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Operator Manual - GE Healthcare Life Sciences

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1. Whal do pou want the wizard to do nstallthe software automatically Hlecommended Install fom a lst or specific location Advanced Check Next to continue 4 Select the Install the software automatically radio button and then press Next 12 ImageQuant RT ECL Capture User Manual 5 The following screen will be displayed Found New Hardware Wizard Please wait while the wizard installs the software Ha GE RT ECL USB Camera 6 After the SBIG Loader has been loaded the following screen will be displayed Found New Hardware Wizard Completing the Found New Hardware Wizard The wizard has finished installing the software for f GE RT ECL USB Camera Chock Finish to close the wizard 7 Press the Finish button to continue 8 You are now ready to operate the software and the camera 1 3 6 Connecting the Printer The Mitsubishi P91DW UB printer connections are color coded for convenience see figure on previous page Plug the USB cable into the back of the printer and into the proper USB connector on the back of the computer Plug in the standard 3 prong power cable to the back of the printer and to the power strip then turn the power on Note The Mitsubishi P91DW UB printer may be connected to any USB port on the computer however the driver may need to be manually reinstalled The Mitsubishi CP700 and CP770 printers connect via the on board parallel LPT1 port Plug in the standard 3
2. PEN STYLE JTExT oRIENT LI Pen Style Selection Tools 4 5 5 Arrows and Straight Lines The LINE ENDS menu specifies the style of the ends of straight lines no arrow single arrow or double arrow Click on the appropriate checkbox for the desired style Note these line ends work with any line thickness pa N olo x a 8 COLOR LINE ENDS PEN WIDTH TEXT STYLE gt PEN STYLE TEXT ORIENT _ _ Line Ends Selection Tools T11 ImageQuant RT ECL Capture User Manual 4 5 6 Text Background and Font The TEXT STYLE menu specifies the style of text Click on the appropriate checkbox to show text with or without a background An opaque background 1s useful if annotations will be made on an image that has wide variations in gray scale By using an opaque background text will not be lost in the background of the image pa N olo x ao l COLOR LINE ENDS R TRANSPARENT BACK PEN WIDTH 2 TEXT STYLE Set Forit m Bd OPAQUE BACK PEN STYLE TEXT ORIENT Text Style Selection Tools This is also the window in which specific font is chosen When the Set Font button is depressed a selection box appears from which text style can be chosen Fon Font Font style Size Italic Bald Bold Italic V Anal Black O Arial Narrow Arial Rounded MT Bok O Arial Unicode MS O Baskerville Old Face F Batang l S
3. To view different regions of a magnified image move the cursor into the inner box Click and hold down the left mouse button The cursor changes to a hand use it to drag the box within the thumbnail until the desired region of the image appears on the screen Alternatively use the scroll bars in the main image window to move the image up down and left right On screen ZOOM tools are also available from the tool bar These buttons duplicate the Zoom tool in Tool Box and also allows for Img Drag to easily pan zoomed images Esa oom Out Zoom In Img Drag Zoom icons in Tool Bar Img Drag Icon in Tool Bar a2 ImageQuant RT ECL Capture User Manual 4 2 The Rotate Flip Tool The Rotate Flip tools are found in the Tool Box This function rotates the image in a clockwise or counterclockwise direction by 1 degree increments up to a maximum of 90 degrees in either direction This is a useful tool if the image is not aligned properly during the capturing process Tool Box Annotate False Color Filters FrameGrabber oom Rotate Flip Histogram Enhance ROTATE 0 UNDO FLIP 90 m g0 K a The Rotate Flip Tool To rotate an image click and hold down on the center sliding bar with the left mouse button and move it left or right until the desired angle of rotation appears in the rotate box Release the left mouse button and image will rotate to the desired angle To undo a rotation just click on the Undo button Al
4. darker Each increasing button click will lighten up the image until button 9 is pressed which will make the image the lightest possible Tool Box Annotate False Color Filters FrameGrabber Zoom Aotate Flip Histogram Enhance Foto Enhance Level BPE Rc fe a ea EA To undo any Auto Enhance Levels just press on the Reset button on the main interface Original Image Auto Enhance Level 2 Image Auto Enhance Level 9 Image seis Tey Je ststelr fe PEE Original Auto Enhance settings Auto Enhance with level 2 Auto Enhance with level 9 75 ImageQuant RT ECL Capture User Manual 4 5 Annotations The annotation tools found in the Tool Box include a number of different options for adding text including Greek symbols drawing arrows and otherwise marking an image Note that these tools are for annotation only Toots Zom i Rotate Flip l TTN Enhance Annotate False Color Filters Frame rabber a 2 N Bl g al E nnmn COLOR _ LINE ENDS FEN WIDTH TEXT STYLE PEN STYLE TEXT ORIENT Annotations Tools 4 5 1 Object Attributes Use the COLOR PEN WIDTH PEN STYLE LINE ENDS TEXT STYLE and or TEXT ORIENT menus to specify object attributes Attributes can be assigned to the cursor before drawing or typing Alternatively they can be assigned to an object while it is in edit mode see the following pages for more details 4 5 2 Annotation Colors Annotat
5. Fesmediabber Bl al BS i Ready to Crop or Copy 54 ImageQuant RT ECL Capture User Manual Once this is completed you can select either the COPY or CROP function in the EDIT menu options COPY will copy the desired area of interest into the Windows Clipboard and allow you to paste into any desktop publishing package 1 e Word Excel Adobe Photoshop etc CROP will display just the region of interest as the active window in the ImageQuant Capture interface Ge p jasp Setup miy e eer iin eee ee rs Lm ary Toda EPP Rea unea Zoom Rotsefip Hitogam Enhance Arnette Falsa Coles I Fae FeeneGretber E 52 N 5 D Frera Cora m a CEREA Zoom 200 intenginy G0 ImageQuant Capture interface after cropped region has been selected 3 3 2 1 Reset and Clear _Feset Clear The Reset option configures the Black White and Gamma settings to default settings Clear removes any annotations that are present on the image 55 ImageQuant RT ECL Capture User Manual 3 3 The Image Menu The Image menu option provides the ability to perform a variety of image processing functions Image Setup Overlay U Equalize Arithmetic Conversion Flat Field Calibrate Resize Image Info Image pull down menu 3 3 1 Equalize The equalize option performs a duplicate function to the EQUAL option in the Contrast Adjustment
6. Fluorescence samples that require epi or transillumination of UV energy should be placed on the purple UV filter glass For colorimetric samples such as protein gels film or blots use the fold down the white light table for your sample For chemiluminescence use the fold down white light table or the adjustable tray if used in conjunction with the fast lens Open the aperture on the camera lens all the way open to the smallest number With the door still open to allow light to enter into the cabinet use the monitor real time readout display to position and focus your sample in the middle of the image acquisition window Focus on the object more details on how to focus on an image are provided in 1 6 1 For fluorescence and chemiluminescence if there is too much light to obtain a good focus setting use the door to the cabinet to adjust the amount of light that enters into the cabinet Do not adjust the aperture to do this For colorimetric samples it is necessary to decrease the aperture to acquire the image Hint Business cards and other pieces of paper with small text often are the easiest objects to obtain optimal focus settings with g If using a zoom lens adjust the zoom setting so that the area of interest on the sample takes up all of the image size on the screen 3 Capturing a bright sample like fluorescently labeled gels colorimetric samples and film a b ae Close the cabinet door Choose the appropriate opt
7. Original Image ee ee f 3 D Contour Level 5 Horizontal Edge Vertical Edge 90 ImageQuant RT ECL Capture User Manual 4 8 FrameGrabber Mode If kinetic multiplex color or chemiluminescence experiments are desired where you wish to have the system automatically capture several images at preset exposure times preset time delay between images preset lighting sources and preset filter choices the FrameGrabber tab can be clicked in Tool Box FrameGrabber is also accessible in the camera setup acquisition screen To access this screen select the acquire button on the tool bar and then select FrameGrabber on the acquisition screen ImageQuant Capture stand alone software does not include FrameGrabber acquisition tools Image Tool Box Noise Reduction Enhancement Tools oom Rotate Flip Histograrn Enhance Annotate False Color Filters Framelrabber Exposure Time i Eil Ea gt 4 4 4 MiliSecs a O F Image o Auto Expose Auto Expose Setup Display Chemi Display M Show Saturation Auto Contrast Reverse REW RE REC STOP PAUSEPLAY FiO DOEL OPEN SAVE Sensitivity A esalutior Normal High Tee tect FrameGrabber Setup in the Tool Box Enhancement Tools High Medium High Low Super Senstivity FrameGrabber oom AOI ar 1 0 camera setup acquisition Tei 1 I 1 1 I 1 I I 1 Aw screen FrameGrabber button in the Ex
8. the save icon on the FrameGrabber tab in the tool box 4 8 2 Loading Saving an Entire Sequence You can load or save an entire sequence by using the load and save icons in LF the FrameGrabber tab in the tool box When a sequence is loaded the loaded frames will be seen as blue in the frame reel When a sequence is saved a dialog box will appear prompting you to select the location and name for the sequence that you would like to save the images as OPEN SAWE 93 ImageQuant RT ECL Capture User Manual 4 9 FrameGrabber Save Load FrameGrabber setup routines Two buttons Save Setup and Load Setup allow you to save and load all FrameGrabber setup parameters Files are saved as mvf files Camera Setup amp Preview m Frame Option Normal Sequence Stack Frames Total frames 1 ree a 1 zi a Exposure Time lt lt B 2 gt I I Auto Expose Hours Minutes Seconds MiliSecs o i o 120 Auto Expose Setup Display I Chemi Display I Show Saturation J Auto Contrast Reverse High Low Dark Field Method Dynamic Correctior Y Shutter f Open Closed Gain Settings ean High Low Sensitivity Resolution Normal High Medium Medium Super Sensitivity 4 9 1 Frame Stacking At the top of the FrameGrabber setup window is an option for stacking frames If this selection w
9. the monitor s video cable to the video card connector Connect the monitor s power cable I 29 ea to the power strip U i LPT1 port E w N i for parallel ort printers The mouse and keyboard connectors e are color coded and icon identified Attach the mouse and keyboard cables to the connectors on the back of the computer by matching the colors The computer is now ready to be turned on Turn the computer on by pushing the power button on the wee USB ports l for printers front of the unit and other devices ImageQuant RT ECL Capture User Manual 1 3 3 Cabinet Assembly When you remove the light cabinet from its shipping carton it is already partially assembled The camera mounting assembly is packed separately in the same container The UV transilluminator and cabinet top are both packed in separate boxes Make sure you have received all the hardware before discarding the shipping carton Start by placing the cabinet on a level flat surface in a position where you can easily reach the top front and back Attaching the Camera to the Cabinet Take the following steps to attach the camera to the cabinet I Remove the camera and lens from the packaging and attach the lens to the camera Attach the camera to the camera stand using the fastening knob Make sure lens is fully extended and that the tip of the lens is level with the bottom of the stand Attach the cables to the camera Carefully low
10. you can click the Img Drag button again to deactivate it Note Img Drag is only active when the image is zoomed in beyond 1X greater than 100 The button is grayed out in other zoom modes The Print button allows for quick and easy image printing with the active default printer Detailed instructions on image printing are available in Chapters 2 and 3 as printing can also be accomplished via traditional Windows File menu options The Notepad button opens up a dialog box to allow the user to quickly track experimental conditions comments and any other details to be saved as an electronic copy for future reference Detailed instructions are available in Chapter 3 as this Notepad function is duplicated in the Utilities function in the upper header bar Clicking Reset returns the image to the system defaults as specified in the active default file This is detailed later in Chapter 3 4 of the manual 44 ImageQuant RT ECL Capture User Manual Clear removes any overlays currently displayed on the image This function can be useful if annotations or other displays obscure parts of the image Once an image is displayed it can be printed on the default printer by clicking the Print button in the Tool Bar window display Most printers can be configured through the Windows operating system to be the default printer Refer to your Windows operating manual for more information on installing a default printer Sample Printouts 45
11. 66 Iy 2 LO AGING an OY Cll AY seiycaied ssa taiotcn brakes TA 67 DO AEG HUET ISS MEN ict see ta nctaetinseihia etcer R T 68 m6 al Il wp PIO Cc nem rer Rare Sener nro ee a nT ee ter re eee ante arr ee ee te een eee eer 68 IOL INOUE DA den a tenis ERAS a E alendeastaaeiecaceness 68 SF The Vew NICOW exces scsivastandeeassnsdatvaeeuscaedebpeseaatendeetaseateavervancandeeputeaedendseaaeeseeaserrncmeteeds 69 Di wl ZOOM ke UNC WONG is eesti tata A 70 Di 2 SOW ANNO IONS a EOT nee wes 70 3o heH D M 0 Reman te re mae a a Aen ene ee Te a 71 2S li es WN tom NOE a A Rene ne nn ee roa tet ror een eee ere ae ae 71 KO ADON e a gee maauaanstateatsdbasuaa mien cee maceanseceteune 71 Chapter 4 The Tool BOX ss cacccsasecsiivaecasissavacanteasatenciavensubsaccaacesa tacactessadenstnvacsusvaccaccssasstaucnsancanets 72 il 4i The Zoom Wl CO 0 Caeeamee rene ere re nam Tete E rd Se ae Ree ty a AEST re cd ee aE 72 Ao The Rotate7 FI Tobaran E 73 MS TEM OS AU Vaca paaisc una neson E E E OIN 74 A UOmatc Enhancement 2h isscuiatiek tat pasuuee raha haba cuuier a dabin arene updecesreauaeneetucaeiahaies 75 AD Annota ONS a a bwedlaeehaacaxaniu tide duiad aaa 76 AS CD JSC CE PACE UVC ae csst als Saca cc seater decide ested oases ered teres eth ca vasecsenetietadons 76 Ae PATO CAO ON ONS erst oars taetonireress ited va Dasiee E belrareessteucs 76 bs Dice Di Len E E 95 cre ince Shs ea E in aga N A E AAEE OE E S 11 nE Ml BD E NA E E E A A E AA N L I E E A E T EE A D Ar
12. 8 2 Loading Savine an Entre SCQUeN CC siini sreisii iea aii 93 4 9 FrameGrabber Save Load FrameGrabber setup routines sssseeeessssssseerrrsssssseee 94 4 9 T Eram U cA HL eo se ts a enn ated eased 94 4 9 2 Capturing a Color Image Using the FrameGrabber Function eee 95 Chapter Se Analy Zine TMVASES on E G 96 Appendix A Opening ImageQuant Capture Files in Other Software Programs 97 Appendix B Security Feat reS srira o Erne E ESEE NONE EEE r 99 il ImageQuant RT ECL Capture User Manual Chapter 1 Introduction and Setup 1 1 ImageQuant Capture Imaging System The ImageQuant Imaging System is a powerful digital imaging system ideal for instant photography of a wide variety of samples The CCD camera allows imaging of low light samples in UV illuminated and fluorescent applications The instrument is controlled by ImageQuant Capture software which is designed with ease of use in mind ImageQuant Capture handles image acquisition and archiving and can be used to prepare images for desktop publishing ImageQuant Capture includes tools to optimize the image display by adjusting contrast automatically or manually Hard to see portions of the image can be clarified by converting the image from positive to negative using digital filters or applying a false color map Notes labels arrows lines and other drawing tools can be recorded directly on the image using the annotation functions available in
13. Chemi Display A Normal High Show Saturation F Auto Contrast Medium Medium Filter Wheel _ Position FrameGrabber Super Sensitivity Chemiluminescenc Zoom ROI j 5 Door Sensor Temperat j CLOSED 22 ImageQuant RT ECL Capture User Manual 1 6 3 Acquire and Transfer the Image Once the appropriate image is displayed on the screen click on the Capture button to acquire and display the image The acquired image will be automatically adjusted by a zoom factor based on the resolution setting to display the image optimally From there the image can be enhanced using ImageQuant Capture s enhancement tools and or transferred to an ImageQuant TL module for analysis Once a satisfactory image has been captured we suggest saving it as an original file However if you do not save before transferring the image to an ImageQuant TL module for analysis then you will be prompted to save as part of the transfer process 1 6 4 Auto Exposure and Auto Exposure Setup The acquisition window for ImageQuant Capture includes a check box and setup window for Auto Exposure x Auto Espora Compentaton Seating Preview E Hamal exposure for image caturstion Under epore for chemndumineccence Over expocune fee faint band detection W Noise Reduction Auto Exposure setup Tae 0 a Se a descriptions E e EE 280 gt 70 00 1 20 gt Clo lt 0 178 EV Exposure Time lt l
