info History Image Name ENDATANotate gel Scan Date 1990 Scan Time 9 23 8 4 41 Pixel Size 87 Model Scan Resolution 114 dots em Make 400 Image Type Single Channel User Comments Figure 5 2 The Scan Info tab of the ImageP roperties window History tab The History tab figure 5 3 displays a list of all the operations performed on the original image if any This list provides an audit trail of all the operations performed on the image that can impact quantitation The current image name appears in the Image N ame box Note M odifications you make to the image display using the Gray Color Adjust feature do not appear in the Sequence of O perations list E ImagePropetties Lx File Info Scan Info History Image Name C DATA rotate gel Sequence of Operations Perfomed on the Original Image CADATA S35w_ph Rotate 180 5 13 1997 10 23 Figure 5 3 The History tab in the ImageProperties window p5 4 ImageQuant Utilities User s Guide Chapter5 File menu 5 9 Mostre
13. ImageProperties x File Info Scan Info History Image Name C DATA rotate gel Sequence of Operations Perfomed on the Original Image FileName Operation Parameter Date__ Time CADATANS35w ph Rotate 180 5 13 1337 10 23 Figure 1 1 The History tab in the ImageProperties window The Operations list shows that the original image was rotated 180 using the ImageQuant Tools utility Theimage name at the top of the window is the name of the image displayed in the Image window Below the name is a list of each processing operation applied to the image You create a new image file each time you apply an operation to the image The Sequence of O perations list provides the following information File Name The name of the image to which you applied the operation N ote The file name is not the name you entered when ImageQ uant Tools performed the operation Operation and Parameter The type of image processing applied to the image plus appropriate parameters For example the image referred to in figure 1 1 was rotated operation 180 parameter DateandTime The dateand time when ImageQ uant Tools performed the operation If no operation has been performed on the image the Sequence of O perations list is blank N ote M odifications you maketo the image display using the Gray Color A djust feature do not appear in the Sequence of O perations list ImageQuant Utilities User s Guide p1 3 Pa
14. a dataset file from single images section 3 2 Building a two channel dataset file from separated image files section 3 3 After creating the dataset you view the multichannel image in ImageQ uant or ImageQ uant Tools 3 1 Creating the images To build a dataset successfully from single image files gel you must create the images using the same Instrument Sample do not move the sample between scans e Scan area Digital resolution FluorSep prompts you if you select files that do not match 3 2 Building a dataset file from single images You can create a dataset from two to four image files gel To build a dataset from single images 1 Start the FluorSep utility and choose Build Dataset from the File menu The DS Builder window appears figure 3 1 2 Makesurethe Separated DS for analysis box is not selected ImageQuant Utilities User s Guide p3 1 Parttwo FluorSep utility mM BEL Sources Wr HE ul WES EIE mcs ft Hep m E IE Separated DS for analysis Cancel Figure 3 1 The DS Builder window 3 Click the Browse button for channel 1 The O pen window appears 4 Locate and select the image file gel that contains the image with the shortest wavelength Important You must assign the image files to the channels in sequence Start with the image file that contains the shortest wavelength and end with the image file that contains the longest wavelength 5
15. addition you can add a local or global background filter an impulse filter or both filters to improve the uniformity of the background If the result for any of the filters is not acceptable you can repeat the separation process without using the filter ImageQuant Utilities User s Guide p2 1 Parttwo FluorSep utility After FluorSep removes the cross contamination the utility creates new separated files for each channel and stores the new files in the existing dataset folder dir The dataset ds file structure was created by the Scanner Control software figure 2 1 The dataset folder contains one separated and one unseparated image file gel for each channel Each channel represents one designated fluorochrome In addition you see one object file dox for each channel T he object file contains the rectangles used by FluorSep during the fluorochrome separation process amp 4color ml xj File Edit View Help 3 File Edit View Help Aclr1 dir c A A u t j 4clr1 ds Sep1 gel Sep2 gel 4cir1 ds B a B lobject s sele Sep3 gel Sep4 gel Unsepl dox linsepl gel 3 D a p Unsep2 dox Unsep2 gel Unsep3 dos Unsep3 gel a 8H Unsep4 dox Unsep4 gel 14 object s 1 41MB Figure 2 1 Sample of the dataset file structure You can view the channels in the dataset overlaid side by side or one at a time After the separation FluorSep displays the channels in the O verlay vie
16. and then paste the individual pixel values in Excel for three dimensional 3D charting You can copy a portion of a single image or a portion of one channel of a multichannel dataset image that is displayed in the side by side mode After you pastethe values in Excel you can create a 3D surface chart that shows the spot contours of the image To copy the selected image data to Excel 1 Choose Define Region of Interest 3 from the Tools menu or the Image toolbar 2 Dragthe pointer through the area you want to chart Excel has a limitation on the number of columns that fit on a worksheet If the area you select contains too much data Excel will truncate the data ImageQuant Utilities User s Guide 8 9 Chapter 4 Using ImageQuant Tools Choose Copy Image Data for Excel 3D Chart al from the Edit menu or the Image toolbar Start Excel and display a new worksheet Choose Paste from the Edit menu or the toolbar The data appear in the worksheet Choose Chart from the Insert menu The Chart Wizard appears Choose Surface as the chart type and click Next O ptional Continue filling in information in the Chart Wizard Click Finish The chart appears 10 Optional Save the chart and data ImageQuant Utilities User s Guide p4 15 Part four Reference Chapter 5 File menu TheFile menu contains Open section 5 1 Close section 5 2 Build Dataset FluorSep only section 5 3 e S
17. at the bottom of the screen The status bar displays messages Choose Status Bar to show or hide the status bar 7 3 Actual Size TheActual Size command displays the image at 10096 which is the actual size at which the image was scanned For large images the utilities will not display all the pixels so that the image can approximate the scan size of the original sample W hen viewing an image in Actual Size ImageQ uant displays the image as close to the actual size as possible 7 4 Fitto Window TheFit to Window command displays the entire image in the Image window If the image is larger than the Image window boundaries the utility reduces the image so that it fits within the boundaries 7 5 Magnification TheM agnification command displays the image at the magnification you select from the submenu The percentages range from 25 to 800 7 6 Zoom In TheZoom In command doubles the magnification of the entire image ImageQuant Utilities User s Guide lini Chapter 7 View menu 7 7 Zoom Out TheZoom Out command reduces the magnification of the entire image by half 7 8 Gray Color Adjust The Gray Color Adjust command graphically shows how the pixel intensities of the image are displayed in the Image window The Gray Color Adjust window contains onetab for a single channel image and one tab for each channel of a multichannel image Figure 7 2 shows the Gray Color Adjust window for a single channel image Click a tab
18. markers ImageQuant Utilities User s Guide p2 9 Parttwo FluorSep utility Figure 2 8 showsthe fluorochrome marker rectangles correctly sized and centered over the markers If you are using the automatic background correction method go to section 2 7 to perform the separation If you are using the manual background correction method go to section 2 6 to position the background rectangles Figure 2 8 Correctly sized and centered rectangles in the Image window 2 6 Locating and placing the background rectangles Manual method only If you are using the M anual background correction method FluorSep creates threeto five additional rectangles that you useto identify the background You use the B1 through B4 rectangles to enclose a portion of the background near the control marker that ends with the same number For example position the B1 rectangle in the background near the F1 rectangle You usethe B rectangle to enclose a portion of the background that contains values that are lower than the lowest band Alternatively you can enclose a portion of the background that contains the highest background value p2 10 ImageQuant Utilities User s Guide Important Chapter 2 Using FluorSep to separate images The number of rectangles depends on the instrument and the settings you selected in the Scanner Control software for that instrument For example if you scanned a sample that included two fluorochrome markers you
19. this feature in detail p5 2 ImageQuant Utilities User s Guide Chapter5 File menu 5 7 Export Gel Files From Dataset to Folder ImageQuant Tools for Windows only The Export Gel Files From Dataset to Folder command allows you to export the images that make up a multichannel dataset image as individual uniquely named images In the Export Gel Files From Dataset to Folder window you type new names for each image and then use the Browse button to select a new folder If you do not want to export the image for one of the channels you can leave the file name for that channel blank After you save the new images you can view them using analysis software such as the D eCyder software H owever the new image files are no longer a dataset image and cannot be viewed in ImageQ uant using the M ultichannel feature Note You can reconstruct the dataset file using the Build Dataset command in FluorSep section 3 2 Section 4 8 describes this feature in detail 5 8 Image Properties Thelmage Properties command displays information about the image Alternatively you can double click in the Image window to display the ImageProperties window The ImageProperties window contains three tabs FileInfo Scan Info and History Click a tab to display the contents File Info tab The File Info tab displays the information shown in figure 5 1 E ImageProperties x File Info Scan Info History Image Name EADATANotate gel