14. ImageQuant RT ECL Capture User Manual 2 3 Tool Box The Tool Box contains an intuitive interface for performing all image enhancement functions Tool Box oom 4 Rotate Flip i a Enhance Annotate False Color Filters Framelrabber aleio oj fg al CTY f COLOR _ LINE ENDS PEN WIDTH TEXT STYLE PEN STYLE TEXT ORIENT Tool Box The Tool Box contains the controls for enhancing and adjusting the image This includes software filtering false colors zoom factors and other unique features The Tool Box options are detailed in chapter 4 of this manual 2 4 Status Bar The Status Bar is located on the bottom of the monitor and provides a real time display of the mouse cursor x y position the image zoom factor and the grayscale intensity at the mouse cursor x y position Pe 89 2 Zoom 200 Intensity 416 46 ImageQuant RT ECL Capture User Manual Chapter 3 Pull Down Menus Across the top of the screen is a Windows menu bar containing several system operation functions These include file saving and loading edit image setup overlay file utilities view and help functions Il ImageQuant Capture File Edit Image Setup Overlay Utilities View Help ImageQuant Capture Menu Bar 3 1 The File Menu Use this menu to save an image as a file retrieve a previously saved image select different printers print an image to a parallel printer overlay multiple images in RGB color channels c
15. Insert the knob into this hole and turn it clockwise until it is firmly seated Close up of the fold down Release the white light tray by Once the tray is released white light tray located at the unscrewing the knob which will screw the knob back into the back of the cabinet This tray Is allow the tray to swing down threaded hole underneath the secured by a detachable knob freely light tray where it will serve as a handle ImageQuant RT ECL Capture User Manual Attaching the Hood Take the following steps to attach the cabinet hood and connect the hood fan 1 Place the hood on top of the cabinet so the holes for screws are centered over the corresponding holes in the top of the cabinet 2 From within the cabinet attach the hood fan s power cord to the temperature board socket on the top right side of the cabinet 3 Fasten the hood to the cabinet using M4x16 socket head screws The cabinet hood is Once the hood is positioned over Close up of socket positioned over the top of the the top of the cabinet plug the cabinet fan cable into the socket on the top right side of cabinet ImageQuant RT ECL Capture User Manual 1 3 4 Camera Installation 1 Connect the USB cable and power supply barrel connector to the back of the camera 2 Connect the standard three prong end to the power strip Power Cable to camera 10 ImageQuant RT ECL Capture User Manual 3 Complete the installation by connecting the
16. Level set at 255 White Level set at 150 2 1 4 Gamma Setting Adjustment Changing the Gamma setting affects the image brightness by adjusting the linearity of the image on the screen and printouts but does not affect quantitative data The camera sees objects linearly while the human eye does not When the Gamma setting is set to a value of 1 the image is displayed as the camera sees it This however is different from what the human eye detects By adjusting the Gamma setting the user can make the image on the screen correspond to what is seen when he she looks directly at the object We recommend a Gamma setting of 0 55 for best visual representation 37 ImageQuant RT ECL Capture User Manual Gamma set at 1 0 Gamma set at 0 55 2 1 5 The Auto Contrast Selection Auto Contrast The Auto Contrast feature will automatically scale the black and white values of an image to more tightly fit the gray scale intensity profiles histogram This selection will use different black and white values for different images depending upon their unique histograms A more dramatic visual change will take place for low light level images such as chemiluminescence where smaller portions of the histogram are used This selection can be turned on or off and will adjust differently for each image 38 ImageQuant RT ECL Capture User Manual 2 1 6 The Reverse Button The Reverse button inverts the gray levels of the displayed image converting a posi
17. Sharpen level 5 has an intermediate effect Sharpen 1 low has the most subtle effect on the image 9 different sharpening levels are available for optimization of the image 4 7 3 Noise Filters This filtering process uses the values of the pixels contained in the area surrounding a pixel to determine the new value given to the pixel of interest The noise filter sorts the pixels in the neighborhood into ascending order and picks the middle or median pixel value as the new value for the pixel of interest 3 levels of noise reduction are available 4 7 4 Despeckle Filters The despeckle filter is a type of smoothing filter based on data rejection Pixels in the neighborhood usually the adjacent pixels are used as a data set upon which the average and standard deviation of the set are calculated If the pixel of interest the center of the neighborhood is different from the neighborhood average either greater or lesser by a threshold a multiple of the standard deviation it is replaced by the average value The effect of the filter is to smooth pixels that are much different from their neighbors Artifacts such as hot pixels cosmic rays etc are commonly rejected by this type of filter The filter strength is controlled by the threshold factor For large factors very little data is rejected as only very large deviations are required for rejection where as low thresholds result in more smoothing For example setting 1 results in outlier
18. This is especially useful for labeling lanes etc To use this tool draw text on the image select it click the Horizontal Alignment tool then deselect the text The text will now be aligned in a straight line across the image Text after Horizontal Alignment E The Vertical Alignment tool aligns annotations in a straight vertical line This is especially useful for labeling markers etc To use this tool draw text on the image select it click the Vertical Alignment tool then deselect the text The text will now be aligned in a straight line down the image 82 ImageQuant RT ECL Capture User Manual 4 6 False Color These tools consist of eleven pre defined color palettes that can be applied to an image To select a palette simply click on one of the four buttons labeled GRAY PALETTE SATURATION NEXT or PREVIOUS Tool Box rere errr terri rrr terre rere e rire rrr rre errr e terete rrr ttt reir rrr errr eer rre errr er rre rrr t errr rrr rrr rrr Enhancement Tools oom Rotate Flip Histograrn Enhance Annotate False Color Filters FrameGrabber GRAY PALETTE SATURATION NEXT PREWIOUS False Color Selection Box When a palette is selected its range of colors is displayed to the left of the palette buttons and automatically applied to the image To apply a different palette to the image click the NEXT or PREVIOUS buttons Note changes in Black level White level and Gamma setting can
19. USB cable into the back of the computer into one of the USB ports 1 ImageQuant RT ECL Capture User Manual 1 3 5 Software Driver Installation Instructions The following instructions apply only if you have not purchased a pre configured system and need to install the camera card manually The camera driver installation will install two drivers the USB device loader and the USB camera engine Windows normally detects new hardware automatically However if that does not occur it s also possible to install the driver software manually through the Windows device manager Run the application installer 1 Turn on the camera and plug in the camera s USB connector Windows will detect the device and launch an installation wizard Found New Hardware Wizard Welcome to the Found New Hardware Wizard Windows will search for current and updated software by looking on your computer on the hardware installation CD or on the Windows Update Web site with your permission Read our privacy policy Can Windows connect to Windows Update to search for software O Yes this time only O Yes now and every time connect a device Click Next to continue 2 Select the No not this time radio button 3 Press Next the following screen will be displayed Found New Hardware Wizard Tit wizard helpe you instal ofan for GE RT ECL USE Camera If your hardware came with an installation CD on floppy disk insert n now
20. Using the mouse move the cursor to the other end point of the line then release the mouse button The arrow can be adjusted by clicking on one of the boxes at the end the other will serve as an anchor point or by clicking in the middle to drag the entire arrow o The button labeled with a rectangle draws a rectangle or square of any size on the image After clicking on the button move the cursor to the position that should correspond to one of the corners of the rectangle Press and hold the left mouse button Using the mouse move the cursor to enlarge the rectangle When it reaches the desired size release the left mouse button E The button labeled with an oval draws a circle of any size After clicking on the button move the cursor to the position on the image where the circle should be started Press and hold the left mouse button Using the mouse move the cursor to enlarge the circle When the circle reaches the desired size release the left mouse button Hint to draw a perfect circle around a portion of an image first visualize a square surrounding the area of interest Position the mouse in the upper left hand corner of the square Click and drag the mouse down across the area of interest at a 45 angle until the circle encloses the area of interest Sample Annotations 80 ImageQuant RT ECL Capture User Manual text with background eo nes Annotated image showing freehand drawings lines with various cha
21. a zoom ring 21 ImageQuant RT ECL Capture User Manual 1 6 2 Using the Zoom ROI Tool Zoom ROI is a tool that is designed to be used with a fast or fixed focal length lens These lenses typically have lower F stop numbers than zoom lenses but do not contain a zoom function Fast lenses are most commonly used for chemiluminescence to decrease the exposure times however they can also be used for fluorescent gels and other applications as well Since there is not a zoom ring on a fixed focal length lens it is now possible to adjust the region of interest of the acquired image using the ImageQuant RT ECL software interface When the region of interest of the sample is smaller than the acquisition window and the system is not equipped with a zoom lens the zoom ROI tool is the ideal work around without having to switch lenses on the system Focus on the image as explained in 1 4 and 1 6 of the manual and then slide the zoom ROI slider across the screen until the region of interest occupies the entire image area in the acquisition window Use preview and capture to acquire the final image This eliminates a post acquisition step of cropping the image down to the appropriate size Camera Setup amp Preview V Noise Reduction Aperture aj Zoom 2 80 gt 70 00 aj Focus wi 1 80 Exposure Time Es gt p gt gt 0 xl Auto Expose Auto Expose Setup whic Display Sensitivity Resolution 3 il o Te F
22. alter the effect of each of the palettes and can enhance the results produced 4 6 1 Gray Scale Palette 0 This is the default or standard gray scale consisting of different gray levels ranging from black to white 4 6 2 Saturation Palette 1 This is a modified gray scale palette in which black is replaced with green and white is replaced with red Over and under exposed areas of the image are thus shown as green or red while areas within the linear range of the CCD chip are shown in gray scale The Saturation Palette is especially useful during quantitation as areas outside the linear range of the instrument do not give accurate quantitative information This palette allows the user to avoid those areas during quantitative analysis This palette can also be accessed by clicking the Show Saturation checkbox in the Camera Setup and Preview function accessible by clicking on the Camera icon in the Tool Bar window 83 ImageQuant RT ECL Capture User Manual 4 6 3 Palettes 2 through 11 These are color substitution palettes in which the gray levels are translated into different color ranges These palettes can be useful to help distinguish features and highlight details on an image Palette 2 maps the gray scale levels to a red green blue palette Values of 0 are mapped to red saturated to blue and values in between to green Palette 3 maps the gray scale levels to a red green blue palette Values of 0 are mapped to blue
23. an Overlay The Load Overlay function allows Overlay files to be retrieved and applied to the image currently displayed Opening an Overlay after an image has been captured places the annotations on top of the image They can be stored as part of the image by saving the file as a modified file See Save Image As in Section 3 1 for instructions Select the name of the file to be loaded If necessary change the directory or drive The file name is then highlighted in the list and appears in the text box below the Filename prompt Once the file has been selected click on the OK button to load the file Alternatively double click on the file name The dialog box disappears and the annotations in the selected file appear on the image To dismiss the dialog box without loading annotations click on the CANCEL button Overlay Libraries ImageQuant Capture contains a library of overlays that can be accessed through the Load Overlay function described above This library of overlays is stored in the Image folder located in the ImageQuant Capture directory OSWHITE OVR OSBLACK OVR 8 lane labels in white black 1OWHITE OVR L1OBLACK OVR 10 lane labels in white black 12WHITE OVR 12BLACK OVR 12 lane labels in white black ISWHITE OVR ISBLACK OVR 15 lane labels in white black 24WHITE OVR 24BLACK OVR 24 lane labels in white black HINDUIOVR Hind IID label The objects in these overlays can be repositioned resized re colored copied o
24. applied to a single image Tool Box Enhancement Tools OOM Rotate Flip Histogram Enhance Annotate False Color Filters FrameGrabber Sharpen Moise Despeckle 3 D UNDO Lowy High uoo 1l2ls 4 s e zlels MORE The Filter Tools with Sharpen highlighted smogthHigh Edge High Horz Edge UAOO smoothed Edge Low Vert Edge MORE Smooth ow Linearize Custom The Filter Tools with More selected 85 ImageQuant RT ECL Capture User Manual 4 7 1 General Information on How Filters Work To enhance an image filters change the value assigned to each pixel The new value assigned to a pixel is determined based on the values of the other pixels in its local vicinity or neighborhood The neighborhood is a two dimensional matrix of pixel values where each dimension has an odd number of elements The pixel of interest is the one at the center of the neighborhood This is the pixel whose old value is being replaced with a new one as the result of the filtering algorithm The pixels in a neighborhood provide information about the brightness trend This information is important to the filtering process The brightness trend is also referred to as the spatial frequency Images with high spatial frequency content contain large closely spaced changes in pixel values For example on a black and white checkerboard the smaller the squares the higher the frequency content Images wi
25. black pixel values At an edge 88 ImageQuant RT ECL Capture User Manual which is an area with large changes in intensity the subtraction yields light colored pixel values The larger the difference in intensities the lighter the resultant pixels Note After applying the horizontal edge filter the entire image may appear black and might require reducing the White level in order to better visualize the results 4 7 9 Vertical Edge Filter This filter Vert Edge brightens vertical edges using the approach described for the horizontal edge filter see above except that the image is shifted horizontally before the shifted image is subtracted from the original In this case the vertical edges produce light colored pixel values As in the horizontal edge filter it may be necessary to adjust the White level in order to better visualize the results of this filtering process 4 7 10 Custom Filter This function also allows the user to customize filters to his her own specifications Using the weighting factors of the other filters as a frame of reference experiment with new weighting factor values 4 7 11 The UNDO Button The upper right button is labeled UNDO This reverses the last filtering process applied to an image To revert the image to its original state after multiple filters have been applied press the RESET button on the main interface 89 ImageQuant RT ECL Capture User Manual 4 7 12 Examples of Filter Results
26. to access the Saturation Palette during image acquisition The Saturation Palette is a modified gray scale palette in which black gray level 0 is replaced with green and white gray level 255 4 095 or 65 535 is replaced with red With this palette over and under exposed areas of the image are shown as green or red while areas within the linear range of the CCD chip are shown in gray scale During image acquisition note regions of the image that appear red or green Adjust the exposure time and or the camera aperture to minimize the amount of red and green in the image area If an image will be quantified it 1s especially important that the actual sample area be neither red nor green Once you are satisfied with the displayed image you may click off the Show Saturation The other three selections Chemi Display Auto Contrast and Reverse are visualization tools designed to enhance contrast and provide flexibility in regards to how the user wishes to view the image Auto Contrast will display the image with automatic black white and gamma adjustments according to the image histogram information black white levels Reverse will show the sample as a negative by switching the black and white values Chemi Display utilizes both the auto contrast and reverse functions as well as adjusting for gamma It is intended for use with chemiluminescent samples These options are also available in the FrameGrabber portion of the acquisition software It i