20. to view the channel color from the list pixel values changes to the Image window Select a new channel _ Select to invert the Ires Mocks Click to apply the Appi Eiane Cancel Hagan Channa 1 Edit High Low button cx A j Edit Lookup Table button Click to apply the changes and close the Gray Color Adjust window ImageQuant Tools for Windows only Figure 7 2 The Gray Color Adjust window for a single image ImageQuant Utilities User s Guide p7 3 Partfour Reference p 4 e Important ThePreview Channels area displays a small version of the portion of the image displayed in the Image window The histogram plot displays the range lof values for the portion of the image displayed in the Preview Channels area If the entire image is not displayed in the Image window changes you make to the histogram plot can produce unexpected results As you make changes in the H istogram area the change appears in the Preview Channels area You click Apply or OK to seethe changes in the Image window and to savethe changes with the image You can alter the image or each channel of the image using the following controls e Color Changethe color of the image by choosing a new color from the Color list ImageQ uant Tools for Windows only N ote You can select the same color for multiple channels of a multichannel dataset image e
21. 0 cece eee eee 5 3 5 9 Most recently used list llle 5 5 5 TO EXCO QUIE scende md es oon Festes uic Se AER Dem dea ley is De dnd 5 5 Chapter 6 Edit menu ImageQuant Tools only 6 1 Copylmage iiio u c n e eA EE dene RR an 6 1 6 2 Copy Image Data for Excel 3D Chart Windows only 6 1 6 3 Crop Dataset Windows only 0 00 0 cece eee 6 2 6 4 Undo Crop Dataset Windows only 00 cece eee 6 2 Chapter 7 View menu 7 I wm Toolbars ex See tite oan Serle eth Oe ee et ERN te ae 7 1 T2 ISCAS B Ae ae eS I E r m 7 2 43 Actual Sienne rei ra Petes dn CEN Ae ea Pes 7 2 1 4 RIE tO WIndOW sos eese E eve pee Xx ly On tle a 1 2 7 5 Magnification eter he ebur ue tae ee 1 2 TG Zoom nescit OR CORR Eo Qe USER RUE TIER Eee De 7 2 ED S ZOOM QUE ex eade dp S RUE SRS sat ete Y rated 7 3 7 8 Gray ColorAdj st ose erem Sa eR Rm dns 7 3 7 8 1 Changing the high and low display values 7 4 7 8 2 Using the lookup tables to change the display 7 6 1595 a Mapai isset dade wasn einige toate enl druide td 7 7 1 10 Multicharninel cierra De Ee RR CREDE aie dale 1 8 7 11 Flicker Display Channels ImageQuant Tools for Windows only 7 9 7 12 Hide Objects FluorSep only llis 1 9 Chapter 8 Tools menu Bede eS CISC a uM Cua ut At t co crate ola Lu e EO EEA 8 1 8 2 Enlarge ImageQuant Tools only cece ee ees 8 1 ImageQuant Utilities User s Guide v Table of con
22. 4 10 are Color Selection for Channels You can change the default gray scale with white background by selecting a different setting from the Color Selection for Channels list To manually select a color for each channel choose U ser Select Colors e Normalize All channels are displayed using the high and low numbers displayed in the N ormalize area For more information on the display range see section 7 8 1 You can change the defaults by typing a new number in the High and Low boxes When using the flicker effect the software saves the normalized values for each image Advanced Setting For Flicker Display xj Color Selection For Channels Greyscale White Background x Normalize High 44614 17 Low 1933 57 Cancel Figure 4 10 The Advanced Setting For Flicker Display Channels window 4 9 2 Selecting the flicker settings Windows only To select the flicker settings 1 Choose Flicker Display Channels 8p from the View menu or the M ultichannel toolbar The Flicker Display Channels window appears figure 4 9 2 Optional In the Static Channel to Display area select the check box for each channel that you want to remain static always displayed 3 Optional In the Channels to Flicker area clear the check box for a channel that you do not want to flicker 4 Optional In the Channels to Flicker area change the order of the channels by selecting a channel to move Then click t
23. Define Region of Interest Crop Current Dataset Undo Cropping and Restore to Previous Size 0 x ej s le aean el rj ap Gray Color Adjust Fit Image to Window Magnifier Previous Frame Create Frame Pixel Distance Pixel Locator Select Pan pA 4 ImageQuant Utilities User s Guide Appendix A Quick reference for Windows A 5 Extensions that identify file types The following extensions identify the types of files ImageQ uant Tools and FluorSep create dir Identifies the folder associated with an image dataset and contains the image file and the object filefor that dataset M ultichannel image datasets contain an image file and an object file for each channel The dataset folder also contains a copy of the dataset file which you can use if you accidentally delete the original dataset file ds Identifies an image dataset Each dataset file has an associated dataset folder dox Identifies a file containing image objects gel Identifies a single image created on an Amersham Biosciences instrument ImageQuant Utilities User s Guide pA 5 Appendix B Quick reference for Macintosh This appendix provides a quick reference for the M acintosh version of IQ Tools and FluorSep The topics in this appendix are Keyboard shortcuts section B 1 Mouse key combinations section B 2 Toolbar functions section B 3 e Icons that identify file types section B 4 B
24. Invert M ode Invert the display of the pixel values in a single image by selecting or deselecting the Invert M ode box You can invert gray scale and color images EditHigh Low Change the high and low display values to exclude background and saturated data section 7 8 1 e Edit Lookup Table Change the image display by applying different lookup tables to the image section 7 8 2 ImageQuant Tools for Windows only Advanced button Click to view the Advance Setting For Flicker Display Channels window which allows you to select settings to use with the flicker feature Section 4 9 describes this feature in detail For a step by step description of how to use the Gray Color A djust features see the mageQ uant U ser s G uide 7 8 1 Changing the high and low display values After you create an image of your sample using an Amersham Biosciences instrument the image has no defined display range The first time you open the image ImageQ uant Tools or FluorSep automatically sets the range of values to include the lowest and the highest data values collected by the instrument The utility saves the display range with the image when you close the Image window ImageQuant Utilities User s Guide Chapter 7 View menu pamm You can changethe high display values to remove saturated pixels from the image and you can remove low display values to eliminate the background pixels To change a display value drag the slider that ap
25. aces the value with the median value of all the pixels in the kernel Figure 4 4 shows how the median filter affects a 3x3 kernel 100 100 300 200 600 200 300 100 100 200 Figure 4 4 How the median filter affects a 3x3 kernel The median value in this kernel is 200 ImageQuant Utilities User s Guide Chapter 4 Using ImageQuant Tools To reduce the noise in an image using the median filter 1 Choose M edian Filter from the Image menu The M edian Filter window appears figure 4 5 Select 3x3 or 5x5 in the Kernel area Click OK The Save As window appears Type a name for the image file Optional Choose a different folder or drive Qv SOL Gee NS Click OK The filtered image appears in a new Image window Median Filter x Kernel 3x3 Cancel C 535 Figure 4 5 The Median Filter window 4 7 Cropping and uncropping a multichannel image Windows only You can crop a multichannel dataset image to exclude portions of the image from analysis If you are cropping a multichannel image all the individual images that make up the dataset are cropped to the same size and can be used by other analysis software such as the DeCyder software If you want to change the cropped area you can return to the previous view of the image After cropping the image you can savethe image as a new image overwrite the original image or close the window without saving the cropped image ImageQ
26. age as desired This document is furnished for informational use only and is subjectto change without notice Amersham Biosciences assumes no responsibility or liability for any errors or inaccuracies that might appear in this book Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences SV Corp 928 East Arques Avenue Sunnyvale CA 94085 4520 USA Amersham Biosciences Corp 2002 All rights reserved May 2002 Table of contents Preface Aboutthiis gulde i e ru rp ERR UR EVER YE ix Related publications 0 0 0 cece ix ASSUIMPUONS 33 suele hm Reim YER ae AR UR ee ix Assistarice nci aati tes E E A ow PLLCLSUBEUD ELE IUE X Partone Introduction Chapter 1 The ImageQuant utilities 11 Overview ofthe FluorSep utility lisse 1 1 1 2 Overview of the ImageQuant Tools utility llle 1 2 13 Checking the audit trail liliis 1 2 Parttwo FluorSep utility Chapter 2 Using FluorSep to separate images 21 Abo tEl orSep is scere edu e ed RR RR dene 2 1 2 2 Displaying the image lsseseeeess mE 2 3 2 3 Selecting Automatic or Manual background correction 2 3 2 3 1 Determining the uniformity of the background
27. alues for a selected area to Excel After you paste the values you can create a three dimensional 3D surface chart that shows the spot contours of the image Section 4 10 describes this feature in detail ImageQuant Utilities User s Guide p6 1 Partfour Reference 6 3 Crop Dataset Windows only gr The Crop Dataset command allows you to crop a multichannel dataset image to exclude portions of the image that you do not want to analyze All the images in the dataset are cropped to the same dimension Section 4 7 describes this feature in detail 6 4 Undo Crop Dataset Windows only TheUndo Crop Dataset command allows you to uncrop the multichannel image displayed in the Image window Section 4 7 describes this feature in detail p6 2 ImageQuant Utilities User s Guide 7 Chapter 7 View menu The View menu contains Toolbars section 7 1 Status Bar section 7 2 Actual Size section 7 3 Fit to Window section 7 4 Magnification section 7 5 Zoom In section 7 6 Zoom Out section 7 7 Gray Color Adjust section 7 8 Map section 7 9 Multichannel section 7 10 Flicker Display Channels ImageQ uant Tools for Windows only section 7 11 HideObjects FluorSep only section 7 12 7 1 Toolbars The Toolbars command allows you to change the toolbar configuration In the Toolbar Configuration window figure 7 1 choose which toolbars to display by clicking the chec