27. xi 1 aj Zoom aj 2 80 g 70 00 80 gt Exposure Time lt j Ea gt i gt Hours a Minutes Seconds a MliSecs a 0 x 0 x 3 x 0 x Auto Expose Auto Expose Setup Display F Chemi Display Normal High Show Saturation F Auto Contrast Medium Medium Filter Wheel Position FrameGrabber Super Sensitivity Chemiluminescence Zoom ROI j S Door Sensor Temperature 31 9 C CLOSED Sensitivity Resolution 20 ImageQuant RT ECL Capture User Manual 1 6 1 Focusing on an Image Focusing on an image in the ImageQuant RT ECL Capture software is easy with the real time display Simply adjust the parameters on the manual lens or if the lens is controlled via the software click on the appropriate software control button 1 TLL TERE POUR E 2 3 To open the aperture turn the lens s aperture ring counter clockwise for more light Conversely if the image is too bright close the aperture by turning it clockwise until an image is visible on the monitor Then zoom in on the image so that the area of interest on the sample takes up the entire field of view on the screen Last if the image appears blurry turn the focusing ring lower ring on the lens until the sharpest image is obtained Note This protocol may vary slightly with different accessory lenses For example the fast lens may have an aperture setting of F1 8 and will not have
28. 1 Chemiluminescence samples no filter Filter Position 2 Ethidium Bromide colorimetric stains film SYPRO Orange 595nm Filter Position 3 optional SYBR green 557nm Filter Position 4 optional Fluorescein SYBR Gold 520nm Filter Position 5 optional SYPRO Red Texas Red 630nm optional Hoechst Blue 460nm Note Each Filter has an approximate bandwidth of 40nm to allow for use with other fluorescence stains as they are developed If you have a custom application please feel free to contact us directly to explore having a custom filter designed for your application 25 ImageQuant RT ECL Capture User Manual 1 6 6 Sensitivity Resolution Controls Sensitivity Resolution Normal High Medium Medium High Lowy Super Sensitivity Sensitivity Resolution Settings ImageQuant Capture allows you to acquire images depending on your critical needs Are you looking for the shortest possible acquisition time the highest resolution or a combination of both Acquisition modes Normal Sensitivity High Resolution Recommend for Fluorescence and Colorimetric Imaging This mode captures the image using the CCD sensors full 2048x2048 pixel resolution generating high quality vibrant images that are publication quality Medium Sensitivity Medium Resolution Recommend for Chemiluminescence This mode captures the image performing a 2x2 pixel bin to decrease the exposure time of the image yet
29. 2 instead of DOSA Must be typed in all CAPS as shown here Close the window and save changes Quit ResEdit After this procedure the icon will change to a TIFF icon and the file may be opened in Canvas 98 ImageQuant RT ECL Capture User Manual Appendix B Security Features This feature should only be used by the purchaser or supervisor of the system Remove this page from the manual and store it in a safe place When the system is shared by a number of users security features may be useful for regulating or maintaining a log of instrument use To Change the Security Setup When you initially receive the system all security features are inactive and the user name and password to access security settings are both set to MASTER To change the security setup select the Setup pull down menu and click on Security A dialog box will appear asking for the system password Login Dialog Uzer Mame Password Security Dialog Box Enter the user name and password and then click on the Login button A new dialog box will appear allowing you to configure your security preferences By clicking on the options in this dialog box it s possible for a supervisor to change the system password set the security mode add and delete names from the authorized list and activate the Auto Logout feature When the desired options have been selected click the OK button Follow this procedure any time changes to the security mode are ne
30. 5 F 21 417 31 84 Russia CIS amp NIS T 7 095 232 0250 F 7 095 230 6377 South East Asia T 60 3 8024 2080 F 60 3 8024 2090 Spain T 935 944 950 F 935 944 955 Sweden T 018 612 1900 F 018 612 1910 Switzerland T 0848 8028 12 F 0848 8028 13 UK T 0800 616 928 F 0800 616 927 USA T 1 800 526 3593 F 1 877 295 8102 Italy Technical Support ts usa ge com T 02 27322 1 Customer Service cS uS ge com F 02 27302 212 Japan T 81 3 5331 9336 F 81 3 5331 9370 For Service Agreements labcrew us ge com CE Conformity The following Gel Imaging Digital Systems Models ImageQuant 100 ImageQuant 300 ImageQuant 400 ImageQuant RT ECL Are in conformity with the provisions of the following EC Directives including all amendments and national legislation implementing these directives Low Voltage Directive 73 23 EEC EMC Directive 89 336 EEC And that the following harmonized standards have been applied EN61010 1 2001 EN61326 1 1997 A1 1998 A2 2001 Class A EN55011 Class A EN61000 3 2 EN61000 3 3 EN61000 4 2 EN61000 4 3 EN61000 4 4 EN61000 4 5 EN61000 4 6 EN61000 4 11 Copyright and Trademark Information All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them GE Healthcare reserves the right subject to any regulatory and contractual approval if required to make changes in specifications and features shown herein or disconti
31. GE Healthcare ImageQuant RI ECL Version 1 0 1 Lastoce segs itia seerers sere Waste Electrical and Electronic Equipment WEEE ENG H FRA F GER F ITA F SPA DZA This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of your equipment Ce symbole indique que les d chets relatifs l quipement lectrique et lectronique ne doivent pas tre jet s comme les ordures m nag res non tri es et doivent tre collect s s par ment Contactez un repr sentant agr du fabricant pour obtenir des informations sur la mise au rebut de votre quipement Dieses Symbol kennzeichnet elektrische und elektronische Ger te die nicht mit dem gew hnlichen unsortierten Hausm ll entsorgt werden d rfen sondern separat behandelt werden m ssen Bitte nehmen Sie Kontakt mit einem autorisierten Beauftragten des Herstellers auf um Informationen hinsichtlich der Entsorgung Ihres Ger tes zu erhalten Questo simbolo indica che i rifiuti derivanti da apparecchiature elettriche ed elettroniche non devono essere smaltiti come rifiuti municipali indifferenziati e devono invece essere raccolti separatamente Per informazioni relative alle modalit di smantellamento delle apparecchiature fuori uso contattare un
32. IDUAL IMAGES PREVIOUSLY CAPTURED IN NORMAL CAPTURE MODE SAVE Save a sequence of images 1 6 11 Saving an Individual Image from a Sequence After you load play and stop a sequence at the desired image it is possible to save the individual image seen on the screen Use the SAVE AS button located on the tool bar to save the image in the desired location and file format on the local or network drives ob Note This will save the current image only To save the entire sequence select the save icon on the FrameGrabber tab in the tool box 1 6 12 Loading Saving an Entire Sequence You can load or save an entire sequence by using the load and save icons in SB EB the FrameGrabber tab in the tool box When a sequence is loaded the loaded frames will be seen as blue in the sequence reel When a sequence is saved a dialog box will appear prompting you to select the location and name for the sequence that you would like to save the images as 32 ImageQuant RT ECL Capture User Manual 1 6 13 FrameGrabber Save Load FrameGrabber Setup Routines Two buttons Save Setup and Load Setup allow you to save and load all FrameGrabber setup parameters Files are saved as mvf files Camera Setup amp Preview Cabinet Controls Disconnected 1 Frame Option i Frame Ry xi F a Normal Sequence C Stack Frames Look in desktop i i e V Noise Reduction E Ow TTI Pi My Computer Copy to Next my Networ
33. ImageQuant Capture Annotations are superimposed on the image when a hard copy is printed and can either be saved as a template file or as part of the image All of these features are accessible via convenient on screen buttons and menus controlled by the mouse ImageQuant Capture can also be used to transfer an acquired image to one of the ImageQuant TL modules for detailed analysis The ImageQuant System is a complete package including all the hardware and software needed for image capture enhancement and analysis The system s computer need not be solely dedicated to ImageQuant Capture and can also be used to run other software such as word processing programs spreadsheets and desktop publishing software Images can be printed using a 256 level gray scale thermal printer or any printer with a Windows driver The low cost high quality prints are ideal for record keeping in lab notebooks or for publication Images can be archived to the system s hard disk floppy disk Zip or optional Jazz drive or to a network drive if applicable In section 1 4 there is a condensed instruction sheet which will be useful if you are familiar with the program but need to quickly refresh your memory We suggest you photocopy the appropriate instruction sheet s and post them near the instrument for easy reference ImageQuant RT ECL Capture User Manual 1 1 1 Mouse Functions The mouse supplied with the ImageQuant System has two buttons The left bu
34. Window This is a useful function for detecting faint bands on a sample 3 3 2 Arithmetic The Arithmetic function is used to add subtract average and divide several images together to generate a compiled image File Edit Image Setup Overlay Utilities View Help C Progr Equalize Arithmetic Conversion Flat Field Calibrate Subtract Resize Quotient Image Info Image Arithmetic dialog box To average a set of images together open one of the images in the set and then select Average a Set under the Image pull down menu A prompt will appear allowing the user to select all of the images that for the set Itis possible to browse the directories looking on the network drives and removable media if necessary Once all of the images have been selected click on the open button to finish the set The resulting image is an average of all of the images together This is a useful function for extending the dynamic range on a Set of similar images by allowing bright spots and faint spots to be seen on the same image The other functions are adding subtracting and dividing images together Adding together images is frequently used for colorimetric markers run together with chemiluminescent samples Subtracting images is often used to remove noise from a sample by running dark images first and subtracting them out of the final image The 56 ImageQuant RT ECL Capture User Manual most common application for quotien
35. a is saved in an unaltered form This option should be selected if the image will be analyzed later If the Black level White level or Gamma settings have been adjusted the new values are saved but the pixel values are not altered When this file is opened at a later time ImageQuant will display it with the values that were displayed when the image was saved however it is still possible to revert to the original raw image file by selecting Reset on the Tool Bar Annotation information cannot be saved with the Original image option It can however be saved as an Overlay See section 3 5 for more information An image that is saved as a Modified file permanently retains the changes to the image s Black level White level and Gamma setting Annotations and any filtering performed are also saved with the image replacing original image information with the new information Note If the image is saved as a Modified file it is converted to an 8 bit image 51 ImageQuant RT ECL Capture User Manual 3 1 5 Printer Setup This function displays a dialog box in which the settings for the parallel printer are specified When all the pertinent printing preferences have been specified click on the OK button If you purchased a printer with ImageQuant this will be preset from the factory Page Setup RIES zixl Printer Name SAVIN 2560 PCL 6 Properties Status Ready Type SAVIN 2560 PCL 6 Where 216 132 94 4002560 Co
36. ample Effects l Strikeout Underline Color Lo white Script wester Font Selection Window Note To choose Greek symbols such as a B m and 0 choose the Symbol font abcdefghijklmnopqrstuvwxyz apyoevOyntgnKrAUvVOTOPDGOtvDaAEWC 78 ImageQuant RT ECL Capture User Manual 4 5 7 Text Angles In the TEXT ORIENT window select whether text should be oriented vertically horizontally or at an angle in 15 increments pa N ojo x a 8 coon LNEENDS gt ioceamise ANGLE JPEN WIDTH _ TEXT STYLE J ANTICLOCKWISE efo gt FEN STYLE TEXT ORIENT Text Orient Selection Tools Note only rotate fonts that are True Type indicated by TT in front of the name other fonts such as Courier and Fixedsys do not re scale properly giving unpredictable results 4 5 8 The Drawing Tools Once the object attributes have been defined click the cursor on any of the drawing tool buttons to assign the function associated with that button to the mouse The cursor will change from an arrow to a cross indicating that ImageQuant Capture is in drawing mode After selecting a drawing tool move the cursor to the correct position on the image to begin drawing Press and hold the left mouse button and move the mouse to the end point of the object to be drawn Release the left button and the object should now appear Boxes will appear at the corners of the new object and the cursor will
37. are version number Use this information when contacting technical support upgrading software etc ImageQuant Capture x Copyright 1993 2005 All Rights Reserved GE Healthcare ImageQuant Capture About Help Dialog Box To close the box click on the OK button 71 ImageQuant RT ECL Capture User Manual Chapter 4 The Tool Box Image enhancement tools are contained within the Tool Box as indicated This tool set allows the user to zoom the image rotate flip the image show the image histogram perform automatic image enhancement annotate on the image display false colors apply software filters and activate the FrameGrabber function 4 1 The Zoom Tool Annotate False Color Filters Zoom Rotate Flip Histogram Enhance soc vf aae m The Zoom Tools The Zoom tool is found in the Tool Box This function magnifies an image making details easier to see and allows movement around the magnified image An image can be displayed 4x 2s 1x 2x 4x or 8x larger than the original display by clicking on the appropriate buttons To return to the original magnification click on the 1X button When an image is magnified only part of it can be displayed on the screen at any one time To see different parts of the magnified image use the Pan Control box within the thumbnail The outer box shows a thumbnail of the entire image while the small inner box represents the portion currently displayed on the screen
38. as chosen during acquisition of the image Stack Frames will use all previous exposure information to sequentially add images to one another Normal Sequence will not perform this addition Please note that stacking frames will increase the noise level in acquired images Sample case Ej Copy to Next Copy to End Go Load Setup Save Setup Open BX Look irx E Desktop t Files oftype Movie Setup Files mv Load Setup and Save Setup buttons Cabinet Controls Disconnected Capture 5 frames at 1 5 sec exposure for total time elapsed 15 sec Display after summation of following frames Frame 1 1 sec Frame 2 1 2 sec Frame 3 1 2 3 sec Frame 4 1 2 3 4 sec Frame 5 1 24 3 4 45 sec 94 ImageQuant RT ECL Capture User Manual 4 9 2 Capturing a Color Image Using the FrameGrabber Function A color image can be generated by acquiring three images each taken with a red green and blue emission filter Once saved these images are then combined in the Overlay pull down menu Open the image captured with each of the three filters as instructed and a RGB red green blue true color image will be generated For color imaging you will need to use the following optional filters Red Filter SYPRO Red Filter Blue Filter Hoechst Blue Filter Green Filter SYBR Green Filter 95 ImageQuant RT ECL Capture User Manual Chapter 5 Analyzing Images Once you have acquired a
39. ation In some instances all software peripheral drivers and operating systems have been pre installed at the factory During the system installation all components should only need to be plugged into the correct ports If the software is not installed or the camera is not responding see section 1 3 4 for software driver installation instructions 1 3 1 Setting up the Power Strip Surge Protector Turn the power switch on the power strip surge protector off Plug the power strip surge protector into a wall outlet preferably a dedicated circuit and then turn the power on CAUTION Do not plug the light cabinet into the same power strip as the PC Use a separate circuit if possible The cabinet needs to be turned on after the operating system has been completely loaded in order for the software to function optimally ImageQuant RT ECL Capture User Manual 1 3 2 Setting up the haa Monitor Mouse and Keyboard The computer will need to have the 1 monitor mouse and keyboard ee Keyboard Mouse y connections color z 4 coded purple and connected to it in the correct ports see the descriptions below Also a 3 prong standard power cable should be plugged into the back of j the computer and the power strip aes COMI port for DE aS _sC SOD00 Light Cabinet Connect the monitor s video cable to the monitor port on the back of the computer If after market video cards were preinstalled into your computer system connect
40. aturation option should be selected for these applications e For low light applications such as chemiluminescence a longer exposure time may be appropriate and it is best to go to the CAPTURE button to directly acquire the image once the desired exposure time is set Alternatively if a good exposure time is unknown try selecting Auto Expose and using the super sensitivity selection Auto Expose will generate an image showing the signal intensity usually in less than 3 minutes Afterwards a different sensitivity binning mode can be selected to generate a better resolution image and the software will automatically calculate the new exposure time for you Note When the system is switched to PREVIEW mode the image may flash or change brightness because the camera collects photons from the image for a longer period of time before sending the image to the computer s display readout 2T ImageQuant RT ECL Capture User Manual 1 6 8 Optimizing the Gray Scale for Saturation and Contrast Displays F Chemi Display T Show Saturation T Auto Contrast Reverse If an image will be analyzed it is important that it not be over saturated too light or under exposed too dark Using Show Saturation the user can see the areas of the image that are assigned to each end of the gray scale spectrum and can adjust the imaging controls accordingly The Show Saturation checkbox found below the camera control functions allows the user
41. authorized users To do this click the Add Remove Users button A dialog box will appear with a list of all the current users Option 5 Closed System with User Name and Password Log In and Out The system only accepts authorized names and their passwords at log in The system supervisor can add or remove names from a list of authorized users as described above Only one password can be assigned to each name A user log is generated If User Logging Active Jw Closed System Auto Logout f 5 minutes 10 minutes 20 minutes Change Master Passyvrord AddRemove Users OK Cancel Note passwords are case sensitive The login dialog defaults to uppercase but the user can override this by pressing the shift key while typing 102
42. can be loaded later and applied to any image SaveAs ee 21x Save in E ImageQuant do A e Es Save as type def files def Cancel Save Defaults Dialog Box Once any of the system settings have been changed a new default file can be created To save a default file enter a file name by typing in the text box below the File Name prompt If it is necessary to change the directory or drive to which the file will be saved select a different directory under the Save In pull down menu ImageQuant Capture will automatically add the appropriate 3 character extension Click on the SAVE button The Save As dialog box disappears and a new file is created To exit this function without loading a default file click on the CANCEL button 3 4 3 Load Defaults This function retrieves system default settings from a saved file To open a default file enter the name of the file by typing its name in the text box below the File Name prompt If it is necessary to change the directory or drive to which the file will be saved select a different directory under the Save In pull down menu Once the file has been selected click on the OK button to load the file The Load Defaults dialog box disappears and the image controls are adjusted to reflect the settings in the file loaded To exit this function without loading a default file click on the CANCEL button 61 ImageQuant RT ECL Capture User Manual 3 4 4 Security When the sy
43. cessary x Closed System Passwords Auto Logout 5 minutes 10 minutes 30 minutes Change Master Password Add Remove Users OK Cancel Security Features Dialog Box 99 ImageQuant RT ECL Capture User Manual NOTE You must exit and re launch ImageQuant Capture in order for any changes to take place If you decide not to edit the security settings at this time simply click on the EXIT button to dismiss the dialog box Setting Various Security Levels A variety of security levels are available Each is described below in increasing order of security Also shown are the proper settings in the dialog box to choose each security level Option 1 No Security Functions No log in or log out is required and anyone can use the system SS t RI P dosed System Paesvvords Aute Logout 5 minutes 10 minutes 20 minutes Change Master Passvrord AddRemove Users OK Cancel Option 2 Open System with User Name Log In and Out This security level keeps the system open but requires users to log into the system by typing a name In this mode a list of users with their log in and log out times is generated Closed System Passwords Auto Logout f 5 minutes f 10 minutes 20 minutes Change Master Passyvrord AddRemove Users OK Cancel 100 ImageQuant RT ECL Capture User Manual Each time a new name is used it is added to the master user list Si
44. creen To dismiss the dialog box without loading an image click on the Cancel button 48 ImageQuant RT ECL Capture User Manual 3 1 2 File Overlay T To superimpose images use the OVERLAY function under the File menu This function will display separate multiplexed images or a RGB color image as a compiled image with the appropriate color channel images added together A simple way to acquire multiple images for this function is to use the FrameGrabber function in image acquisition and acquire a series of identical images The Overlay Images option allows you to overlay up to three different images with three different color channels You can select the BROWSE button for each color channel and select the appropriate images to be used for generating a color image For example 1f you have a saved grayscale images of an identical gel taken with a SYPRO red filter for the red stain and a SYBR green filter for the green stain you can choose these images in the appropriate Red and Green Channels to generate a composite image with the red and green colors mapped onto the compiled image Note The images must be the same bit depth and resolution for the software to overlay the images 3 1 3 File Close This function closes the image currently displayed on the screen 49 ImageQuant RT ECL Capture User Manual 3 1 4 File Save and Save As Save allows original images to be saved in several different formats Save As allows images
45. e for colorimetric gels and film 2 Ethidium Bromide 3 5 Optional filters 18 ImageQuant RT ECL Capture User Manual 1 5 System Information To display system information select the About option in the Help menu This button accesses a pop up box About ImageQuant ECL Capture Eg ImageQuant Capture Version 1 0 1 GE Healthcare Copyright 21993 2006 All Rights Reserved GE Healthcare ABOUT Pop Up Dialog This box shows the instrument serial number where appropriate and the Software version number Use the information specific to your instrument and software when calling for technical support software upgrades etc To close the dialog box click on the OK button 19 ImageQuant RT ECL Capture User Manual 1 6 Acquiring an Image In the TOOL BAR window click on the Camera Acquire icon Acquire Once the Camera Acquire Icon is pressed the CAMERA SETUP AND PREVIEW window is activated to provide exposure control of the camera sensitivity resolution controls all lighting and filter wheel position controls contrast display options and cabinet door open close indicator using a software virtual control menu Also Movie Setup is controlled and activated from this window This window will open in the Focus control mode activated blue button provide a near real time readout to allow for easy sample positioning and optics adjustments Camera Setup amp Preview V Noise Reduction Aperture a aj Focus
46. e is captured Overlay Utilities w Load Overlay Save Overlay Overlay pull down menu An overlay is any set of annotations text boxes arrows etc that have been drawn on the image They can be saved as a group and opened later If repetitive samples are being imaged an overlay eliminates the need to re enter the same information such as lane numbers standard sizes etc continually 3 5 1 Saving an Overlay Once annotations have been made select Save Overlay from the Overlay menu D autorad 1 6bit tif 1280x1024x16 fre ee Be ae BR GS te Oy Sole ow oo oe leet mw ee wm z kperstd120 Save AS ex Save in E Image oe c i Demotutr a 1 2white owr fa O8black ovr fa 1Sblack ovr la O8white ovr fa 1 Swhite ovr fa 10black ovr a 24black ovr a 1 Owhite ovr a 24white ove la 12black ovr fa Hindiii ovr File name kperstr Save as type Overlay Fies cov Cancel i j Save Overlay Dialog Box Enter a new file name in the text box below the File Name prompt ImageQuant Capture will automatically give the appropriate 3 character extension 66 ImageQuant RT ECL Capture User Manual The current directory is the one in which the new overlay file will be saved If necessary change the directory or drive as described in Section 3 1 Once a name has been entered and the appropriate directory has been accessed click the SAVE button to save the overlay file 3 5 2 Loading
47. e rrr errr rrr errr rrr rire tr rrr rrr Enhancement Tools oom Rotate Flip Histograrn Enhance Annotate False Color Filters Framelrabber Image REW RE REC STOP PAUSEPLAY FiO DOEL OPEN SAVE Once all images have been captured the above FrameGrabber display box will become displayed The remaining buttons will perform the following tasks REC Opens the Camera Setup and Preview screen to record a sequence PLAY Display a continuous loop of all of the captured images STOP Stop the sequence at the current frame display PAUSE Pause the playback of the sequence at a user defined image REW rewind Rewind the sequence to the first image REV reverse Play the sequence in a continuous loop in reverse FWD forward Forward the sequence to the last image DEL delete Delete the sequence on the display THIS FUNCTION WILL NOT DELETE A SEQUENCE SAVED TO THE HARD DRIVE LOAD Load a previously saved sequence THIS FUNCTION WILL NOT LOAD INDIVIDUAL IMAGES PREVIOUSLY CAPTURED IN NORMAL CAPTURE MODE SAVE Save a sequence of images 4 8 1 Saving an Individual Image from a Sequence After you load play and stop a sequence at the desired image it is possible to save the individual image seen on the screen Use the SAVE AS button located on the tool bar to save the image in the desired location and file format on the local or network drives px Note This will save the current image only To save the entire sequence select
48. e the entire field of view for an image Creating flats can be art in itself there are many documents on the internet that can help users interested in this arena to create the ideal flat for the application However some useful flats that have been created in the past involve very simple tools like a piece of 8 5 x 11 regular low quality copy paper higher quality paper contains watermarks that will show up in the final image It is essential that both the flat and the gel images be identical including the aperture zoom if applicable and focus settings on the lens Step by Step Flat Field Calibration 1 Place the gel or other application in the cabinet or dark room 2 Adjust the aperture zoom if applicable and focus on the lens 3 Use auto expose set to the normal selection and acquire an image of the gel Alternatively it is possible to select show saturation and then use preview mode and adjust the exposure time manually to just under saturation 4 Save the image of the gel 5 Next remove the gel from the UV transilluminator or white light tray and clean and or dry off the surface if necessary using glass cleaner 6 Place the white piece of paper onto the appropriate surface For example if the UV transilluminator was used place the paper onto the UV transilluminator 1f the white light tray was used place the piece of paper onto the white light tray 7 Turn on the appropriate light source used white light UV trans
49. e turned off in the View menu by just deactivating the check mark next to the item that you would like to remove from the screen Also since these items are floating tools you can click on Default Tools Position to move all tools to the default locations for more intuitive operation Lastly except for the status bar it is possible to select and move any of the other tools to a custom location c Ca 3 7 1 Zoom Functions oom Out Zoom In Additional options provide the ability to Zoom In and Zoom Out on the image Zoom to 1X and to Fit to Screen Note Zoom In and Zoom Out are duplicate functions for the Zoom In and Zoom Out icons in the Tool Bar and the Zoom options in the Tool Box 3 7 2 Show Annotations There is also an option to display not display any annotations associated with the current image To display the annotations place a check mark next to the Show Annotations option in the View menu Otherwise remove the check mark to remove the annotations from the image viewing area the annotations are not deleted by selecting this option 70 ImageQuant RT ECL Capture User Manual 3 8 The Help Menu Help On Line Mote Abouk Help pull down menu 3 8 1 On Line Note On line help is available from the ON LINE NOTE section of the help menu 3 8 2 About To display system information select the About option in the Help menu This button accesses a pop up box This box shows the system serial number and softw
50. efe sjzjejsi tet moe Original Filter E Lower right TE Upper middle EN 4 7 6 Smoothing Filters These filters are very useful for reducing the visual noise present in an image When a smoothing filter is applied to an image rapid changes in intensity are averaged out with the remaining pixels in the neighborhood thereby decreasing the high frequency content The visual result is a slight smoothing of the image because sharp pixel transitions are averaged with their surroundings Smooth High has the largest effect on the image Smooth Med has an intermediate effect Smooth Low has the most subtle effect on the image 4 7 7 Edge Filters These filters use Laplacian enhancement to highlight edges regardless of direction All edge enhancement operations attenuate the low frequencies of the image Regions of constant intensity or linearly increasing intensity become black as a result of these transformations and regions of rapidly changing intensity values are highlighted Note The White level may need to be adjusted after using the Edge filters in order to see the result of this filtering process 4 7 8 Horizontal Edge Filter This filter brightens horizontal edges This can be useful in pinpointing bands on a gel The horizontal edge filter Horz Edge enhances image edges by shifting an image vertically by one pixel and then subtracting the shifted image from the original In an area of constant pixel intensity the subtraction yields
51. er the camera stand with attached camera through the top of the cabinet placing it over the gasket and positioning the lens directly above the hole that leads to the filter wheel Fasten the camera post to the top shelf of the cabinet using the screws that are provided Adjust the location of the camera on the post so that the fully extended lens is slightly above the filter wheel Make sure that the lens is fully extended when doing this to ensure that the lens will not come into contact with the filter wheel during cabinet operation Camera stand camera and Completed camera camera Camera and stand being attached fastening knobs stand assembly to the cabinet Make sure that the fully extended lens is just above the filter wheel but does not touch ImageQuant RT ECL Capture User Manual Installing the UV Transilluminator Take the following steps to install the UV transilluminator this is a box shaped UV lighting apparatus that rests at the bottom of the cabinet 1 At the bottom of the cabinet there is an extendable metal tray that is held in place by a single screw this is to prevent the tray from being damaged during shipping Start by removing this screw and then pulling the tray out so that it is fully extended 2 With the tray fully extended re insert the screw in its original position underneath the slide out tray This ensures that you will have the screw if you ever have to ship the unit and also prevent ligh
52. expose settings refer to section 1 6 4 of this manual Select the Super sensitivity resolution setting for details on the resolution settings refer to section 1 6 6 Wait for the exposure bar to indicate that the proper exposure time has been found and that there is no saturation in the image the exposure bar will turn green when this is complete If the exposure bar is pink in color saturation 1s still present in the image Select Medium Medium resolution Capture Select Note The exposure time will vary depending on which resolution setting is selected in step 4 h If the exposure time calculated by the auto expose setting is too long it is possible to select the High Low or High Medium setting instead Alternatively if the exposure time is short enough omal High may be selected for a full resolution 4 million pixel chemiluminescent image 5 Save the original image a b C d Click on the Save Image function in the FILE menu or click on the SAVE or SAVE AS icon in the Tool Bar Enter a file name and select the directory to which it should be saved the directory path should be less than 100 characters Specify the file format TIF BMP PCX MAC color TGA Click OK to save the file 6 Enhance the display optional a b C Adjust the black white and gamma levels by moving the slider bars at the right of the image in the Contrast Adjust window or select auto contrast Apply digita