28. ance command Both markers are displayed in the Image window Because the utility saves the last value for each command you can recall the value by selecting the command again Click the Select button 8 to end the mode 8 8 Pixel Distance The Pixel Distance command displays the distance between two pixels Choose Pixel Distance and then move the pointer into the Image window The pointer turns into a cross Drag the pointer across the area you want to measure The data displayed in the status bar arethe pixel coordinates of the origin pixel Po the pixel coordinates of the final pixel Pc and the distance in millimeters between the two points To add the distance in pixels to the status bar display CTRL drag the pointer across the area you want to measure You can usethe Pixel Locator and Pixel Distance commands sequentially Determine a pixel location using the Pixel Locator command and then determine the distance between two pixels using the Pixel Distance command Both markers are displayed in the Image window Because the utility saves the last value for each command you can recall the value by selecting the command again Click the Select button 8 to end the mode ImageQuant Utilities User s Guide p8 3 Partfour Reference a 8 9 Define Region of Interest ImageQuant Tools only pm The D efine Region of Interest command allows you to specify an area of css the image to copy or crop After you choose the Defin
29. and displays copyright information N ote The About FluorSep command on a M acintosh computer is located in the Apple menu 11 6 About ImageQuant Tools The About ImageQ uant Tools command displays the version number of the ImageQ uant Tools software you are using and displays copyright information N ote T he A bout ImageQ uant Tools command on a M acintosh computer is located in the Apple menu pll 2 ImageQuant Utilities User s Guide Part five Appendixes Appendix A Quick reference for Windows This appendix provides a quick reference for the Windows version of IQ Tools and FluorSep The topics in this appendix are Keyboard shortcuts section A 1 Mouse key combinations section A 2 Toolbar functions for FluorSep section A 3 Toolbar functions for ImageQ uant Tools section A 4 Extensions that identify file types section A 5 A 1 Keyboard shortcuts Menu Key Function File menu CTRL O Open SHIFT F2 Image Properties Edit menu CTRL SHIFT C Copy Image ImageQuant Tools Only View menu F9 Zoom In SHIFT F9 Zoom Out F8 Gray Color Adjust CTRL 1 Multichannel Channel 1 CTRL 2 Multichannel Channel 2 CTRL 3 Multichannel Channel 3 CTRL 4 Multichannel Channel 4 Tools menu CTRL SHIFT S Select CTRL R Define Region of Interest Window menu F5 Cascade SHIFT F5 Tile ImageQuant Utilities User s Guide pA 1 Part five Appendixes A 2 Mouse key combinations
30. ate the image in 0 5 increments in either direction To rotate the image in 0 5 increments 1 Choose Rotate Image from the Image menu The Image Rotation window appears figure 4 1 2 Select the Arbitrary Angle check box 3 Click the up arrow to rotate the image clockwise or click the down arrow to rotate the image counterclockwise The Destination image rotates so that you can preview the effect of the rotation Alternatively you can typea number ImageQuant Utilities User s Guide p4 3 Partthree ImageQuant Tools utility 8 9 O ptional Select the Anti Alias check box to smooth the edges of the image O ptional If you want to open the rotated image automatically after you save it select the O pen image after rotate check box When the rotation is correct for the Destination image click Rotate The Save As window appears In the Save As window type a name for the rotated image O ptional Choose a different folder or drive Click Save A new file containing the rotated image is created using the name you typed in step 7 If you selected O pen image after rotate in step 5 the rotated image appears in a new Image window 4 4 Inverting the data values in the image The Invert Image command inverts the data values in an image and allows you to save the inverted image Figure 4 2 shows an image before and after inverting By inverting the data values in an image thetroughs in a negative image are in
31. ave As ImageQ uant Tools only section 5 4 Save Region of Interest As ImageQ uant Tools only section 5 5 Save Cropped Dataset over Original ImageQ uant Tools for Windows only section 5 6 e Export Gel Files From Dataset to Folder ImageQuant Tools for Windows only section 5 7 e Image Properties section 5 8 Mostrecently used list section 5 9 Exitor Quit section 5 10 5 1 Open The O pen command provides access to any stored image To open an image Windows O pen the folder that contains the image and then double click the name in the box M acintosh Double click the image name in the File N ame box The utilities use several file types Each typeis identified with a different extension or icon Windows See appendix A for a description of each extension M acintosh See appendix B for a description of each extension 5 2 Close TheClose command closes the active window Choosing the Close command is the same as clicking the Close box in the window 5 3 Build Dataset FluorSep only TheBuild Dataset command allows you to create a multichannel dataset image file from separate image files Section 3 3 describes this feature in detail ImageQuant Utilities User s Guide p5 1 Partfour Reference 5 4 Save As ImageQuant Tools only The Save As command allows you to save the selected image and associated files using a new name In the Save As window type the name to use for savi
32. cate the markers automatically The Find Fluors feature locates the fluorochrome marker for each channel with better than 8096 accuracy The Find Fluors feature evaluates the entire image pixel by pixel using a 2 pixel by 8 pixel area FluorSep looks at the continuity of the pixels in each band figure 2 5 FluorSep calculates the brightest band in each channel with the least amount of bleed into other channels Channel 4 y X Pixel 1 Channel 3 y gt Channel 2 y Channel 1 y Brightest pixel in channel 1 UH Figure 2 5 To place the fluorochrome markers FluorSep compares pixel 1 in all four channels and then compares pixel 2 in all four channels After evaluating all the pixels in all the channels FluorSep places a fluorochrome marker on the brightest pixels in each channel FluorSep uses the width of the rectangles to define the width of the bands and then applies the histogram algorithm using the same width to determine the background FluorSep assumes the bands are in lanes that run from the top to the bottom of the image Before locating the markers using the Find Fluors feature you might want to use the instructions in section 2 5 to position and size the rectangle for the largest fluorochrome marker Doing this can help FluorSep locate the brightest pixel in each marker and can help you identify which is the brightest pixel To locate the fluorochrome mar
33. cently used list TheFile menu displays the last four most recently used files You can open one of these files by choosing the name from the File menu 5 10 Exit or Quit The Exit Windows or Quit M acintosh command closes ImageQ uant Tools or FluorSep You will be prompted to save any changes ImageQuant Utilities User s Guide p5 5 Chapter 6 Edit menu ImageQuant Tools only The Edit menu contains e Copy Image section 6 1 Copy Image Data for Excel 3D Chart Windows only section 6 2 Crop Dataset Windows only section 6 3 Undo Crop Dataset Windows only section 6 4 6 1 Copy Image The Copy Image command allows you to make a copy of the entire image or a portion of the image To copy a portion of the image you define the portion using the Define R egion of Interest command or toolbar button section 8 9 before using the Copy Image command The copy of the image is placed on the clipboard as a 24 bit RGB image You can paste the copy into another application such as M icrosoft Word Excel or Adobe Photoshop You can copy the following images A single channel image displayed in gray scale or color A multiple channel image displayed in the overlaid color mode e Theselected channel of a multichannel image displayed in the side by side gray scale mode 6 2 Copy Image Data for Excel 3D Chart Windows only The Copy Image Data for Excel 3D Chart command allows you to copy the pixel v
34. ck the Select button sp to end the Reduce mode ImageQuant Utilities User s Guide p8 1 Partfour Reference S E p8 2 e 8 4 Zoom Area TheZoom Area commands create a stack of enlarged images that you view in reverse order Choose C reate Frame from the submenu and then draw by dragging a box around the area to enlarge R epeat to create additional frames Choose Previous Frame from the submenu and then click in the Image window to display the previous frame Continueto click in the Image window to recall all the stored frames After you view a stored frame the utility deletes the frame Choose Reset from the submenu to delete the stored frames without viewing them Click the Select button 8 to end the Zoom Area mode but leave frames for later viewing The utility deletes the frames when you close the Image window 8 5 Magnifier The M agnifier command doubles the magnification of the portion of the image directly under the pointer Choose M agnifier and then move the pointer into the Image window The pointer turns into a magnifying glass amp M ove the pointer to the desired area and hold down the mouse button A box appears displaying the enlarged portion of the Image window Release the mouse button to remove the box You can select a different area for enlargement by repositioning the pointer and holding down the mouse button or you can select an area by dragging the enlargement box around the Image window W
35. d image that contains bright distinct bands but an uneven background p2 14 ImageQuant Utilities User s Guide Chapter 2 Using FluorSep to separate images SSS 2 9 2 Global background correction filter The Global background filter figure 2 11b reduces the overall intensity of the image while maintaining the relative intensity of the bands to the background The Global filter uses an algorithm to locate the lowest background value and then subtracts this value from the entire image ae 2 Line Graph 1 B x 2 Line Graph 2 B x 2 Line Graph 3 n x mm 18 70 mfu Z 0 000 mm 18 20 rfu 3 153 mm 17 10 ru z 14 105 m LINE 1 mE LINE 1 ae LINE 1 a Local filter b Global filter c No background filter Levels out the variations Locates the lowest Displays the original in the background relative background value and intensity of the fluorochrome to the bands to create a subtracts this value bands relative to the uniform background from the entire image background Figure 2 11 Comparison of the effects of the background filters used to separate the same dataset file a Local filter b Global filter and c No background filter ImageQuant Utilities User s Guide p2 15 Parttwo FluorSep utility 2 10 Using the impulse filter during separation N ote The impulse filter is a directionally applied filter des