53. frames images and a minimum of 1 frame that can be captured with each sequence Frame Option The FRAME selection is used for setting up the conditions for each frame image For example if three 3 images are to be captured you would choose FRAME 1 and setup all of the desired lighting and filter requirements You can then click on FRAME 2 and repeat the above Or you can click on COPY TO NEXT to help speed up the setup process COPY TO NEXT copies all settings from the previous frame to the current frame Usually for chemiluminescence imaging all lighting is off and the filter wheel is positioned for the chemiluminescence position for all frames Thus the only variable that is changing from one frame to the next is the exposure time In this situation COPY TO NEXT is a useful tool to save time in the setup process If you are performing kinetic experiments where you want to have a predetermined delay between captured images then you can use DELAY to configure this function The default DELAY is set for the shortest possible delay 19 milliseconds but can be configured up to 50 minutes between each image Also if your exposure delay and or exposure time and or lighting options filter position are consistent for the entire sequence then once you set up the first frame you can select the COPY TO END selection to automatically choose the first frame settings for the entire sequence of frames images Once the frame sequence is set up t
54. g over and over at that exposure time until a different mode is selected If Capture is selected after the software has achieved the correct exposure time in Preview mode the software will begin acquiring at the last exposure time calculated during the preview it does not start the calculation over from scratch Similarly if a different sensitivity binning mode is selected after the exposure calculation has been calculated it will do a quick conversion and does not have to start the calculation from scratch 24 ImageQuant RT ECL Capture User Manual 1 6 5 Cabinet Controls Activating the Light Source and Selecting a Filter Use the mouse to click on the desired light source in the virtual cabinet control interface or you can use the controls on the cabinet itself Both mechanical and software controls are linked and communicate display setting selections Note There is a slight delay when the button is depressed until the light source is fully activated Standard lighting choices include Transillumination White For protein gels autorads film plates flasks Transillumination UV For fluorescent gels such EtBr SYPRO Red etc Reflective White For colorimetric blots and membranes Reflective UV optional For SYBR green TLC plates and Chemifluorescence No Light Selected For chemiluminescence You can also select your FILTER to correspond with your sample staining The options include Filter Position
55. ges Using the FrameGrabber Option ccccceeeeeeeeeeeeeees 30 1 6 11 Saving an Individual Image from a Sequence cccccecececesseeeeeeceeeeeaeeeeees 32 1 6 12 L ading Saving an Entire Sequence areon ea a a aaa 32 1 6 13 FrameGrabber Save Load FrameGrabber Setup Routines 0008 33 FOIA Brame SACKING eurae a a ea neniuaie anata 33 1 6 15 Capturing a Color Image Using the FrameGrabber Function 00 34 Chapter 2 Getting Started Basic Imaging Functions cccccccccssssssssssssscccccsccsssssssccsoees 35 AEC t Adjustment ese E N 35 2 1 1 Using the Contrast Adjustment Tools for Grayscale Images cccccce 36 2e Blac ke le Adus imen aa aa aA 36 ZA WW ATS Level Aduse it as reac arte xtra head date ett Bed abba tirew acetates 37 2 Wiel Gamma Seung AdSense acs ome tial astra eae sie Sena cees 37 213 Lhe Auto Contas Selecionar a aieeteledcecee secre 38 ALO The Revene BUON hnn n A sreasisesaeentnaeieetaaee 39 ImageQuant RT ECL Capture User Manual PRS Noe he Gid BONON een pee rcer ee rene ee ae oe Rn rene t a ent aera ere Reo 40 2 1 8 Making Linear Log or Equal Adjustments 2 0 0 0 cceeecseeecceceeesseeeeeceeeeeeaeas 4 2 1 9 Using the Contrast Adjustment Tools for Color Images cceeeeeecceeeeeeees 42 2D TOLD Al annaa a ek bes ete g ie ewes E E ial sits iat ta satay 43 23 TOOLBOX nerasit e TEE ATUA E TEE A OETA 46 A OAS D AT arue A ated 46 Chapter 3 Pull Dow
56. gle unit increments or the user may type in a specific unit 2 1 2 Black Level Adjustment The number beneath the Black Level scale corresponds to a gray level There can be 256 4095 or 65 536 possible gray levels depending on the system type For the example below an 8 bit image will be used with 256 total gray scale values When the Black slider is at the very top of the scale the number is 0 As the slider is moved downwards along the scale the number increases and the image becomes progressively darker This is because all pixels at the specified gray level and lower are shown on the screen as black pixels If the slider is set to O all the pixels whose gray levels are at 0 are shown as black If the setting is then changed to 60 all the pixels between 0 and 60 are shown as black and the image appears darker 36 ImageQuant RT ECL Capture User Manual Black Level set at 0 Black Level set at 60 2 1 3 White Level Adjustment The number beneath the White Level scale also corresponds to a gray scale value When the slider is at the very bottom of the scale this number is 255 As the slider is moved upwards along the scale the number decreases and the image becomes progressively lighter This is because all pixels at the specified gray level value and above are shown on the screen as white pixels For example if the slider is set to 150 all the pixels between 150 and 255 are shown as white and the image appears lighter White
57. he acquisition screen ImageQuant Capture stand alone software does not include FrameGrabber acquisition tools FrameGrabber Setup in the Tool Box Image Tool Box Enhancement Tools eee OOM Rotate Flip Histograrn Enhance Annotate False Color Filters Frame Capture Aperture a ZOOM Focus Ihi GE Exposure Time Aa E AE q e Ii gt D gt i B Ba Hours Minutes a Seconda MiliSecs REW REY REC STOP PAUSEPLAY FWO DEL OPEN SAWE 0 0 3 0 Auto Expose Auto Expose Setup Display Sensitivity A esalutiorn Chemi Display Normal High Show Saturation Auto Contrast Medium Medium Reverse High Low FrameGrabber button FrameGrabber in the camera setup acquisition screen oom A I 1 0 c lai rv o j I moro By Sensor Temperature 31 9 C 30 ImageQuant RT ECL Capture User Manual Once FRAMEGRABBER is selected a display box will appear for independent control of all lighting filters and exposure delay for each image frame 4 a 4 f Normal Sequence f Stack Frames 1 El kW Noise Reduction Copy to Next Exposure Tine l e 2 j gt gt Copy to End A Minutes Seconds a Mlisecs a D x D 3 xj oO Auto Expose Auto Expose Setup The TOTAL FRAMES setup provides you with the ability to determine how many individual frames images you want for the sequence of frames There is a maximum of 50
58. ical filter for your sample type a Position 1 for colorimetric gels and film no filter b Position 2 for ethidium bromide gels c Positions 3 5 for other fluorescently labeled gels optional filters Turn on the illumination source UV or white light using the touch panel or virtual software controls Preview Select the button Select Af Show Saturation Select Mf Auto Expose for details on how to adjust the auto expose settings refer to section 1 6 4 of the manual Select the Momal High resolution setting for details on the resolution settings refer to section 1 6 6 of the manual 16 h ImageQuant RT ECL Capture User Manual Once the preview mode image does not contain any saturation red false color palette for white bands green for dark bands select pe The exposure bar will turn green when this is complete If the exposure bar is pink in color saturation is still present in the image For really bright images particularly in white light applications it may be necessary to reduce the aperture setting until the saturation is removed from the image 4 Capturing a low light image like chemiluminescence oF mh Oo AO Close the cabinet door there should not be any lights on in the cabinet Choose Position 1 in the filter wheel for no filter Preview Select the button Select Af Show Saturation Select W Chemi Display Select Mf Auto Expose for details on how to adjust the auto
59. ies View Help Load Defaults Save Defaults Adjust input level Security Prink Info Print Mode la w Full Image Print Date Screen Dump Preferences Image Window Setup Print Image Info Dialog Box Printing can be achieved using three different methods Full Image Prints the original image Does not print zoomed images or images overlaid with data screens Screen Dump Prints the imaging area Well suited for printing images overlaid with data screens and or graphs zoomed images etc Image Window Prints the highlighted window 3 4 7 Print Date Under the Setup menu there is a selection labeled Print Info This allows the user to change the format in which the date is printed The choices are MM DD YYYY and DD MM YYYY 3 4 8 Preferences In order to change the preferences of the system you will need to find the administrator of the ImageQuant Capture software program to log in If you do not have an administrator for ImageQuant Capture see Appendix B in this manual Login Dialog User Mame Password Login Dialog box for Preferences 63 ImageQuant RT ECL Capture User Manual There are four tabs in the Preferences menu ImageQuant Capture standalone software does not contain the Image Acquire or Cabinet Settings tabs 1 General Configure prompts and file saving opening formats 2 Image Acquire Inverts the image seen by the camera and adjusts the ROI values Preference
60. ifferent software versions and or hardware configurations TIF BMP GIF MAC PCX Adobe Photoshop 2 51 LE Mac Open no Open Open no As TIFF Adobe Photoshop 3 0 Mac no Open Open Canvas 3 5 Mac use no no use no ResEdit ResEdit Microsoft Word 6 0 Mac Insert Insert no Insert no Picture Picture Picture Microsoft Word 6 0a Win Insert Insert no Insert Insert Picture Picture Picture Picture Microsoft Excel 5 0 Mac no no no No No Microsoft Excel 5 0c Win Insert Insert no Insert Insert Picture Picture Picture Picture NIH Image 1 59 Mac Open no no Open no For instructions on using ResEdit see next page Additional packages such as Claris Works and PowerPoint have also been tested For these systems it is necessary to save the file with a TIFF extension in order for them to recognize the file as a TIF format 97 ImageQuant RT ECL Capture User Manual Using ResEdit l 2 Save image as a TIFF or MACTIFF Obtain a copy of the freeware ResEdit by downloading from Apple Computers through the Internet Open ResEdit An animated startup display will show up and continue until you click on the mouse or any key A dialog box will appear Open the TIFF image Another dialog box will appear asking if you want to add a resource fork click on OK Next go to the File pull down menu and click on Get Info for This File In the File Info window change the Type to TIFF instead of TEXT and the Creator to DAD
61. illuminator epi lights etc 8 Without changing anything on the lens acquire another image of the Flat image following step 3 again 9 Save the Flat image 10 Open the original gel or other application image 11 Select Flat Field Calibrate from the Image pull down menu 12 Browse the directories for the Flat image created 13 Click on open Make sure to save the flat field calibrated image for future use 58 ImageQuant RT ECL Capture User Manual 3 3 5 Image Resize The image resize function can be used to resize an image to a specific dimension for use in graphical presentations You have the option to Preserve aspect ratio to avoid image dimensional distortion or you can deactivate this function and configure the image resolution to the desired Width and Height dimensions Original dimensions Width 1280 Height 1024 Cancel IW Preserve aspect ratio Image Resize dialog box Note It is recommended that you DO NOT perform quantitative analysis on resized images 3 3 6 Image Info The Image Info function provides a dialog box with all detailed image properties An example is shown below Click on the OK button to close this dialog box x Image idth 1136 Image Height B92 Image Type Grayscale Color Planes 1 Bits per Pixel 16 Acquisition Date 13 6 2003 Acquisition Time 3 57 07 pm Machine ID 960334 Image ID an Modified no Exposure Time OhOmOs 18ms Sensitivity Fac
62. ions can be displayed in a variety of colors The color options are displayed by clicking the COLOR checkbox To select a color simply click the cursor on the button labeled with the desired color The color button appears depressed indicating that it is selected Any annotations subsequently entered will appear in that color It should be noted however that annotations are printed in gray scale on the video printer Further when an image is saved as a modified image the annotations are saved in gray scale not color ajea ae 6 l COLOR LINE ENDS PEN STYLE TEXT ORIENT Pen Color Selection Tools 76 ImageQuant RT ECL Capture User Manual 4 5 3 Line Thickness The PEN WIDTH menu specifies the thickness of lines when using the freehand lines box and circle drawing tools Click on the appropriate checkbox for the desired width All annotations subsequently entered will appear at that width enan HJA COLOR LINE ENDS lt PEN WIDTH TEXT STYLE je PEN TYLE TEXT ORIENT L I I Pen Width Selection Tools 4 5 4 Line Types The PEN STYLE menu specifies the style of lines when using the freehand lines box and circle drawing tools Click on the appropriate checkbox for the desired style Al annotations subsequently entered will appear in that style Note these pen styles only work with a thin line see Line Thickness above ja EES EER COLOR LINE ENDS PEN WIDTH TEXT STYLE rs lt
63. k Places rar Exposure Time lt lt By Ba gt Ea Le Coon End Minutes a a Miisecs o fe o wl 3 e o r Auto Expose Auto Expose Setup j i A Filename O Display Sensitivity Resolution Files of type Sequence Setup Files mve ny Cancel Chemi Display Normal High F Show Saturation F Auto Contrast Medium 7 Medium Go Reverse High Low ala Cancel FrameGrabber Super Sensitivity Load Setup L oa d S etu p an d Save Setup Save Setu p butto ns Chemiluminescence Zoom ADI Door Sensor Temperature 32 1 C CLOSED 1 6 14 Frame Stacking At the top of the FrameGrabber Setup window is an option for stacking frames If this selection was chosen during acquisition of the image Stack Frames will use all previous exposure information to sequentially add images to one another Normal Sequence will not perform this addition Please note that stacking frames will increase the noise level in acquired images Sample case Capture 5 frames at 1 5 sec exposure for total time elapsed 15 sec Display after summation of following frames Frame 1 1 sec Frame 2 1 2 sec Frame 3 1 2 3 sec Frame 4 1 2 3 4 sec Frame 5 1 24 3 4 5 sec 33 ImageQuant RT ECL Capture User Manual 1 6 15 Capturing a Color Image Using the FrameGrabber Function A color image can be generated by acquiring three images each taken with a red green and blue emission filter Once saved the
64. l filters found in the Tool Box under the FILTERS to stop a filter hit any key on the keyboard to reverse the effects of a filter click UNDO Add text boxes arrows etc to the image using the annotation tools in the Tool Box under the ANNOTATE tab 7 Print the image using the large PRINT button in the Tool Bar or the pull down FILE menu option 8 Analyze the sample by using the analysis options in the toolbar 1D Array Colony and Toolbox When one of these buttons is pressed the image is transferred to the appropriate ImageQuant TL module for analysis 17 ImageQuant RT ECL Capture User Manual 1 4 1 Cabinet Controls UV p erride UV Transillumination Controls These two buttons are used to toggle the UV transilluminator and the reflective white light table on and off Reflective Light Controls These two buttons are used to toggle the reflective UV and white light sources on and off UV Override Button Normally if UV light is being used the cabinet automatically shuts off the UV light source when the cabinet is opened This button can be used to override this behavior the button should be held down 5 7 seconds before opening the cabinet Filter Wheel Position These controls are used to set the filter that will be utilized for acquisition Use the arrow buttons to cycle through the available filter settings The LED indicates the number of the filter that 1s currently being used 1 No filter us
65. ll allow users to double click TIFF files from Windows Explorer and automatically launch the application on any machine that has ImageQuant Capture loaded on it Users may customize this in the preferences section covered in section 3 4 of the manual if they wish to change the default file type Mac TIFF is the Apple Macintosh version of the TIFF file format Mac TIFF files have the extension MAC so they can be easily distinguished from Windows TIFF files Most software can distinguish between Mac and Windows TIFF formats and can accept either ImageQuant Capture offers the option of both formats in the event that only one of the two is acceptable GLP is a proprietary file format that allows changes to only be made in ImageQuant Capture programs It will accept 8 bit and 16 bit images and can not be opened in any other software program BMP PCX TGA PIC JPG GLP are additional graphic file formats which may be useful when saving an image for desktop publishing These file formats can be imported directly into many Macintosh and PC programs See Appendix A for more information Do not use these formats to save images that will be analyzed later since pixel data can be lost or altered when saving files in these formats Note Not all of the file types listed above can be saved as a 16 bit file Some may require you to convert the image to an 8 bit file first Original versus Modified Files An Original image file is one in which the dat
66. lose an image log off of the system or exit the system OEM Overlay Close Save Save Modified Save As Prine Printer Setup 1 Co Images Sample tif 2 Cf Images Testpatn tif 3 Cf Images coomassiel 6 tif Exit Alt F4 Logoff ImageQuant Capture File pull down menu 47 ImageQuant RT ECL Capture User Manual Tu Lar 3 1 1 File Open This function opens an image that has been previously saved as a TIF GLP BMP PCX TGA PIC JPG or Macintosh TIFF MAC file ll ImageQuant Capture File Edit Image Setup Overlay Utilities View Help 2 xX ey E Look ir E Images d E w coomassie 16 tif w Sample tit w Testpatn tif lk Testpatt tif File name Biwi tt Files of type Tit Files tif Cancel Good Laboratory Practices files glp Paint Brush files pcx Color T arga files tga Headerless files pic JPEG format joa Mac Tift Files mac All image files File Open Dialog Box Using the left mouse button click on the name of the file to be loaded That name is then highlighted in the list and appears in the text box below the File Name prompt Alternate disk drives can be accessed using the Look In dialog box Once the file has been selected click on the Open button to load the file Alternatively double click on the file name The dialog box disappears and the selected image appears in the image window on the s
67. m with log Contrast Adjustments selected log provides minimum and maximum adjustment tools from 0 to 100 Log performs a logarithmic adjustment to the grayscale range of the displayed image Image of film with equal Contrast Adjustment selected Equal automatically adjusts the image display for maximum contrast which is beneficial for faint band detection 41 ImageQuant RT ECL Capture User Manual 2 1 9 Using the Contrast Adjustment Tools for Color Images ImageQuant Capture can also import RGB color images ImageQuant Capture automatically detects this process and the Contrast Adjustment tools are configured for color image adjustments Contrast Adjustment J Gamma Linear Log Equal tow _ 9 Reverse Auto Contrast rid jw Thumbnail gt Contrast Adjustment display with Show thumbnail clicked for color image The Black White and Gamma bars can now be adjusted individually for each of the three RGB color channels by selecting on the Red Green or Blue button and making the appropriate B W G adjustments Also by clicking on the Show thumbnail option a thumbnail display of each of the three color channels is displayed These thumbnails also display any black white or gamma adjustments for each color channel 42 ImageQuant RT ECL Capture User Manual 2 2 Tool Bar The Tool Bar window provides intuitive buttons for the most common functions in ImageQuant Captu
68. mD sipeetateds cave recsiteds T a paver ce vaeda nares 6 We Cate ra Tis Calan iO asc aces cco Secrtn a anand a a N 10 1 3 5 Software Driver Installation INStructions ccccccsssesssecceeceeeeeeeeeeeceeeeeeaaas 12 5 5 OC ONNE CLINGS NE Printer uiers tactes teen awsenu sue educa patentee tedweea tele 13 13 7 starune ImageQuant Capture coruna a EE een aaah 15 lL Aslmace Quant system Quick Gide viisisteteteriaiotenis ioiaierinh Oa 16 VA A Capiinet COMMONS innnan eetadecadanmesedeveg E aaah vacaxanmetedeven dieu 18 MSY SCCM TINE OF ALI ON ss cesestenectint at a reciaeaigurswiueninl ad ceca a eaa 19 DG PNG UIE AMINA Cheat siec Get bos cris sa acetate Ceara E 20 ROT POCUSI 2 ON an NaS Crs AE chante a tetecaneigpadsen 21 1 6 2 Using the Zoom ROI Tool ssisaccencsescsiessiencenchlandentuevsareadvandasteeesineadiandewnsisseizadineds 22 1 6 3 Acquire and Transfer the Image i0 cisscssccsccsarsssrderea cater cdescasrecs etaieeevorsanea canes 23 1 6 4 Auto Exposure and Auto Exposure Setup ccccccccssssssseceeceeceeeeeseseeeeeeeeeas 23 1 6 5 Cabinet Controls Activating the Light Source and Selecting a Filter 25 1 6 6 Sensitivity Resolution Controls wig casei ced chews ante dai ia beanie 26 126 7 Use Preview to set Exposure Time sssi ie eanne ke a Eer EEA ET OESE Zi 1 6 8 Optimizing the Gray Scale for Saturation and Contrast Displays 28 16 9 Acquire and Fransfer the Mace seran E 29 1 6 10 Capturing Ima
69. mment Network Cancel Source Auto Tray Select Margins inches Right f Top Bottom f Cancel Printer Printer Setup Dialog Box Printer Dialog Box i Portrait Lett r C Landscape For more information on using the Print menu see the Windows manual 52 ImageQuant RT ECL Capture User Manual Ej 3 1 6 Print aL This function sends the image to the default printer specified in Print Setup 3 1 7 Logoff This function logs the current user out of ImageQuant Capture when security features are in use 3 1 8 The Exit Function The Exit function closes ImageQuant Capture To restart ImageQuant Capture from Windows double click on the ImageQuant Capture icon on the desktop 53 ImageQuant RT ECL Capture User Manual 3 2 The Edit Menu The Edit menu provides the ability to copy crop and remove any annotations or filters that have been added to the original image Edit Image Setup w Edit Activation Copy Crop Reset Clear Edit pull down menu To activate the Copy and Crop functionality place a check mark next to EDIT ACTIVATION This will turn the mouse cursor into a sign that will allow you to highlight the region of interest for the image After Edit Activation is highlighted the desired area of interest is drawn using the mouse irwrt PC bescsmigew Lame Ail atte bbe Zoom ReaeFip Hitngam Enhance Arcia Fahe Coke Fien
70. n Men s sexs ceseccaasusd cone daseivasedentaanveedvxacesensnacesddexacecoceaanusesevanedcneanaesedusasecectanse 47 Ps bad Bo A oa 270 eer erence ne ene E eee ec eee ee neo Neder ne Oe re A ae 47 cla i oe werner ere nner et nT Se eo eI See 48 PE TEO a aE E a a 49 ed a a EIC TOSE aia E A E A N eee ee ee eee 49 Sel AE PIC DAYS mAd SIVE AS r a E esse OEE NEN 50 ALIP Seprona E N O 52 LOR eseu a E E eee radiwediee 53 FELEO ON on a maida sede aac res ase 53 ALO LEE EENE O a N OET ONO 53 ITeC Edt MeN naa r E E E T O 54 Died ERES OCE sie as a E AA 55 Di The Maire MeNi srs EN 56 ILEUS A itis eae te eaehenanetens 56 Di Dye ANNE C ede wr E E E E tiie TO awd wex taba tans dade a a 56 Se i CONVE SO a ses yesia eek eaaey R ind ee 57 JRP eld C aB ea a E A E 58 T E S126 aa E ae rom aren a ane Rede na hy 59 PREA A I E EE etree eet fear Oe ere EA fear Oem A 59 S24 ne Setup Meni xigoctane Wont a dt naelegin dian aiid dastakediia te nutiuese tah eatenes 60 SL Dert Paramete Sea E a R E E 60 DL AVS De Uls E a E A E T T 6l IA Load DeSean a a a N 6l JAA Oe CUMIN euse a e a s 62 O REA Ea U0 E ET E N E E ET E E E ETE 62 F ROPTDEMOO E aen ee nee nee es eee E eee eee 63 i ee PON oe cer nee en E E er een ee 63 Fo od Aor a Rod NG oi eer REET re noe ean RE ne ern eee Ere PRAT eee er eee 63 DOC Tl Me A as cerned age as T na ec iae E annigparants 66 Dip AV MINS ANN OY CUA ciel catia eisai etree ection detan eae Me eect ui ueaididelgat eee asaualnnds teenie
71. n image using ImageQuant Capture you can transfer it to one of ImageQuant TL s analysis modules using the toolbar options The ImageQuant Capture toolbar contains 4 buttons 1D Array Colony and Toolbox 1D x This is a 1D electrophoresis gel or TLC plate analysis T E module Acquire Overlay GE Array The Array module to analyze microplate images gridded arrays and dot and slot blot images Colony The Colony Counter module can be used to analyze images by detecting and measuring image features 2D spots or colonies You can display and print measurement data from the detected colonies Toolbox The Toolbox module allows you to perform analysis on a wide variety of images The flexible nature of this module means that you are not restricted to a single type of analysis when it is used If the acquired image has been saved the selected module is immediately accessed when the appropriate button is selected If the image hasn t been saved there will be a prompt to save it before the image is transferred from ImageQuant Capture to the appropriate ImageQuant TL module 96 ImageQuant RT ECL Capture User Manual Appendix A Opening ImageQuant Capture Files in Other Software Programs Files generated by ImageQuant have been tested in the software packages below For successful imports the command line is given Programs for the Macintosh operating system were tested on a PowerMac 8100 1O0AV Results may vary for d
72. nce this list requires a password in case the system is later changed to a closed password protected system ImageQuant Capture will automatically assign the user s name in uppercase as the password and the password field is disabled When finished using ImageQuant the user can choose Logoff from the File menu or can log out by exiting the system Otherwise he she will remain logged in unless Auto Logout is active Option 3 Open System with User Name and Password Log In and Out This security level keeps the system open but requires users to log into the system by typing their names and passwords When someone enters a name and assigns a password to that name the same password is needed whenever that name is used to log in Only one password can be assigned to a name A list of users with their log in and log out times is generated W User Logging Active Closed System Auto Logout 5 minutes 10 minutes 20 minutes Change Master Passyrord AddRemove Users OK Cancel 101 ImageQuant RT ECL Capture User Manual Option 4 Closed System with User Name Log In and Out A user can log in only by entering an authorized name A user log is generated IY User Logging Active Jw Closed System Auto Logout f 5 minutes 10 minutes 20 minutes Change Master Passyvrord AddRemove Users OK Cancel In closed systems the system supervisor may want to add or remove names from a list of
73. nue the product described at any time without notice or obligation Contact your local GE Healthcare representative for the most current information GE and GE Monogram are trademarks of General Electric Company ECL ImageQuant Amersham and Amersham Biosciences are trademarks of GE Healthcare Limited 2006 General Electric Company All rights reserved All third party trademarks are the property of their respective owners SYPRO and SYBR are trademarks of Invitrogen Windows is a trademark of Microsoft Corporation Macintosh and ResEdit are trademarks of Apple Computer Inc ChromaLight is a trademark of Alpha Innotech Corporation GE Healthcare Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK Table of Contents Chapter L Mmtrod cion and Setup e ike Aaa eee ee Make ee 1 1 TnaeeOuant Capture Imasine Systemene eA l LI MOUSE Funcions sienen E E AA EA E 2 ELEA Dout EMIS Manta ber R N becad nessa 2 LSS Im cOn Cape aE eeaeerhe eens iauenbeineeuness 2 1 2 ImaseOuant masing S yS En Se UP a E een Mua a ad 3 L2 ESY eme C Omon MS e E A T lcwawuieds 3 t22 System Plien E 3 12 gt Cape C Onne ONS cesa A 3 1 3 Computer Camera and Peripheral Installation ee eccccccccccecesseeeeeeeeeeeeeeaeeeeees 4 1 3 1 Setting up the Power Strip Surge Protector i cecccccccccecceeeeeeeseeeceeeeeeeeeeeees 4 1 3 2 Setting up the Computer Monitor Mouse and Keyboatd ccccccssseeeees 5 13 3 CaBIncl ASSE
74. o the desired configuration click on the GO button The software will then begin the image acquisition for each frame of the image When it is complete the Camera Setup and Preview window will disappear and the TOOL BOX will automatically display the FrameGrabber tab This will allow you to play back the sequence save or load the sequence or record a new sequence 31 ImageQuant RT ECL Capture User Manual Tool Box Peretti etree errr errr errr rer errr e rer rre rrr ttre irre rrr errr rrr errr rrr rire tr rrr rrr Enhancement Tools oom Rotate Flip Histograrn Enhance Annotate False Color Filters Framelrabber Image REW REY REC STOP PAUSEPLAY FiO DOEL OPEN SAVE Once all images have been captured the FrameGrabber tab will be displayed in the Tool Box The buttons on this tab perform the following tasks REC Opens the Camera Setup and Preview screen to record a frame sequence PLAY Display a continuous loop of all of the captured images STOP Stop the sequence at the current frame display PAUSE Pause the playback of the sequence at a user defined image REW rewind Rewind the sequence to the first image REV reverse Play the sequence in a continuous loop in reverse FWD forward Forward the sequence to the last image DEL delete Delete the sequence on the display THIS FUNCTION WILL NOT DELETE A SEQUENCE SAVED TO THE HARD DRIVE LOAD Load a previously saved sequence THIS FUNCTION WILL NOT LOAD INDIV
75. posureCompLimits 4 0 4 0 Notepad Display Window 68 ImageQuant RT ECL Capture User Manual 3 7 The View Menu The View function provides the ability to control the display of the on screen control tools as well as provide image enhancement abilities view Help w Status Bar W Tool Bar Contrast Window w Tool Box Default tools position Zoom In Ctrl zoom Cut Ctrl zoom lx Fit In Screen w Show Annotation View pull down menu Four main tools exist within ImageQuant Capture Contrast Adjustment Tool Bar Status Bar and the Tool Box E Acquire Overlay Ch Ch Zoom Out oom Ir FF y Saturation Img Drag sy Contrast Adjustment Gamma Linear Log Equal Low o j gt i High J gt Gamma 0 55 Reverse Auto Contrast Grid Thumbnail Contrast Adjustment Window Tool Bar Window 69 ImageQuant RT ECL Capture User Manual ee eee Enhancement Tools smn oom L Rotate Flip i oa Enhance Annotate False Color Filters FrameGrabber i COLOR LINE ENDS PEN WIDTH TEXT STYLE PEN STYLE TEXT ORIENT aleja ojoj xima E mone AE 89 2 Zoom 200 Intensity 416 Status Bar Tool Box These tools are automatically displayed when ImageQuant Capture is launched for additional ease of use and to generate a common look and feel However if you would like to hide any of these tools they can b
76. prong power cable on the back of the printer and into the power 13 ImageQuant RT ECL Capture User Manual strip then turn the power on Set the printer up for color or black and white printing following the directions in the owner s manual supplied with the printer Power on the cabinet after the Windows operating system has fully loaded The ImageQuant Imaging System is now ready for use 14 ImageQuant RT ECL Capture User Manual 1 3 7 Starting ImageQuant Capture To start ImageQuant Capture from Windows double click the ImageQuant icon on the windows desktop Touant Capture RT ECL 15 ImageQuant RT ECL Capture User Manual 1 4 ImageQuant System Quick Guide Note This is intended as a quick reference guide for acquisition For more detailed information on the individual features reference section 1 6 of this manual 1 Power on the system a b C 2 Positioning and Focusing on the Sample a Turn on the computer monitor and optional printer After the computer has booted up completely to the Windows desktop turn on the power to the cabinet aaa Activate ImageQuant Capture software by double clicking on the ECL ImageQuant Capture icon In the Tool Bar select the Acquire icon to activate the image acquisition software features Setup In the Image Acquisition window select the button Open the door to the cabinet and position your sample on the preferred illumination source
77. r deleted as needed Note Overlays are specific to the resolution of the image that they were created on Therefore if an overlay was created on an image with a different resolution than the image that the overlay is being loaded onto the overlay may not match the original image 67 ImageQuant RT ECL Capture User Manual 3 6 The Utilities Menu A number of functions are now handled by Windows programs To access many of these programs while in ImageQuant Capture open the Utilities menu and select the program of choice Utilities View Hel Explorer Lab NotePad Utilities pull down menu 3 6 1 Explorer This option can be used as a shortcut to Windows Explorer which allows access to files and other information saved on the local machine or the network 3 6 2 Notepad The Notepad is a blank screen that allows the user to make notes about the experiment and save them as an ASCII file The Notepad is useful for saving any imaging comments or experimental conditions with the saved image for future reference lox File Edit Format Help File Type AppDets Versian 2003 02 HardwareContig LensCtrlEnable U CabinetType 1 Devices Cabinet COM1 9600 n 6 1 LensCtr COM2 9600 n 0 1 AutoExposeContral AutoExposeLevel U 1 AutoExposeNoise 0 01 AutoExposesat U 0 AutoExpaseDrift U 125 AutoExpaseF ilter1 AutoExpasevalidate 0 RecalcLiveOverDrnit 0 PrecalcOverBoost 1 Precale TimeLimit 0 ExposureCompoteps o Ex
78. racteristics circles squares text with various characteristics 4 5 9 The Editing Tools When the cursor is in edit mode it can be clicked on an object to select it Note the cursor can be toggled between edit and drawing modes by clicking the right mouse button When an object is selected small square boxes appear at the corners Selected objects can be resized copied deleted or moved e To resize an object click on one of the gray boxes at the corners of its perimeter and drag the box until the object reaches the desired size e To copy an object use the Copy tool e To delete an object use the Cut tool or the Eraser e To move an object click within its boundary and drag it into the desired location To select more than one object outline them with the mouse any objects that fall completely within the outline drawn will be selected Note The entire object must be enclosed by the cursor s movement in order to be selected A Selected Object 81 ImageQuant RT ECL Capture User Manual Once an object or group of objects has been selected clicking on the Cut tool deletes it from the image ol The Copy tool makes an exact copy of the selected object The new object becomes the selected object and can be repositioned by placing the cursor within the object s boundary and moving it to the desired location 500 The Horizontal Alignment tool aligns annotations in a straight horizontal line