36. e A step by step guide for all major functions of the ImageQ uant software mageQ uant Reference A reference by menu item for each function of the ImageQ uant software e mageQ uant Tutorial A tutorial that highlights certain features of the ImageQ uant software A user s guide for the instrument Assumptions The software related instructions in this guide assume you have basic computer skills You should be familiar with either the M icrosoft Windows or the Apple MacOS graphical user interface If you do not have these skills refer to the Windows or M ac O S documentation or refer to the H elp for these operating systems ImageQuant Utilities User s Guide ix Preface x e Assistance If you have problems contact technical support using one of the numbers listed below Asia Pacific Latin America Tel 4852 2811 8693 Tel 55 11 3667 5700 Fax 4852 2811 5251 Fax 455 11 3667 87 99 Australasia M iddle East and Africa Tel 461 2 9899 0999 Tel 30 1 96 00 687 Fax 461 2 9899 7511 Fax 30 1 96 00 693 Austria N etherlands Tel 01 576 0616 22 Tel 0165 580 410 Fax 01 576 0616 27 Fax 0165 580 401 Belgium Norway Tel 0800 73 888 Tel 2318 5800 Fax 03 272 1637 Fax 2318 6800 Canada Portugal Tel 1 800 463 5800 Tel 21 417 70 35 Fax 1 800 567 1008 Central East and Southeast Europe Te 443 1 982 3826 Fax 43 1 985 8327 Denmark Tel 45 16 2400 Fax 45 16 2424 Fin
37. e R egion of Interest command the pointer changes shape Position the pointer at one corner of the area you want to copy and then drag the pointer until the area is enclosed in a dashed box N ext you can e Choose the Copy Image command from the Edit menu to copy the area and paste the copy into another application such as M icrosoft Word Excel or Adobe Photoshop section 6 1 Windows only Choose the Copy Image Data for Excel 3D Chart command from the Edit menu or Image toolbar to copy the pixel values and paste them into Excel section 4 10 Windowsonly Choose the Crop Dataset command from the Edit menu or M ain toolbar to crop a multichannel dataset image to exclude portions of the image that you do not want to analyze section 4 7 p8 4 ImageQuant Utilities User s Guide Chapter 9 Image menu ImageQuant Tools only Thelmage menu contains e Rotate Image section 9 1 Invert Image section 9 2 Impulse Filter section 9 3 MedianFilter section 9 4 9 1 Rotate Image The Rotate Image command allows you to rotate flip or rotate and flip the image See sections 4 2 and 4 3 for a complete description of the Rotate Image command 9 2 Invert Image The Invert Image command allows you to invert the pixel values in the image Inverting the pixel values changes a black background to white See section 4 4 for a complete description of the Invert Image command 9 3 Impulse Filter The Im
38. ects to identify thefluorochrome markers and to identify portions of the image as background The fluorochrome marker designates the brightest location of each channel relative to the other channels Accurate sizing and placement of the rectangles are important for proper functioning of the background correction methods To select the M anual background correction method click M anual in the Background Correction M ethod area Depending on the number of fluorochromes in the sample five or more rectangles appear on the image figure 2 4 The F rectangles are used to locate the fluorochrome markers and the B rectangles are used to locate the background used in the background correction calculations ImageQuant Utilities User s Guide p2 5 Parttwo FluorSep utility You usetheFind Fluors featureto place the fluorochrome marker rectangles section 2 4 N ext you recenter or resize the fluorochrome marker rectangles section 2 5 Finally you place the background rectangles in the correct areas of the background section 2 6 Figure 2 4 The rectangles that appear when you use the Manual background correction method on an image containing four fluorochrome markers p2 6 ImageQuant Utilities User s Guide Chapter 2 Using FluorSep to separate images 2 4 Locating and placing the fluorochrome marker rectangles In both the automatic and manual background correction methods you use the Find Fluors feature to help you lo
39. el image 4 11 Image Rotation window 4 2 image displaying in 4 1 impulse filter 4 5 Impulse Filter window 4 6 invert data values 4 4 median filter 4 6 Median Filter window 4 7 overview 1 2 rotate image 1 to 15 4 3 rotate image 90 4 2 rotate using anti alias feature 4 3 uncrop multichannel image 4 7 Impulse Filter command 9 1 impulse filter in ImageQuant Tools 4 5 Impulse Filter window in ImageQuant Tools 4 6 impulse filter in FluorSep 2 16 Index command 11 1 Invert Image command 9 1 Index K keyboard shortcuts Macintosh B 1 L list of open windows 10 1 local background correction filter in FluorSep 2 14 lookup tables in Gray Color Adjust window 7 6 M Macintosh keyboard shortcuts B 1 Magnification command 7 2 Magnifier command 8 2 Map command 7 7 Median Filter command 9 1 median filter in ImageQuant Tools 4 6 Median Filter window in ImageQuant Tools 4 7 menu Edit 6 1 File 5 1 Help 11 1 Image 9 1 Tools 8 1 View 7 1 Window 10 1 Molecular Dynamics Home Page command 11 1 most recently used list in File menu 5 5 Multichannel command 7 8 multichannel image 3D charting 4 14 crop 4 8 export as individual images 4 10 save cropped image 4 10 uncrop 4 10 using flicker effect 4 11 view one channel 7 8 view overlaid 7 8 view selected channels 7 8 view side by side 7 8 ImageQuant Utilities User s Guide Index 3 Index Multichannel submenu Overlay command 7 8 Side by Side command 7 8 vie
40. er s Guide p4 11 Partthree ImageQuant Tools utility p4 12 4 9 1 Default settings Windows only The default flicker settings in the Flicker Display Channels window figure 4 9 are Static Channels to Display Y ou can change the default N one to select the channel images that you want to remain static During the flicker sequences the software always displays the static images e Channels to Flicker You can change the defaults so that not all the channels flicker To do this clear the check box for any channel you do not want to flicker In addition you can change the order the channels appear by selecting a channel and then clicking the Up or Down button to move the selected channel e Flicker Rate You can change the default flicker rate 500 ms to increase or decrease the flicker rate The flicker range is 1 ms to 2 000 ms To do this drag the slider to the right to slow down the flicker rate or drag the slider to the left to speed up the flicker rate Alternatively you can type the milliseconds in the box Flicker Display Che xj Gray Color Adjust Static Channels to Display ve m e a None 1 2 3 4 Channels to Flicker Flicker Rate 500 ms Close Figure 4 9 The Flicker Display Channels window ImageQuant Utilities User s Guide Important Chapter 4 Using ImageQuant Tools The default flicker settings in the Advanced Setting For Flicker Display Channels window figure
41. he U p or Down button ImageQuant Utilities User s Guide p4 13 Partthree ImageQuant Tools utility p4 14 e Important 5 Optional In theFlicker Rate area changethe flicker rate by dragging the slider to increase or decreasethe rate Alternatively you can type a number in the box 6 Optional To change the advanced settings click the Gray C olor Adjust button The Gray Color Adjust window appears Click Advanced The Advanced Setting For Flicker Display Channels window appears figure 4 10 Select a different image color combination from the Color Selection for Channels list If you select User Select Colors you choose a color for each channel from the Color list in the Gray Color Adjust window e Type different values in the High and Low boxes to change the normalization Click OK to return to the Gray Color Adjust window If you are manually selecting colors you select a color from the Color list for the channel currently displayed in the window Then click the next Channel tab you want to change and choose a color for that channel Continuefor the remaining channels Click OK to return to theFlicker Display Channels window 7 Click Start The images flicker according to the settings you selected 8 After viewing the animation sequence click Stop and then click Close to close the Flicker Display Channels window 4 10 Copying image data to Excel for 3D charting Windows only You can copy the image data
42. hilethe enlargement box is displayed you can increase the magnification by pressing the plus key on the numeric keypad or you can decrease the magnification by pressing the minus key The status bar displays the magnification of the enlargement box It also displays the upper left X Y pixel coordinates P and thelower right X Y pixel coordinates Py of the enlarged area 8 6 Pan The Pan command moves the image in any direction Choose Pan and then move the pointer into the Image window The pointer turns into a hand Drag the pointer in any direction to move the image When the entire image is displayed in the Image window the Pan command is inactive ImageQuant Utilities User s Guide Chapter 8 Tools menu 8 7 Pixel Locator ThePixel Locator command displays information about the selected pixel Choose Pixel Locator and then move the pointer into the Image window The pointer turns into a cross Click the mouse button at the desired pixel location The pixel information appears in the status bar The data include the pixel coordinates P and the intensity value of the pixel in the measurement units of the instrument To add the raw pixel value to the status bar display CTR L4click at the desired pixel location You can usethe Pixel Locator and Pixel Distance commands sequentially Determine a pixel location using the Pixel Locator command and then determine the distance between two pixels using the Pixel Dist
43. igned to improve images scanned on the Typhoon Fluorlmager and Storm instruments To improve the separation you can usethe impulse filter to remove artifacts caused by contaminants and imperfections such as dust or bubbles in the sample matrix The impulse filter reduces 1 pixel impulses positive only by comparing 3 vertical pixels at a time For each 3 pixel group pixels A B and C the impulse filter compares and resolves the middle pixel B as follows If pixel B gt filter weight x A C then B 2 A4C 2 The filter weight is a factor used to compare pixel B against pixels A and C You can choose one of three filter weight levels from the Impulse Filter list box Low is 0 75 and allows the greatest discrepancies between the pixel values in the three pixel set and removes the least number of one pixel impulses e Medium is 0 6 and provides a midrange setting Highis0 55 and allows the fewest discrepancies between the pixel values and removes the most impulses FluorSep applies the impulse filter to the unseparated files and then separates the image p2 16 ImageQuant Utilities User s Guide Chapter 3 Using FluorSep to build a dataset file TheFluorSep utility includes a feature that allows you to build a dataset file You can build a dataset file from single images gel or from images created as part of a dataset ds Thetopics in this chapter are Creating the images section 3 1 Building