79. rappresentante autorizzato del fabbricante Este s mbolo indica que el equipo el ctrico y electr nico no debe tirarse con los desechos dom sticos y debe tratarse por separado Contacte con el representante local del fabricante para obtener m s informaci n sobre la forma de desechar el equipo Denna symbol anger att elektriska och elektroniska utrustningar inte f r avyttras som osorterat hush llsavfall och m ste samlas in separat Var god kontakta en auktoriserad tillverkarrepresentant f r information ang ende avyttring av utrustningen imagination at work Contact Information Asia Pacific T 852 2811 8693 F 852 2811 5251 Australasia T 61 2 9899 0999 F 61 2 9899 7511 Austria T 01 57606 1619 F 01 57606 1627 Belgium T 0800 73888 F 03 272 16 37 Canada T 1 800 463 5800 F 1 800 567 1008 1 514 457 7000 Technical Support ts canada ge com Customer Service csdcanada ge com Central East amp Southeast Europe T 43 1 982 3826 F 43 1 985 8327 Denmark T 45 16 24 00 F 45 16 24 24 Eire T 1 800 709992 F 44 1494 498231 Finland amp Baltics T 358 0 9 512 3940 F 358 0 9 512 39 439 France T 01 69 35 67 00 F 01 69 41 96 77 Germany T 0761 4903 490 F 0761 4903 405 Latin America T 55 11 3933 7300 F 55 11 3933 7304 Middle East and Africa T 30 210 9600 687 F 30 210 9600 693 Netherlands T 0165 580 410 F 0165 580 401 Norway T 815 65 555 F 815 65 666 Portugal T 21 417 70 3
80. re zj Acquire Overlay Cy oom Out oon In Saturation Img Drag 2 Tool Bar Display Window Cei The Open button functions identically to the File Open function in the upper menu bar This function is used to open previously saved images Detailed instructions are available in Chapter 3 oom Out Zoom Iri The Zoom Out and Zoom In buttons provide easy zooming ability while you are active in image enhancement functions providing increased versatility Detailed instructions are available in Chapter 4 as this function is also available in the Tool Box Note The Status Bar always displays the image zoom setting in real time Tit Saturation The Saturation button allows for a quick image display of saturation Completely saturated black regions gray scale 0 will turn green and saturated white regions ie gray scale 255 4095 65 535 will turn red 43 ImageQuant RT ECL Capture User Manual Da Img Drag This is a useful tool to check for linearity of an image before analysis occurs Saturation is a feature that is most important during the acquisition stages and is thoroughly detailed in the acquisition features of the system manuals The Img Drag button is useful for to pan with a zoomed image To activate this function click on the button and move the mouse cursor to the image The cursor will have changed to a small hand Click the left mouse button and drag to move the image When you are done
81. revert to an arrow indicating that ImageQuant Capture is now in edit mode At this point the object can be resized or repositioned The color pen thickness line type etc can also be changed simply by clicking on the desired choice as described in Object Attributes above To draw another object click the right mouse button to return to draw mode or click on one of the drawing tool buttons a The button labeled with an A adds text to the image Place the cursor at the location on the image where the left edge of the text should appear Click the left mouse button and begin typing To place another piece of text click where it should be placed Once all text is entered click on the right mouse button To edit text double click on it An edit window will appear in which changes can be made To change fonts see Text Background and Font above The button labeled with a pencil icon allows the user to draw lines freehand After clicking on the pencil move the cursor to the correct position on the image to begin drawing Press and hold the left mouse button Using the mouse move the cursor as if it were a pencil When finished drawing release the mouse button 79 ImageQuant RT ECL Capture User Manual gt The button labeled with a diagonal arrow draws arrows and straight lines After clicking on the button move the cursor to the position on the image where the line should begin Press and hold the left mouse button
82. rows and Strate nt LANES erien TI 4 5 0 Text Ba cksro ndand Font sserunieiinismn ea A EET 78 Bed OKT NING CS aa a tonsa abe ieee aeaha lance 79 dao THe Drawins L00 Bisera aaea E E AS 79 A D TENG ECan Tools sesser E EEE 81 Ah SO ae SO LO Be pete ssa tte eae valance atneanie dc neta sede wena ance eaten nce venetian 83 Onl Grav S Cale Pa te OV ara E ahah ee ent tuae edea nae neha ei tah 83 AO Sarao Pee T enaa ra a eee a eae 83 40 Paletes 2 1ntOusn al Esera a a S 84 Aoi Dna eF HOTS ernen i sasacucansoruateurecndauavena mie nsaceandiecs stews 85 4 7 1 General Information on How Filters Work cceeccccccccceessseeeeeceeeeeeeeeeeees 86 A oe SNarpenine PALES rsrsr i a T 87 Db Tes SIN ONS Fleis e cache sta ess sae herald estate hs seasons chemists estasen cage 87 AAP Despec ke PIMC Siete tudes etc caatta tnd telat aantaahs dan ted tatiaueias ban taunadald tea ouial 87 A fs RD C ONON lL os Coe et ee et a et Se Ree een a i 87 ATOS MOO MILI FINO S He tuau dawnt aat esa T 88 A lat GOS Tillers iaseskanenetcoct anes r Manet anal atneal MeRs yaaa taal al Maney aaesWineal aes 88 AT S FLOTIZ OUTLAY FOSS LC eias a EA Ea 88 Ae NCR al e Ueto ssi zden ema taale A idence laters 89 BOGUS OMI TLC saeco oeiaia serie a E sapiens 89 A Nl Fhe UNDO BUM serseri a a 89 gl 2 Examples of Fer RESUS raae a A 90 do Framer DEE Mode erenean a e deb tenbat eae nates t asain 91 4 8 1 Saving an Individual Image from a Sequence ccccccccccesseeeeeeeeeeeeeeaeeeeeees 93 4
83. s Mormal High Medium f Medium High t Lowe Super sensitivity 64 ImageQuant RT ECL Capture User Manual 3 Cabinet Settings Used for adjusting the port settings and customizing filter positions on the cabinet General Image Acquire Cabinet Settings Auto Enhancements Devices Cabinet COM1 9600 n 8 1 Filters Selection Jeter Colorimetric crei eo 4 Auto Enhancements Used to customize the Auto Enhance Levels located in the tool box General Image Acquire Cabinet Settings Auto Enhancements Enhance Tonal Adjustment selection Black Level 0 00 white Level 0 35 Gamma Lewel 1 00 Device Corrected Negative V Enable Auto Levels Negative Change caroet_ anol _ To make changes to the preferences select your change by typing in a new value or select de select the appropriate box with a check mark and then select apply Some settings may require that the software be restarted before the change will take effect 65 ImageQuant RT ECL Capture User Manual 3 5 The Overlay Menu The Overlay menu provides a means of saving and retrieving annotation overlays This is especially useful when a standard gel format is run repeatedly Lane numbers molecular weight marker sizes and other pertinent information can be stored as an Overlay file and retrieved at a later date This eliminates the need to re enter the information each time a new imag
84. s operating system preinstalled ImageQuant RT ECLimage processing and analysis software preinstalled and calibrated with computer and hardware system e ImageQuant light cabinet with UV transilluminator and white light fold down transilluminator e Optional epi illuminating UV lights e Optional printer Check the packing list included with the system to verify that all components have been received 1 2 2 System Placement As with all electrical instruments the ImageQuant System should be located away from water solvents or corrosive materials on a table or bench top that is dry and stable Further the system should be placed away from interfering electrical signals and magnetic fields If possible a dedicated electrical outlet should be used to eliminate electrical interference from other instrumentation in your laboratory 1 2 3 Cable Connections The cable ends and the ports into which they are inserted are keyed or unique for each connection to eliminate confusion The connections are pictured and described in the next few sections WARNING Make sure the power is OFF and all power cords are disconnected while connecting the cables and setting up the instrument The ImageQuant light cabinet weighs 37kg 81 4 lbs It is recommended that two people lift the cabinet from the shipping container to the desired location in the laboratory ImageQuant RT ECL Capture User Manual 1 3 Computer Camera and Peripheral Install
85. s important to note that these Contrast Display options are only visualization tools and are NOT changing the image data that is being acquired by the CCD camera 28 ImageQuant RT ECL Capture User Manual 1 6 9 Acquire and Transfer the Image Once the appropriate image is displayed on the screen click on the Capture button to acquire and display the image The acquired image will be automatically adjusted by a zoom factor based on the resolution setting to display the image optimally From there the image can be enhanced using ImageQuant Capture s enhancement tools and or transferred to an ImageQuant TL module for analysis Once a satisfactory image has been captured we suggest saving it as an original file However if you do not save before transferring the image to an ImageQuant TL module for analysis then you will be prompted to save as part of the transfer process 29 ImageQuant RT ECL Capture User Manual 1 6 10 Capturing Images Using the FrameGrabber Option If kinetic multiplex color or chemiluminescence experiments are desired where you wish to have the system automatically capture several images at preset exposure times preset time delay between images preset lighting sources and preset filter choices the FRAMEGRABBER tab can be clicked in the Tool Box FrameGrabber is also accessible in camera setup acquisition screen To access this screen select the acquire button on the tool bar and then select FrameGrabber on t
86. s of 1 standard deviation greater or lesser than the neighborhood average to be corrected for setting 2 results in outliers of 2 5 standard deviations to be corrected for and setting 3 results in outliers of 5 standard deviations to be corrected for These filters are particularly effective in eliminating random noise contained in an image and produce less blurring than the Noise filter described above 4 7 5 3 D Contour Filters These filters are particularly useful for visualizing faint bands They produce a 3 D effect defining edges and making details easier to see 3 D level 9 high has the largest effect on the image 3 D level 5 has an intermediate effect 3 D level 1 low has the least effect on the image 9 different 3 D levels are available for optimization of the image 87 ImageQuant RT ECL Capture User Manual In addition 3 D allows the user to control the direction of the 3 D shadowing effect Once the level is refined just click on a direction arrow to visualize the shadowing adjustment The default shadow direction is in the lower right hand corner direction and 9 different directions are available LW YT i il Original Image 3 D Image Level 5 amp lower right 3 D Image Level 5 amp upper shadow middle shadow Sharpen Noise pesson Gane Sharpen Noise resect M sane Sharpen Noise pese eE disa aJ2ajslslsizielsl a pss ajll slzlsls H Tee tfall
87. saturated to green and values in between to red Palette 4 maps the gray scale levels to a red green blue palette Values of 0 are mapped to green saturated to red and values in between to blue Palette 5 maps the gray scale levels to a cyan magenta yellow palette Values of 0 are mapped to cyan saturated to yellow and values in between to magenta Palette 6 maps the gray scale levels to a cyan magenta yellow palette Values of 0 are mapped to yellow saturated to magenta and values in between to cyan Palette 7 maps the gray scale levels to a cyan magenta yellow palette Values of O are mapped to magenta saturated to cyan and values in between to yellow Palette 8 maps the gray scale levels to a red palette Values of 0 are mapped to dark red saturated to white and values in between to shades of red Palette 9 maps the gray scale levels to a blue palette Values of 0 are mapped to dark blue saturated to white and values in between to shades of blue This palette may be useful when printing an image of a Coomassie stained protein gel onto a color printer Palette 10 maps the gray scale levels to a green palette Values of 0 are mapped to dark green saturated to white and values in between to shades of green This palette may be useful when printing an image of a SYBR Green I stained protein gel onto a color printer Palette 11 maps the gray scale levels to an orange palette Values of 0 are mapped to dark orange saturated
88. se images are then combined in the Overlay pull down menu Open the image captured with each of the three filters as instructed and a RGB red green blue true color image will be generated For color imaging you will need to the following optional filters Red Filter SYPRO Red Filter Catalog 63005664 Blue Filter Hoechst Blue Filter Catalog 63005665 Green Filter SYBR Green Filter Catalog 63005661 34 ImageQuant RT ECL Capture User Manual Chapter 2 Getting Started Basic Imaging Functions 2 1 Contrast Adjustment The Contrast Adjustment window allows for the best visualization possible of a sample utilizing the black white and gamma adjustments as well as image reverse and auto contrast The image on the screen is made up of picture elements pixels in an array Each pixel is assigned a brightness or a gray scale value level between black and white A very bright image has most of its pixels registering high gray level values and conversely a very dark image has most pixels registering low gray level values approaching zero The distribution of these gray values to the image is determined by the Contrast Adjustment Controls These controls regulate the Black level White level and Gamma setting brightness linearity allowing adjustment of the display to obtain the best image possible Note These enhancement features modify the image display on the monitor only and do not change the original quanti
89. so a Flip option allows for the image to be rotated 180 degrees in a vertical or horizontal fashion The Reset button on the main software interface will also remove any rotations or image flips and return to the display to the original image Image rotated 12 degrees Image rotated 12 degrees Counterclockwise and flipped vertically ROTATE UNDO FLIP ROTATE UNDO FLIP ROTATE UNDO FLIP sia sia Pie Rotate Flip box at 12 degrees Rotate Flip box at 12 degrees Rotate Flip box at 12 degrees and vertical flip button pressed 73 ImageQuant RT ECL Capture User Manual 4 3 Histogram The histogram is a graphical display of the proportion of pixels assigned to each of the image s gray levels This tool is found in the Tool Box Tool Box Ue Enhancement Tools Annotate False Color Filters FrameGrabber Zoom Rotate Flip Histogram Enhance E Linear Seale Log Scale EJix 4 libs 4x Histogram display in the Tool Box The image is made up of picture elements pixels having brightness levels ranging from black to white A very bright image will have most of its pixels registering high gray levels and conversely a very dark image will have most pixels with low gray levels approaching zero The histogram is displayed in the lower left corner of the screen below the image window The horizontal axis represents the gray scale range black at the left end and whi
90. stem is shared by a number of people or laboratories a method of keeping track of its use may be helpful The Security feature allows various levels of security and user log functions We suggest one user be designated as the supervisor of the system This individual should refer to Appendix B which describes the security features in detail Note It is strongly recommend that you remove Appendix B from the manual to avoid unauthorized users changing the password and the security settings 3 4 5 Print Info When printing an image basic image information is included on the print This includes the exposure time the Black level White level and Gamma setting the date and time the image file was generated an image ID number and the name of the file to which the image is stored File Edit Image Setup Overlay Ukilities View Help Load Defaults Save Defaults Adjust input level Security Print Mode t ow Bottom Print Date OFF Preferences Setup Print Image Info Dialog Box To print this information at the top of the print choose Top from this menu To print at the bottom of the print choose Bottom Note Printing image information at the top or bottom of a print may obscure a small portion of the image To print the image with no information on it choose Off 62 ImageQuant RT ECL Capture User Manual 3 4 6 Print Mode ImageQuant Capture provides custom printing options File Edit Image Setup Overlay Ukilit
91. still maintain a publication quality 1024x1024 pixel resolution image Medium Medium sensitivity selection will decrease the exposure time needed over a full resolution image by approximately a factor of 4X High Sensitivity Low Resolution Recommend for Chemiluminescence This mode captures the image performing a 4x4 pixel bin increasing the sensitivity by a factor of 16X Super Sensitivity Low Resolution Recommend for Chemiluminescence This mode captures the image performing a 8x8 pixel bin increasing the sensitivity by a factor of 64X 26 ImageQuant RT ECL Capture User Manual 1 6 7 Use Preview to Set Exposure Time Once the sample has been positioned and the camera has been focused close the door to the ImageQuant light cabinet and make sure that the appropriate illumination source is turned on Also make sure the cabinet door indicator in the Cabinet Control software interface indicates CLOSED Click on the blue PREVIEW button and select the desired exposure time in the menu options to give the desired image intensity quality Individual adjustments for 1 30 seconds seconds minutes and hours are available e For most white light applications 50 millisecond exposures are sufficient with final adjustments of the aperture for best image quality e For UV fluorescence applications usually an 8 milliseconds to 4 seconds 1s sufficient and the aperture should be adjusted fully open for the standard zoom lens The show s