44. k box next to each toolbar you want displayed a FluorSep and ImageQuant Tools for Macintosh b ImageQuant Tools for Windows Do r ToolTips r Color Buttons ToolBars ToolTips Main c c E mue Show Color di Show C Hide C Biw Multichannel C Hide Cancel Multichannel owes Figure 7 1 The Toolbar Configuration window a FluorSep and ImageQuant Tools for Macintosh and b ImageQuant Tools for Windows ImageQuant Utilities User s Guide p7 1 Partfour Reference p7 2 Important A ToolTip is a brief description of the function of a button If you select Show you see the tip when you rest the pointer on the button In FluorSep and ImageQuant Tools for the M acintosh you can select whether you want the toolbar buttons displayed in color or in black and white Click OK to confirm your choices and close the window If you reposition a toolbar ImageQ uant saves the new position when you close the application Leave the Multichannel toolbar in the horizontal orientation Changing the orientation to vertical causes the View One Channel button to disappear See appendix A for a description of the toolbar buttons in the Windows version of the FluorSep and ImageQ uant Tools software See appendix B for a description of the toolbar buttons in the M acintosh version of the FluorSep and ImageQ uant Tools software 7 2 Status Bar The Status Bar command displays or hides the status bar which is
45. kers click the Find Fluors button in the Fluorochrome Separation window FluorSep compares the channels in the image pixel by pixel FluorSep uses the F1 through F4 rectangles to enclose the control markers of the fluorochromes that you included in your experiment and collected through emission filters 1 through 4 respectively ImageQuant Utilities User s Guide p2 7 Parttwo FluorSep utility SSS After FluorSep finds the brightest fluorochrome markers FluorSep places each rectangle so that the brightest pixel within the marker is centered in the rectangle figure 2 6 Because the brightest pixel might not be at the center of the marker you might need to reposition the rectangles section 2 5 Contaminants such as dust or a poor signal to noise ratio can prevent Find Fluors from accurately locating the optimal fluorochrome markers If Find Fluors cannot locate the control marker for a specific fluorochrome a window appears with the message Cannot Find F 1 2 3 or 4 position Use the instructions in section 2 5 to place the rectangle on the fluorochrome marker for this channel manually Brightest pixel in band a UL B ann 4 i PTT jae te Figure 2 6 The new positions of the rectangles after using Find Fluors to locate the fluorochrome markers p2 8 ImageQuant Utilities User s Guide Chapter 2 Using FluorSep to separate images 2 5 Sizing and centering the fluorochr
46. kup table and alter the slope of the sigmoidal lookup table definition Click Reset in the M ode area to return to the default linear lookup table definition 7 9 Map TheM ap command displays the M ap window which is a smaller version of the Image window The M ap window allows you to select a different portion of the Image window for viewing An outline depicts the area currently displayed in the Image window To change the image display drag the outline in the M ap window The Image window displays the new area when you release the mouse button To move the M ap window move the pointer into the M ap window SH IFT 4drag the M ap window to a new location To move the M ap window outline and the image display together CTRL 4drag the outline The image display changes as you move the outline To return the M ap window to the upper left corner of the Image window Windows M ovethe pointer into the M ap window Right click and choose Reset from the pop up menu e Macintosh M ove the pointer into the M ap window SHIFT CTR L click and choose Reset from the pop up menu To close the M ap window click the M ap command in the View menu ImageQuant Utilities User s Guide p7 7 Partfour Reference 7 10 Multichannel The M ultichannel command contains the display commands for multichannel images figure 7 5 The multichannel command and toolbar are active when you display a multichannel image in the Image window
47. l Keyboard shortcuts Menu Key Function File menu 360 Open SHIFT F2 Image Properties 36Q Quit Edit menu 3C Copy Image ImageQuant Tools Only View menu F9 Zoom In SHIFT F9 Zoom Out F8 Gray Color Adjust CTRL 1 Multichannel Channel 1 CTRL 2 Multichannel Channel 2 CTRL 3 Multichannel Channel 3 CTRL 4 Multichannel Channel 4 Tools menu CTRL S Select CTRL R Define Region of Interest Window menu F5 Cascade SHIFT F5 Tile ImageQuant Utilities User s Guide pB 1 Part five Appendixes B 2 Mouse key combinations Mouse key Function Image window Mouse Pull down menus Drag Commands Click Toolbars Click Activate window SHIFT CTRL mouse Pop up menus Magnification CTRL click Switch between Enlarge and Reduce modes Mouse keypad plus Enlarge while in Magnifier mode Mouse keypad minus Reduce while in Magnifier mode Map window SHIFT click Move Map window CTRL4click Move Map window in sync with Image window pB 2 ImageQuant Utilities User s Guide AppendixB Quick reference for Macintosh B 3 Toolbar functions Main toolbar Multichannel toolbar 2 2 lets T4 Cla Help Open View One Channel Overlay Side by Side Channel 4 Channel 3 Channel 2 Channel 1 Image toolbar v e aaan oj Define Region of Interest Gray Color Adjust Map Fit to Window Magnifier Previ
48. land amp Baltics Tel 4358 0 9 512 39 40 Fax 4358 0 9 512 17 10 France Tel 01 69 35 67 00 Fax 01 69 41 96 77 Germany Tel 0761 4903 291 Fax 0761 4903 405 Italy Tel 02 273221 Fax 02 27302 212 Japan Tel 481 3 5331 9336 Fax 481 3 5331 9370 Web site http w w w amershambiosciences com ImageQuant Utilities User s Guide Fax 21 417 31 84 Russia amp other C I S amp N I S Tel 7 095 232 0250 956 1137 Fax 7 095 230 6377 So Tel utheast Asia 460 3 8024 2080 Fax 460 3 8024 2090 Spain Tel 93 594 49 50 Fax 93 594 49 55 Sweden Tel Fa 018 612 1900 x 018 612 1910 Switzerland Tel Fa UK Tel Fa 01 802 81 50 x 01 802 81 51 0800 616928 x 0800 616927 USA Te 1 800 526 3593 Fa x 1 877 295 8102 Part one Introduction Chapter 1 The ImageQuant utilities ThelmageQ uant Utilities U ser s Guide explains how to use the FluorSep and ImageQ uant Tools utilities This chapter provides a brief description of each utility Thetopics in this chapter are Overview of the FluorSep utility section 1 1 Overview of the ImageQ uant Tools utility section 1 2 Checking the audit trail section 1 3 1 1 Overview of the FluorSep utility The FluorSep utility uses a fluorochrome separation process to separate the multichannel images you create using the Typhoon Fluorlmager or Storm
49. lues with a light background low values Windows only Flicker Y ou can use the flicker feature on a multichannel dataset image to simulate an animation sequence Windows only Crop You can crop a multichannel dataset image to remove portions of the image from analysis All the channels images of the multichannel image are cropped to the same size e Windows only Copy pixel values for 3D charting You can copy a portion of an image and paste the pixel values in Excel to create a three dimensional 3D chart of the image features Windows only Save multichannel image as individual images Y ou can save the channels of a multichannel dataset image as individual uniquely named images See part 3 for a description of the ImageQ uant Tools utility 1 3 Checking the audit trail Because both the FluorSep and the ImageQ uant Tools utilities modify the original image the software provides an audit trail of all the image processing operations applied to the image You can check the audit trail from FluorSep ImageQ uant Tools or ImageQ uant To check the audit trail 1 Open the image in FluorSep ImageQ uant Tools or ImageQ uant 2 Choose Image Properties from the File menu ThelmageProperties window appears and displays the File Info tab 3 Click the History tab The contents of the History tab appear figure 1 1 pl 2 ImageQuant Utilities User s Guide Chapter 1 The ImageQuant utilities E
50. m select the M anual method section 2 3 3 After you select the background correction method use sections 2 4 through 2 6 to place the rectangles ImageQuant Utilities User s Guide p2 3 Parttwo FluorSep utility 2 3 1 Determining the uniformity of the background If you cannot determine the uniformity of the background visually you can use the Pixel Locator tool to examine the uniformity of the background by pixel value To use the Pixel Locator tool 1 Choose Pixel Locator xp from the Tools menu or the Image toolbar 2 Movethe pointer to the background near thetop left of the image Click the mouse button and make a note of the value 3 Movethe pointer to a background location farther down and to the right Click the mouse button and make a note of the value 4 Repeat step 3 until you reach the bottom of the image 5 Click Select sp to end the Pixel Locator mode 6 Examine the difference between the highest and the lowest relative fluorescent unit rfu values fthedifferenceis less than 2096 of the total values choose the Automatic method section 2 3 2 f the difference is more than 20 of the total values you can either choose the Automatic method section 2 3 2 and the Local or Global filter section 2 9 or choose the M anual method section 2 3 3 and the Local filter section 2 9 1 2 3 2 Selecting the Automatic method Theautomatic background correction method uses rectangle objects
51. mages using ImageQuant or other analysis software 4 1 Displaying an image Before you can process an image using ImageQ uant Tools you display the image in an Image window To display an image 1 In theImageQ uant Tools window choose O pen E from the File menu or the M ain toolbar The O pen window appears showing the active folder and its contents 2 Iftheimage name is not listed choose a different folder and view its contents ImageQuant Utilities User s Guide p4 1 Partthree ImageQuant Tools utility oe 3 Locate and select the image file The Image window appears and displays the image you selected See your Windows or M acintosh documentation for a more complete description of how to use the features in the O pen window 4 2 Rotating the image in 90 increments To rotate the image in 90 increments choose Rotate Image from the Image menu The Image Rotation window appears figure 4 1 The Image Rotation window contains two copies of the image displayed in the Image window The Image Rotation window contains rotation controls for 90 increments and arbitrary increments If you want to rotate the image in increments smaller than 90 see section 4 3 Figure 4 1 The Image Rotation window The Control Image area contains the eight 90 rotation choices Thetop four choices rotate the image clockwise in 90 increments The bottom four choices flip mirror the image and then rotate the image coun
52. mbinations 000 c eee en Toolbar functions for FluorSep saaana Toolbar functions for ImageQuant Tools 0 0 eee eee ImageQuant Utilities User s Guide Table of contents A 5 Extensions that identify file types 0 cece eee A 5 Appendix B Quick reference for Macintosh B 1 Keyboard shortcuts aasa aa B 1 B 2 Mouse key combinations 00 0 cece ee B 2 B 3 Toolbarfunctions sr ana a A TAAA B 3 B 4 Icons that identify file types 0 0 eee ee B 4 ImageQuant Utilities User s Guide vii Preface About this guide ThelmageQ uant Utilities U ser s G uide describes how to usethe FluorSep and ImageQ uant Tools utilities e Part one Introduction Provides an introduction to the mageQ uant utilities e Part two FluorSep utility D escribes how to use the FluorSep utility to separate multichannel images and to create multichannel datasets e Part three ImageQ uant Tools utility D escribes how to use the ImageQ uant Tools utility to modify images e Part four Reference Provides a reference to features in the FluorSep and ImageQ uant Tools utilities that are not discussed in parts two and three Part five Appendixes Provides a quick reference for the FluorSep and ImageQ uant Tools utilities Related publications In addition to the I mageQ uant Utilities U ser s G uide Amersham Biosciences provides the following publications mageQ uant User s Guid
53. nd an unknown in channel 2 and channel 3 you must build two datasets before you can analyze the data in Fragment Analysis One dataset includes the standard in channel 1 and the unknown in channel 2 The second dataset includes the standard in channel 1 and the unknown in channel 3 To build a new dataset file from the separated images in another dataset 1 Start the FluorSep utility and choose Build D ataset from the File menu The DS Builder window appears figure 3 1 2 Select the Separated DS for analysis box The fields for channels 3 and 4 become inactive figure 3 2 3 Click the Browse button 2 for channel 1 The O pen window appears DS Builder x M BEL Sources lew zz EM Aum a CL sse col Figure 3 2 The DS Builder window in the separated dataset mode 4 Locate the filename dir folder that contains the separated image data file you want to usefor channel 1 where filename is the name of the dataset file 5 Doubleclick the folder to display the contents ImageQuant Utilities User s Guide p3 3 Parttwo FluorSep utility um 6 Select the sep gel file where can be 1 through 4 that represents the number of image channels contained in the dataset 7 Click the Browse button 5 for channel 2 The O pen window appears 8 Repeat steps 4 through 6 to select the second image 9 Click Build After the software creates the dataset file choose Save As fr
54. ng the image If you are saving the image to a different folder from the default choose the folder from the folder file name list If you are saving a dataset image ImageQ uant Tools copies the dataset document the dataset folder and the contents of the dataset folder ImageQ uant Tools adds the appropriate extensions to all the files The original image and associated files remain unaltered 5 5 Save Region of Interest As ImageQuant Tools only The Save R egion of Interest As command allows you to save a portion of an image using a new name You select the portion of the image using the Define Region of Interest command on the Tools menu section 8 9 After defining the region of interest the Save Region of Interest As command becomes active In the Save Region of Interest As window type the name to use for saving the image If you are saving the image to a different folder from the default choose the folder from the folder file name list If you are saving a dataset image ImageQ uant Tools copies the dataset document the dataset folder and the contents of the dataset folder ImageQ uant Tools adds the appropriate extensions to all the files The original image and associated files remain unaltered 5 6 Save Cropped Dataset over Original ImageQuant Tools for Windows only The Save Cropped Dataset over O riginal command allows you to overwrite the uncropped dataset files with the cropped dataset files Section 4 7 describes
55. om the File menu The Save As window appears 10 Type a name for the dataset file and click Save Important You cannot use the Undo Separation feature on a dataset file that you create using this method p3 4 ImageQuant Utilities User s Guide Part three ImageQuant Tools utility Important Chapter 4 Using ImageQuant Tools ThelmageQ uant Tools utility allows you to modify a copy of an imagein a variety of ways Thetopics in this chapter are Displaying an image section 4 1 Rotating the image in 90 increments section 4 2 Rotating the image in 0 5 increments section 4 3 nverting the data values in the image section 4 4 Removing noise using the impulse filter section 4 5 e Removing noise using the median filter section 4 6 Cropping and uncropping a multichannel image Windows only section 4 7 e Exporting a multichannel image as individual images Windows only section 4 8 Viewing a multichannel image using a flicker effect Windows only section 4 9 Copying image data to Excel for 3D charting Windows only section 4 10 Before using the features in ImageQuant Tools on an image check that sufficient disk space is available on the computer Each process requires approximately twice as much disk space as the size of the file being processed After you process the image you will have the original image file and the new modified image file You can open and quantitate both i
56. ome marker rectangles The way you placethe fluorochrome marker rectangles determines how accurately FluorSep removes the background Because all the rectangles must be the same size you need to size the rectangle that encloses the largest fluorochrome marker After you size the rectangle you should reposition each rectangle so that the fluorochrome marker is centered in the rectangle To size and center a rectangle 1 Click to select the rectangle that encloses the largest fluorochrome marker Handles appear on the corners of the rectangle 2 Placethe pointer on the rectangle name and drag the rectangle until two perpendicular sides of the rectangle are close to the marker figure 2 7a 3 Placethe pointer on the handle farthest from the marker and drag the handle until the remaining two sides of the rectangle are close to the marker figure 2 7b The rectangle should enclose the entire marker with as little background as possible N ote Because the rectangles must be the same size all the rectangles change size as you change the selected rectangle Handle l aa F I RE OEOENE i F a Click the name and drag the b Drag the handle so that rectangle so that the two sides the remaining two sides are are close to the fluorochrome close to the fluorochrome marker marker Figure 2 7 Positioning and sizing the rectangle over the fluorochrome marker 4 Center the remaining rectangles over the fluorochrome
57. ommand 10 1 list of open windows 10 1 Tile Horizontal command 10 1 Tile Vertical command 10 1 Z Zoom Area command 8 2 Zoom Area submenu Create Frame command 8 2 Previous command 8 2 Reset command 8 2 Zoom In command 7 2 Zoom Out command 7 3 ImageQuant Utilities User s Guide e Index Index 5
58. ous Frame Create Frame Pixel Distance Pixel Locator Select Pan ImageQuant Utilities User s Guide pB 3 Part five Appendixes B 4 Icons that identify file types The following icons identify the types of files ImageQ uant Tools and FluorSep create Identifies the folder associated with an image dataset and contains the image file and the object file for that dataset M ultichannel image datasets contain an image file and an object file for each channel The dataset folder also contains a copy of the dataset file which you can use if you accidentally delete the original dataset file Identifies an image dataset Each dataset file has an associated dataset folder Identifies a file containing image objects Identifies a single image created on an Amersham Biosciences instrument Identifies a single image file that has been converted to an 8 bit TIFF format An 8 bit image can be stored in or transferred to another application pB 4 ImageQuant Utilities User s Guide Index 3D charting 4 14 A About FluorSep command 11 2 About ImageQuant Tools command 11 2 Actual Size command 7 2 anti alias use with rotate 4 3 audit trail checking 1 2 background correction method automatic 2 4 manual 2 5 selecting 2 3 background uniformity determine 2 4 Build Dataset command 5 1 C Cascade command 10 1 charting in Excel 4 14 Close All command 10 1 Close command 5
59. pears above or below the histogram plot or type a new valuein the High or Low box figure 7 3 You can use the Autoscale button to return to the scale determined by the utility N ote If you do not see the H igh box Low box or Autoscale button dick the Edit H igh Low button 4 Bo BoBE Eco Firar Chane Haitzin L hangs 1 o y D krni d oc Drag to change the j Hugh a4614 1 high data value Edit High Low button Type a new high data value Click to have the utility scale the image Type a new low data value Drag to change the low data value 18335 ebid ImageQuant Tools for Windows only Figure 7 3 The High Low controls in the Gray Color Adjust window ImageQuant Utilities User s Guide p7 5 Partfour Reference 7 8 2 Using the lookup tables to change the display The Gray Color Adjust window also provides lookup table definitions that change the image display These changes affect the image display but not the features in the utility To usethe lookup table controls click the Edit Lookup Table button N table controls figure 7 4 The utility replaces the scaling controls with the lookup a Curve mode controls NDemolmgs sypro c cht m Preview Channels 1 rd Aa sni Histogram Channel 1 Color H Invert Mode r Bend Log C Sigmoidal eset 0 00 35984 78
60. pulse Filter command allows you to remove the background noise from the image See section 4 5 for a complete description of the Impulse Filter command 9 4 Median Filter TheM edian Filter command allows you to remove bright noise spikes from the image See section 4 6 for a complete description of the M edian Filter command ImageQuant Utilities User s Guide p9 1 Chapter 10 Window menu TheWindow menu contains Cascade section 10 1 TileHorizontal section 10 2 Tile Vertical section 10 3 e Close All section 10 4 e List of open windows section 10 5 10 1 Cascade The Cascade command overlaps multiple windows The title bar for each window is displayed and accessible To bring a window to the top of the stack click anywhere in the window 10 2 Tile Horizontal The Tile H orizontal command displays all the windows horizontally The windows are reduced to fit the tiled arrangement 10 3 Tile Vertical The Tile Vertical command displays all the windows vertically The windows are reduced to fit the tiled arrangement 10 4 Close All The Close All command closes all open windows You are prompted to save changes to the files 10 5 List of open windows A list of the open windows appears at the bottom of the menu You can select a filename from the list and bring the window to the front if it is hidden or difficult to locate ImageQuant Utilities User s Guide p10 1 Chapter 11 Help menu