92. t gt ee gt gt E 2 Flours a vinutes Seconds MiiSecs 0 0 3 0 Auto Expose Setup Expose Yokoo EV Adanim Auto Expose Auto Exposure options puan Sensitivity Resolution Chemi Display A Normal High Show Saturation Auto Contrast Medium Medium Reverse High Low E tues Super Sensitivity oom AQI Teil 1 1 1 1 1 1 1 1 1 ow Sensor Temperature 31 9 C 23 ImageQuant RT ECL Capture User Manual Auto Exposure Setup Auto Exposure Compensation Setting 4 choices available e Normal Exposure for image saturation Use this choice for normal Colorimetric and Fluorescent imaging e Under Exposure for chemiluminescence e Over Exposure for faint band detection e Custom Exposure Compensation user definable Exposure Value EV The up down arrows allow the user to change the EV value by whole units left up down arrows or by Lge right up down arrows Auto Exposure works in both Preview and Capture modes As the software calculates the correct exposure time you will see the status bar change from red to yellow and then to green When it reaches green the software has achieved the correct exposure time If the bar turns pink this is an indication that there are saturated pixels in the image area at the exposure time calculated In Capture mode the image will be acquired when the bar turns green While in Capture mode the software will continue exposin
93. t 1s changing from one frame to the next is the exposure time In this situation COPY TO NEXT is a useful tool to save time in the setup process Reverse FRAME 2 and repeat the above Or you can click on COPY TO NEXT to help speed up the setup process COPY TO NEXT copies all settings from the previous frame to the current frame If you are performing kinetic experiments where you want to have a predetermined delay between captured images then you can use EXP DELAY to configure this function The default EXP DELAY is set for the shortest possible delay 19 milliseconds but can be configured up to 50 minutes between each image Also if your exposure delay and or exposure time and or lighting options filter position are consistent for the entire sequence then once you set up the first frame you can select the COPY TO END selection to automatically choose the first frame settings for the entire sequence of frames images Once the sequence is set up to the desired configuration click on the GO button The software will then begin the image acquisition process for each frame of the image When capture is complete the FrameGrabber box will disappear and the TOOL BOX will automatically display the FRAMEGRABBER tools This will allow you to play back the sequence save or load the sequence or record a new sequence 92 ImageQuant RT ECL Capture User Manual Tool Box Peretti etree errr errr errr rer errr e rer rre rrr ttre irr
94. t coming through the threaded hole that seats the Screw 3 Note that the extended tray has four holes which match the rubber legs on the transilluminator Place the transilluminator on the tray so that its legs are firmly seated in these holes 4 Plug the power cord at the bottom of the cabinet into the socket at the back of the UV transilluminator 5 Push the tray back into the cabinet Close up of the slide out tray Once the tray is extended Place the UV transilluminator at the bottom of the cabinet replace the locking screw in its on the tray making sure that Note that there is a screw that original position underneath its rubber feet are firmly holds the tray in place for the slide out tray This will seated in the holes on the shipping Remove this screw prevent light from entering into tray and then plug the to allow the tray to slide freely the cabinet through the cabinet s power cord into the threaded hole back of the transilluminator ImageQuant RT ECL Capture User Manual Unfastening the Fold Down White Light Tray The cabinet contains a fold down light tray that is locked for shipping see illustration below Take the following steps to unfasten the light tray 1 The light tray is held in place by a detachable knob that screws into place just above the light tray Unscrew the knob until it is separated from the cabinet and the tray can move freely 2 Note the bottom of the tray has a threaded hole
95. t is for those technical users who run their own flat field corrections This can be done using the Flat Field Calibrate selection under the Image pull down menu which will be described in detail later in this section All three of these arithmetic functions are performed by opening the main image that will be adjusted Next select the appropriate arithmetic function under the image pull down menu Then select the image that is to be added subtracted or divided from the original image and select open The dialog box will disappear and the resultant image will appear Note Images that have been arithmetically altered are ideal for publications and documentation however they are strongly not recommended for analysis as the pixel values have been adjusted 3 3 3 Conversion Since ImageQuant Capture can generate 16 bit files the conversion option is useful when an image is to be imported into a program that only accepts 8 bit images Choosing this option will convert a 16 bit image into an 8 bit image Arithmetic Conversion 68 bit grayscale Flat Field Calibrate Resize 16 bit grayscale Image Info Image Conversion dialog box 57 ImageQuant RT ECL Capture User Manual 3 3 4 Flat Field Calibrate Flat Field Calibrate is a function that is used to flatten the image so that the pixel data is even across the entire image area This is a function that is useful for large gels and other applications that us
96. tative data ImageQuant Capture can also import RGB color images ImageQuant Capture automatically detects this process and the Contrast Adjustment tools are configured for color image adjustments An image can be enhanced using these tools and then saved as a modified file for publications However to preserve the original image information it is recommended that the file be saved as a different file name when using the save modified 35 ImageQuant RT ECL Capture User Manual 2 1 1 Using the Contrast Adjustment Tools for Grayscale Images There are three sliding scales found in the image control area to the right of the image Below each scale is a box displaying a number that corresponds to the position of the slider By adjusting these sliding scales the image display can be optimized Contrast Adjustment Gamma Linear Log Egual Low o bd hi High e _J gt rese Gamma 0 55 Grid J Thumbnail gt Imaging Display Tools Black Level White Level Gamma Setting with B W G Linear Log and Equalize options To adjust any of these settings place the cursor on the slider Click and hold down the left mouse button while dragging the slider to a new setting As the slider is moved along the scale the image display is updated along with the change in numeric value The arrows above and below the scale bars can also be clicked to change the settings in sin
97. te at the right end with levels of gray in between The number of pixels registering a particular gray level determines the height of each bar along the axis A Coomassie blue stained protein gel visualized with a white light box has a histogram reflecting mostly bright pixels Linea Scale Loy Scale 1x he RN 1E Fite Histogram of a typical Coomassie gel Most of the pixels are found in the light portion of this histogram The dark bands represent a small number of pixels and include a variety of gray values and therefore do not show up as a single peak The histogram function is particularly useful to verify that an image spans the maximum range of gray levels When an image is to be used for analysis it is especially important that the gray level range be as large as possible If an image does not include most of the gray levels we recommend repeating the image capturing process 74 ImageQuant RT ECL Capture User Manual 4 4 Automatic Enhancement The Enhance tool is found in Tool Box This function is ideal for new or inexperienced users of the system since it offers 9 levels of automatic image enhancement of the black white and gamma levels simultaneously For an inexperienced user it can be difficult to adjust each black white and gamma buttons to their respective optimal positions By clicking on one of the nine Auto Enhance Level buttons the image is optimized according to a unique level Button 1 will make the image
98. tended OD Ext OD Off 91 ImageQuant RT ECL Capture User Manual Once FRAMEGRABBER is selected a display box will appear for independent control of all lighting filters and exposure delay for each image frame The TOTAL FRAMES setup provides you with the ability to determine how many individual frames images you want for the sequence There are a maximum of 50 frames images and a minimum of 1 frame that can be captured with each sequence The FRAME selection is used for setting up the conditions for each frame image For example if three 3 images are to be captured you would choose FRAME 1 and setup all of the desired lighting and filter requirements You can then click on Total frames Frame Option 1 f Mormal Sequence C Stack Frames Frame Frame 1 Ej al o el Copy to Hest Copy ta End Noise Reduction Exposure Time Ea l lt El gt Seconde MiliSeos a 104 l Auto Expose Setup Hours a Minutes a Auto Expose Display O Chemi Display lf Show Saturation F Auto Contrast Sensitivity Resolution Normal High Medium Medium High Mediurn High Lows Go Super Sensitivity Load Setup oom AU 1 0 Save Setup 1 wol 1 I I 1 I I 1 I I Fi i Usually for chemiluminescence imaging all lighting is off and the filter wheel is positioned for the chemiluminescence position for all frames Thus the only variable tha
99. th low spatial frequency content for example images of clouds contain large areas of slowly changing pixel values Most of the filter options available with the exception of the Noise filters use a weighted summation process to determine the value assigned to the pixel of interest Each pixel in a 3x3 neighborhood is multiplied by a convolution kernel having the same dimensions The resulting sum is assigned to the pixel of interest K1xP1 K2xP2 K3xP3 K4xP4 P1 P2 P3 K1 K2 K3 K5xP5 P4 P5 P6 X K4 K5 K6 K6xP6 P7 P8 P9 K7 K8 K9 K7xP7 K8xP8 3x3 pixel neighborhoodconvolution kernel K9xP9 P5 is being calculated New Value for P5 Each element of the convolution kernel is a weighting factor also called a convolution coefficient The size and arrangement of these weighting factors determine the type of transformation the image will undergo Changing a weighting factor influences the overall sum and therefore affects the value given to the pixel of interest 86 ImageQuant RT ECL Capture User Manual 4 7 2 Sharpening Filters These filters can increase image sharpness and provide edge enhancement However image noise may be enhanced as well These filters accentuate the high frequency details of an image while leaving the low frequency content intact High frequency portions of the image get brighter while low frequency portions become black Sharpen level 9 high has the largest effect on the image
100. that have previously been saved to be saved in a different location or as a different file type without affecting the original image ImageQuant Capture has the ability to save files in several formats see the following figure iil ImageQuant Capture _ File Edit Image Setup Overlay Utilities View Help UU File name ti nave as type Tit Files tif Cancel Tiff Files tif Windows bitmaps bmp Good Laboratory Practices tiles glp tif bmp glp pcx tga Paint Brush files pce pic jpg mac Color Targa files tga E ae i File Formats Headerless files pic JPEG format p Mac Tiff Files mac All image files Enter a new file name in the text box adjacent to the File Name prompt Next choose a file type from the Save As Type list ImageQuant Capture will automatically give the appropriate 3 character extension ImageQuant Capture will also create a file with the same base name and an STP extension This setup file saves information specific to this file such as Black Level White Level Gamma Setting and 1D Multi template placement If the file is accessed later these settings will be recalled 50 ImageQuant RT ECL Capture User Manual File Types TIFF is the default file format for ImageQuant Capture files TIFF is an acronym for tagged image file format and was developed as a flexible and machine independent graphic file format Saving as a TIFF file wi
101. tive image to negative or vice versa For instance an image with black bands on a white background is converted into an image with white bands on a black background by simply clicking the Reverse button Clicking the button a second time returns the image to its original form Original Image Reversed Image Note Reversing an image changes the way it is displayed on the screen but does not change the quantitative data For example the bands in the above gel have the same density regardless of whether the gel is displayed as white bands on a black background or black bands on a light background For information on reversing pixel values see Invert option under Setup Preferences see Chapter 4 for more info about this option 39 ImageQuant RT ECL Capture User Manual 2 1 The Grid Button Grid The Grid button provides an on screen grid after image acquisition to check for proper sample alignment C Program Files Part11FC Images Sample tif 640x480x8 40 ImageQuant RT ECL Capture User Manual 2 1 8 Making Linear Log ordua Adjustments Original image of film with default BWG settings rer ee At pre reer Gomme Urea Log Ena Image of film with linear Contrast Adjustments selected Linear provides minimum and maximum adjustment tools from 0 to 100 Linear stretches the grayscale range of the displayed image to the maximum system dynamic range of 0 65 535 grayscales Image of fil
102. to white and values in between to shades of orange This palette may be useful when printing an image of an EtBr stained gel onto a color printer 84 ImageQuant RT ECL Capture User Manual 4 7 Image Filters ImageQuant Capture includes a variety of enhancement filters that can improve the appearance of an image Some filters sharpen detail others smooth and reduce random noise Still others help visualize edges and separate closely spaced bands or objects Depending upon the unique characteristics of an image the results of each filtering operation vary Assess the characteristics of the image and then select the filter designed to minimize its imperfections Tool Box errr errr rere rere rere rere re rere rrrre rere re terrier iri rrr rrr eer errr reer rere rrrr errr etree rrr Enhancement Tools oom Rotate Flip Histograrn Enhance Annotate False Color Filters FrameGrabber Sharpen Moise Despeckle z Lowy High J h aaas E Filter Tools with 3 D contour selected When an image is filtered the original image information is replaced with the results of the filtering operation As a result the original image information is altered To avoid losing the original image save it as an original TIFF file before applying a filter When the FILTERS button is selected in the Tool Box a pop up dialog appears If the desired filter is not shown choose MORE to display more filters One or many filters can be
103. tor 1 0 Binning ixl ain Factor 1 0 Lights TUY Filter EtBr Colorimetric Display Black D Display White 65535 Display Gamma 0 55 Display Inverted ves Image Info dialog box 59 ImageQuant RT ECL Capture User Manual 3 4 The Setup Menu This menu customizes the system settings by allowing users to save default parameter preferences and customize the software settings Setup Overlay Utilitie Load Defaults Save Defaults Adjust input level Security Print Info d Print Mode d Prink Date Preferences Setup pull down menu 3 4 1 Default Parameters Default parameter files eliminate the need to readjust the system settings each time the program is used and can be especially useful if more than one person works with the program The default file used by ImageQuant Capture when it initially loads is called TQcapture def The system setting defaults are Exposure Time 8 milli sec Black Level 0 White Level up to 255 4095 or 65 535 depending on the system type Gamma Setting 0 55 REVERSE off ARRAY 96 circles 12 x 8 1D MULTI 8 lanes While these parameters can be changed we do not recommend changing TQcapture def Instead we recommend that each user set up an individual default file s reflecting their preferences 60 ImageQuant RT ECL Capture User Manual 3 4 2 Save Defaults Every time a default file is saved all of the settings for the parameters listed above are saved These
104. tton is used to activate functions and otherwise make selections when using the software In some cases the right mouse button is used to recall or reactivate the function that was most recently assigned to the left button 1 1 2 About This Manual Throughout this manual different fonts are used to indicate certain things This font indicates the name of a button a menu or a function found in a menu This font indicates an entry that is typed Letters of words found between lt gt refer to keys on the keyboard NOTE Notes will be used throughout the manual to inform on interesting points and provide useful hints CAUTION Cautions will be used to inform the reader of action that may have the potential to either harm the instrumentation or affect the quality of the data WARNING Warnings are used to provide special notice of actions that have the potential to cause harm to the operator 1 1 3 Starting ImageQuant Capture To start ImageQuant Capture from Windows double click the ImageQuant RT ECL Capture icon on the windows desktop Touant Capture RT eae ImageQuant RT ECL Capture User Manual 1 2 ImageQuant Imaging System Setup 1 2 1 System Components The ImageQuant System includes High performance high resolution CCD camera with lens adaptor USB camera interface cable Camera power supply and power cord Zoom lens fast lens and interference filter Computer with keyboard mouse and SVGA monitor Window

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