61. r compares and resolves the middle pixel B as follows If pixel B gt filter weight x A C then B 2 A4C 2 The filter weight is a factor used to compare pixel B against pixels A and C You can choose one of three filter weights from the Impulse Filter window Low 0 75 and allows the greatest discrepancies between the pixel values in the three pixel set and removes the least number of one pixel impulses Medium 0 6 and provides a midrange setting High 20 55 and allows the fewest discrepancies between the pixel values and removes the most impulses ImageQuant Utilities User s Guide p4 5 Partthree ImageQuant Tools utility p4 6 To remove the noise from an image 1 Choose Impulse Filter from the Image menu The Impulse Filter window appears figure 4 3 Choose Low M edium or High for a filter weight Click OK The Save As window appears Type a name for the image file O ptional Choose a different folder or drive Qv Ur owe Oc SP Click OK The filtered image appears in a new Image window Impulse filter x Filter weight C Low C Medium High Cancel Figure 4 3 The Impulse Filter window 4 6 Removing noise using the median filter Using a median filter you can remove bright noise spikes from the image without blurring the image You can choose a 3x3 or 5x5 kernel The median filter determines the highest pixel value noise spike in the kernel and repl
62. rogress appears in the status bar If you want to cancel the separation press the ESC key Note The larger the image the longer the time needed for the separation function After FluorSep removes the cross contamination a window displays the message Fluorochrome Separation has been completed Click OK The separated image appears in the Image window in the O verlay view figure 2 10 and the Perform Separation button changes to Undo Separation Figure 2 10 Image separated using the automatic background correction method displayed in the Overlay view ImageQuant Utilities User s Guide p2 13 Parttwo FluorSep utility Lm 2 8 Evaluating the results Important To improve the visibility of the image after separation you might need to optimize the image using the Gray Color Adjust feature section 7 8 If you see distinct colored bands in the overlaid image you have completed the separation process You can start your analysis using the ImageQ uant software If the colored bands are not what you expected you can undo the separation Click the U ndo Separation button A window appears with the message Are you sure you want to undo Fluorochrome Separation Click OK The original unseparated image replaces the separated image in the Image window R epeat the separation using one or more of the following options Theaccuracy of fluorochrome separation is very sensitive to the placement of the rectangles Check each rec
63. ropped dataset image Windows only Depending on the command you choose you can save the cropped dataset image as a new dataset image overwrite the original image or save the dataset image as individual images Note If you do not want to save the cropped image close the Image window without saving To save the image as a new dataset choose Save As from the File menu The Save As window appears Type a new name for the dataset image and click Save Both the original dataset and the new dataset appear in the Data folder or the folder you selected e To overwrite the existing dataset image choose Save C ropped D ataset over Original from the File menu A message window appears warning you that you are about to overwrite the original dataset Click Yes to overwrite or click No to close the message window without overwriting To export the dataset image as individual images follow the instructions in section 4 8 4 8 Exporting a multichannel image as individual images Windows only You can export the displayed multichannel image as individually named images After you save the images you can view the data using other analysis software such as the D eC yder software If you export a multichannel image as individual images you cannot view the images in ImageQuant as a multichannel dataset image To export the multichannel image as individual images 1 Choose Export Gel Files From Dataset to Folder Bh from the File men
64. rt two FluorSep utility Chapter 2 Using FluorSep to separate images The FluorSep utility allows you to separate the multichannel images you created using the Typhoon Storm 860 or Fluorlmager instruments The topics in this chapter are e About FluorSep section 2 1 Displaying the image section 2 2 Selecting Automatic or M anual background correction section 2 3 Locating and placing the fluorochrome marker rectangles section 2 4 Sizing and centering the fluorochrome marker rectangles section 2 5 Locating and placing the background rectangles M anual method only section 2 6 e Performing the separation section 2 7 Evaluating the results section 2 8 Choosing a background correction filter section 2 9 e Using the impulse filter during separation section 2 10 2 1 About FluorSep TheFluorSep utility provides two methods to remove the cross contamination Automatic FluorSep determines which pixel values are background by using a histogram peak algorithm Use this method if the image background is uniform e Manual You determine which areas on the image represent the background Use this method if the image background is not uniform You should try the automatic method first FluorSep includes an Undo Separation feature which allows you to if necessary repeat the separation process using different variables If the results are not acceptable you can usethe manual method In
65. tangle to make sure that the marker is centered in the rectangle R esize the rectangle that encloses the largest fluorochrome marker to remove more background pixels section 2 4 e If you do not like the results of the automatic background correction method use the M anual background correction method to define specific areas on the image to use as the background section 2 6 In addition to the automatic background correction method use the Local or Global background filter to help remove the background values section 2 9 e In addition to the manual background correction method use the Local background filter to help remove the background values section 2 9 1 In addition to the automatic or manual background correction method use the Impulse Filter to remove artifacts caused by imperfections in the sample matrix section 2 10 2 9 Choosing a background correction filter To enhance the quality of the image background correction you can use the Local or Global background correction filter with the Automatic method or the Local background correction filter with the M anual method 2 9 1 Local background correction filter Inconsistencies in the sample thickness used during the scan can result in an image background that is not uniform The Local filter figure 2 11a removes the variation in the background values relative to the image bands which creates a uniform background The Local filter works best on an unseparate
66. tents o n 8 3 vi 8 4 8 5 8 6 8 7 8 8 8 9 Reduce ImageQuant Tools only 0 0 00 c eee eee ZOOM ANE haina aa sau ites ore cei m Gia tease uta te a aea e eed t edd Magnifier Lii pa kale hoe atk eh MEX Eh Dea it erede bed Pixel Locatore oia ice De S E ud rere aere RH Pixel DISTANCE a u iu kai ike RRERRIIEEFAXEEENEPEeeqHura Define Region of Interest ImageQuant Tools only Chapter 9 Image menu ImageQuant Tools only 9 1 9 2 9 3 9 4 Rotate Image seien St poete qoe eoe Se sea a Tha ca ad InvertImage tect eee eR pea ee Reed Impulse Filter us oer UEM aks EXE NGA NR Red M dia Filter ru eel ie RD e eviter eine eee Aes Chapter 10 Window menu 10 1 10 2 10 3 10 4 10 5 Cascade uuu umo aac ERR e ose vaca vut dy esee Te tid Tile Horizontal a teach Bet AN XUBMEA TERRE ERN RR E Til amp Vertical ze eee ue ese dete an rede i aede Glose All ET List of open WINdOWS 1 tee Chapter 11 Help menu 11 1 11 2 11 3 11 4 11 5 11 6 Index FluorSep and ImageQuant Tools for Macintosh only Using Help FluorSep and ImageQuant Tools for Macintosh only User s Guide ImageQuant Tools for Windows only Molecular Dynamics Home Page isses esses About Fluors ep nies oro a A b Ga dra About ImageQuant Tools i n Part five Appendixes Appendix A Quick reference for Windows Ad A 2 A 3 A 4 Keyboard shortcuts 0 0 c cette tes Mouse key co
67. terclockwise in 90 increments p4 2 ImageQuant Utilities User s Guide Chapter 4 Using ImageQuant Tools To rotate the image 1 In the Control Image area click the button that represents the rotation you want The Destination image rotates so that you can preview the effect of the rotation 2 Optional If you want the rotated image to open automatically after you save it select the O pen image after rotate check box 3 When the rotation is correct for the Destination image click Rotate The Save As window appears 4 Inthe Save As window type a name for the rotated image 5 Optional Choose a different folder or drive 6 Click Save A new file containing the rotated image is created using the name you typed in step 4 If you selected O pen image after rotate in step 2 the rotated image appears in a new Image window 4 3 Rotating the image in 0 5 increments Thelmage Rotation window figure 4 1 contains two copies of the image displayed in the Image window and the window contains rotation controls for 90 increments and 0 5 increments If you want to rotate the image in 90 increments see section 4 2 You usethe controls in the Arbitrary area to rotate the image in 0 5 increments If you want to smooth the jagged edges of the displayed image select the Anti Alias feature The 90 increment and the arbitrary increment features can be used together For example you can rotate the image 90 and then rot
68. to identify the fluorochrome markers Important The fluorochrome marker designates the brightest location of each channel relative to the other channels Accurate sizing and placement of the rectangles is important for proper functioning of the background correction methods To select the Automatic background correction method click Automatic in the Background Correction M ethod area of the Fluorochrome Separation window Depending on the number of fluorochromes in the sample two or more rectangles appear on the image figure 2 3 Each rectangle is labeled F4 where is the fluorochrome marker number for the emission filter N ote The number of rectangles depends on the number of channels acquired by theinstrument and the settings you selected in the Scanner Control software for that instrument For example if you scanned a sample that included two fluorochrome markers you will see two rectangles labeled F1 and F2 p2 4 ImageQuant Utilities User s Guide Important Chapter 2 Using FluorSep to separate images You usetheFind Fluors feature to place the fluorochrome marker rectangles section 2 4 After placing the rectangles you might need to resize or recenter them section 2 5 Figure 2 3 The rectangles that appear when you use the Automatic background correction method for an image containing four fluorochrome markers 2 3 3 Selecting the Manual method The manual background correction method uses rectangle obj
69. ty 2 4 displaying an image 2 3 DS Builder 3 1 effects of using background filter 2 15 evalute results 2 14 Find Fluors 2 7 fluorochrome markers 2 4 Fluorochrome Separation window 2 3 global background correction filter 2 15 impulse filter using 2 16 local background correction filter 2 14 locating background rectangles 2 10 locating fluorochrome marker rectangles 2 7 manual background correction method 2 5 overview 1 1 performing separation 2 13 sizing and centering rectangles 2 9 Undo Separation button 2 14 G global background correction filter in FluorSep 2 15 Gray Color Adjust command 7 3 Gray Color Adjust window changing display values 7 4 description 7 3 using the lookup tables 7 6 H Help menu About FluorSep command 11 2 About ImageQuant Tools command 11 2 Index command 11 1 Molecular Dynamics Home Page command 11 1 User s Guide command 11 1 Using Help command 11 1 Hide Objects command 7 9 History tab in Image Properties window 1 2 5 4 Image menu Impulse Filter command 9 1 Invert Image command 9 1 Median Filter command 9 1 Rotate Image command 9 1 Image Properties command 5 3 Image Properties window File Info tab 5 3 History tab 5 4 Scan Info tab 5 4 Image Properties audit trail 1 2 Image Rotation window in ImageQuant Tools 4 2 ImageQuant Tools 3D charting 4 14 copy image for publication 6 1 crop multichannel image 4 7 export multichannel image as individual images 4 10 flicker effect on multichann
70. u or the Image toolbar The Export Gel Files From Dataset to Selected Folder window appears figure 4 8 2 Typea unique name for each channel in the boxes labeled 1 through 4 under Gel File Names in Dataset ImageQuant Utilities User s Guide Chapter 4 Using ImageQuant Tools N ote If you do not want to export one of the gel files in the dataset delete the default file name and leave the file name box blank 3 Optional Click Browse and select a new folder from the Browse window Click OK to return to the Export Gel Files From D ataset to Selected Folder window 4 Click Saveto save the images Export Gel Files From Dataset to Sele ted x Current Dataset Folder C Data Gel_33301 Gel_33301_ DIF Gel File Names in Dataset unsep gel files Save Gel Files to Folder Save Cancel i Note f you do not type a file extension the sofware adds gel to the file name f you do not want to export a gel file in the dataset leave the file name blank Figure 4 8 The Export Gel Files From Dataset to Selected Folder window 4 9 Viewing a multichannel image using a flicker effect Windows only Using a flicker effect you can simulate an animation sequence on the displayed multichannel dataset image You choose the images you want to flicker and the flicker rate You can also change the default gray scale to a color and you can change the normalization display values ImageQuant Utilities Us
71. uant Utilities User s Guide p4 7 Part three ImageQuant Tools utility eS 4 7 1 Cropping a dataset image Windows only To crop a dataset image 1 Make sure the image is displayed in the Overlay mode If it is not choose Overlay G IN from the M ultichannel submenu on the View menu or on the M ultichannel toolbar 2 Choose Define Region of Interest gp from the Image toolbar or the Tools menu 3 Dragthe pointer to draw a box around the portion of the image that you want to use for analysis figure 4 6 N ote If you want to changethe portion of the image in the box drag again LU BLUE au el Figure 4 6 Selecting the portion of the multichannel dataset image to crop p4 8 ImageQuant Utilities User s Guide Chapter 4 Using ImageQuant Tools Parce 4 Choose Crop Dataset imp from the Edit menu or the Image toolbar The image changes to display only the area within the box figure 4 7 5 Optional To change the cropped area repeat steps 2 through 4 vw PImageljuant Deals 24 4r small ERIPPED UB ds Figure 4 7 The image after cropping ImageQuant Utilities User s Guide p4 9 Partthree ImageQuant Tools utility p4 10 e Important 4 7 2 Uncropping a dataset image Windows only To return to the previous image choose U ndo Crop of D ataset i from the Edit menu or the Image toolbar You can continue to uncrop until the image returns to the original size 4 7 3 Saving the c
72. uu ImageQuant Utilities User s Guide Microsoft Windows Apple Macintosh lt t Amersham am 375 580 e d DeCyder Fluorlmager FluorSep ImageQuant Storm and Typhoon are trademarks of Amersham Biosciences Limited Amersham and Amersham Biosciences are trademarks of Amersham plc Adobe Acrobat and Photoshop are trademarks of Adobe Systems Incorporated Apple Macintosh and Mac OS are trademarks of Apple Computer Inc Microsoft and Windows are trademarks of Microsoft Corporation Cover Foreground A two color gel image of fluorescently labeled DNA ladders and PCR products scanned with the Fluorlmager 595 and displayed in FluorSep The Fluorochrome Separation window is open in the lower right The image has been processed in FluorSep to facilitate spectral separation of the two labels Channels 1 Fluorescein and FAM Green and 2 TAMRA Red are displayed in the overlay mode Yellow indicates simultaneous signals from both fluorochromes at positions of comigration in the gel Cover Background A three color DNA typing gel short tandem repeats or STR scanned with the Fluorlmager 595 and displayed in ImageQuant Tools The image has been preprocessed in FluorSep to facilitate spectral separation of the three labels Channels 1 Fluorescein Green 2 TMR Blue and 3 CXR Red are displayed in the overlay mode The Image Rotation window in ImageQuant Tools is used to rotate the resulting three color im
73. verted to peaks and the peaks in the negative image are inverted to troughs producing a positive image a Before inverting the image e m y om on v ON e oe oo me ae Ore uw me e aa b After inverting the image Figure 4 2 Inverting the data values in an image a before inverting and b after inverting p4 4 ImageQuant Utilities User s Guide Chapter 4 Using ImageQuant Tools To invert an image 1 Choose Invert Image from the mage menu The Save As window appears Type a name for the image file O ptional Choose a different folder or drive mx GO ch Click OK The inverted image appears in a new Image window 4 5 Removing noise using the impulse filter Using an impulse filter you can remove artifacts caused by contaminants and imperfections such as dust or bubbles in the sample matrix R emoving the artifacts and imperfections improves the quality of the image analysis The impulse filter reduces one pixel impulses positive only by comparing three vertical pixels at a time table 4 1 Table 4 1 How the impulse filter affects pixel B using the Low Medium and High weights Pixel Original pixel value Low weight Medium weight High weight A 250 250 250 250 B 350 350 275 275 C 300 300 300 300 For each three pixel group pixel A B and C the impulse filte
74. w p2 2 ImageQuant Utilities User s Guide Chapter 2 Using FluorSep to separate images 2 2 Displaying the image Before using the FluorSep utility you display the image you want to separate in an Image window Note The Scanner Control software for your instrument might open FluorSep and display the image automatically To display an image 1 Choose Open amp from the File menu or the M ain toolbar The O pen window appears and displays the active folder and its contents 52 If the dataset name is not listed choose a different folder and view its contents Mx Locate and select the dataset file ds Theimage appears in an Image window and the Fluorochrome Separation window appears figure 2 2 See your Windows or M acintosh documentation for a more complete description of how to use the features in the O pen window Fluorochrome Separation Background Correction Method s mE M Local Background Filter He Global Background Filter Impulse Filter High hd Find Fluors Figure 2 2 The Fluorochrome Separation window 2 3 Selecting Automatic or Manual background correction The FluorSep utility provides two methods to remove the background correction To determine which method to use you need to evaluate the uniformity of the background section 2 3 1 If you determinethe background is uniform select the Automatic method section 2 3 2 If you determine the background is not unifor
75. w combination of channels 7 8 view one channel 7 8 0 Open command 5 1 Overlay command 7 8 P Pan command 8 2 Pixel Distance command 8 3 Pixel Locator command 8 3 Q Quit command 5 5 R Reduce command 8 1 Rotate Image command 9 1 S Save As command 5 2 Save Cropped Dataset over Original command 5 2 save cropped multichannel images 4 10 Save Region of Interest As command 5 2 Scan Info tab in Image Properties window 5 4 Select command 8 1 Side by Side command 7 8 Status Bar command 7 2 Storm 860 1 1 T Tile Horizontal command 10 1 Tile Vertical command 10 1 Toolbar Configuration window 7 1 Toolbars command 7 1 Index4 ImageQuant Utilities User s Guide Tools menu Define Region of Interest command 8 4 Enlarge command 8 1 Magnifier command 8 2 Pan command 8 2 Pixel Distance command 8 3 Pixel Locator command 8 3 Reduce command 8 1 Select command 8 1 Zoom Area command 8 2 Typhoon 1 1 U uncrop multichannel image 4 10 Undo Crop Dataset command 6 2 Undo Separation button in FluorSep 2 14 User s Guide command 11 1 Using Help command 11 1 V View menu Actual Size command 7 2 Fitto Window command 7 2 Flicker Display Channel command 7 9 Gray Color Adjust command 7 3 Hide Objects command 7 9 Magnification command 7 2 Map command 7 7 Multichannel command 7 8 Toolbars command 7 1 View Status Bar command 7 2 Zoom 7 2 Ww Web site connection 11 1 Window menu Cascade command 10 1 Close All c
76. will see five rectangles labeled F1 F2 B1 B2 and B 2 6 1 Positioning the B1 through B4 rectangles After you size and center the fluorochrome marker rectangles you place each background rectangle in a portion of the image background near the corresponding fluorochrome marker rectangle To position the background rectangles 1 Click to select the B1 rectangle 2 Drag the B1 rectangle to a portion of the background near the control marker for fluorochrome 1 3 Repeat steps 1 and 2 for the B2 through B4 rectangles and the control markers for fluorochromes 2 through 4 Note The number of background rectangles depends on the number of fluorochromes used to scan the image 2 6 2 Determining the location for the B rectangle Choosing the correct location for the B rectangle improves or optimizes the separation results If your image has weak bands you should place the B rectangle in an area that contains values that are lower than the weakest band you want to see Placing the rectangle in an area with the highest background value can cause weak bands to disappear from the separated image You can visually determine the weakest band on the image If your image does not have weak bands you can place the B rectangle in an area that contains the highest background value found on the image N ote Placing the B rectangle at the lowest background value is similar to using the Global background filter

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