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Chapter 1 - GE Healthcare Life Sciences

1. Sensitivity Resolution I Chemi Display Normal High whi MV Show Saturation Medium Medium Auto Contrast High Medium Reverse High Low FrameGrabber Super Sensitivity ric m Zoom AOI L 1 0 Chemiluminescence Wei OG Ce De th he 15x a gt m Extended OD Ext OD Off US SO i Oe ale 1 E i 1 Door CLOSED 29 ImageQuant 400 User Manual 1 6 9 Acquiring a Standard Image Without Extended OD ImageQuant Capture is capable of acquiring images in standard mode single image exposure and in Extended OD mode To acquire a standard image slide the Extended OD slider bar to the far left until the display shows Extended OD Off Clicking the Acquire button with Extended OD acquisition turned off will acquire a standard image according to the exposure time set by the user or by Auto Exposure 1f chosen 1 6 10 Acquire and Transfer the Image Once the appropriate image is displayed on the screen click on the ACQUIRE button to capture and display the image in ImageQuant Capture 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 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
2. 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 single 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 b
3. 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 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 atthe unscrewing the knob which screw the knob back into the back of the cabinet This tray will allow the tray to swing threaded hole underneath the is secured by a detachable down freely light tray where it will serve as Knob a handle ImageQuant 400 User Manual Attaching the Hood Take the following steps to attach the cabinet hood and connect the hood fan l 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 From within the cabinet attach the hood fan s power cord to the temperature board socket on the top right side of the cabinet Fasten the hood to the cabinet using M4x16 socket head screws The cabinet hood is positioned Once the hood is positioned over over the top of the cabinet the top of the cabinet plug the fan cable into the socket on the top right side of cabinet ImageQuant 400 User Manual 1 2 4 Cable Connections The Cable ends and the ports into which they are inserted are keyed or unique f
4. ImageQuant 400 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 iP ImageQuant 400 Capture i iojxj i Edt mage Setup Cherlay tities View Help Files Part 1 Lf C Inages coomassic 16 1 128i 12400 NUPAGE 10 GEL W MOPS BUFFER Tr EEE VEX PH 800 556 6839 FAX 619 45266 f Rotate Fip Histogram Enhance I Fale Colt Fies FramaGrabbes a es lo el fjcoton ONEEN PEN WIDTH JTEKT STYLE PEN STYLE TEXT ORIENT Ck YY 662 12 Zoom 50 Intensity 53376 ij Ready to Crop or Copy 55 ImageQuant 400 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 Captu
5. 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 62 ImageQuant 400 User Manual 3 4 4 Security When the system 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 Bla
6. 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 process of image acquisition for each frame of the image When the process is complete the Camera Setup and Preview window will disappear and the TOOL BOX will automatically switch to the FrameGrabber tab This will allow you to play back the sequence save or load the sequence or record a new sequence 93 ImageQuant 400 User Manual Too ED oom Rotate Flip Histogram Enhance Annotate False Color Filters FrameGrabber ulame u gt a ail REY REC STOP PAUSEPLAY FWO OEL OPEN SAVE Once all images have been captured the FrameGrabber tab will be displayed in the Tool Box The remaining buttons will perform the following tasks REC Opens the Camera Setup and Preview window 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 Moves forward in 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 FUN
7. fa 10black ovr fa 24black ovr a 1 Owhite ovr ja 24white ovr ja 1 2black ovr fa Hindiii ovr File name kperstr Save as type Overlay Files ovr 7 Cancel E p m i 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 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 67 ImageQuant 400 User Manual 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 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 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 o
8. 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 quantitative 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 36 ImageQuant 400 User Manual
9. 401 Norway T 815 65 555 F 815 65 666 Portugal T 21 417 70 35 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 Technical Support ts usa ge com Customer Service cS uS ge com For Service Agreements labcrew us ge com CE Conformity The following Gel Imaging Digital Systems Models ImageQuant 100 ImageQuant 300 ImageQuant 400 ImageQuant 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 discontinue the product de
10. Grid Thumbnail Contrast Adjustment Window Tool Bar Window 70 ImageQuant 400 User Manual emma erases Enhancement Tools smn oom Rotate Flip on Enhance Annotate False Color Filters Frame Capture COLOR _ LIHE ENDS _ PEN WIDTH TEXT STYLE _ PEN STYLE TEXT ORIENT ajea 5 ol sioa E MN 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 be 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 Zoom 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 d
11. 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 96 ImageQuant 400 User Manual Chapter 5 Analyzing Images Once you have acquired an 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 ToolBar E Acquire s Overlay 1D This is a gel or TLC plate analysis module 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 1s used If the acquired image has been saved the selected module is immediately accessed when the appropriate button 1s selected If the image hasn t been saved there will be a prompt to save i
12. 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 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 103
13. Setup Reflective UM m Display Sensitivity Resolution I Chemi Display Normal High whe MV Show Saturation Medium Medium Auto Contrast High Medium Reverse Taye Fs eee Super Sensitivity j g a Zoom AOI Peer 1 0 Chemiluminescence 1 Melle i i T CERT Fy a D Extended OD Ext OD Off Si et ie Ie S E 1 Door CLOSED This window contains an Extended OD slider bar located to the left 26 ImageQuant 400 User Manual Acquiring in Extended OD Acquisition Mode The user may acquire a Extended OD image with a factor from 2 to 500 Choose the Extended OD factor to use by sliding the Extended OD slider bar to the right Extended OD acquisition is activated whenever the Extended OD slider is in any position other than Off As the slider bar is moved to the right the Extended OD Factor will increase and be displayed ey Extended OD Factor 8 0 1 Time x 544 In addition to the Extended OD Factor the software also shows a time estimate for the total acquisition time This time estimate is displayed as Time x value where Time is the initial exposure time see below and the value is the multiplier to achieve the chosen Extended OD Factor In the example above the Factor 1s set to 8 The total acquisition time will be 544 times the initial exposure time If the initial exposure time is 10 milliseconds t
14. Super Sensitivity FrameGrabber button in the camera setup oom Al 1 0 I 1 1 I I 1 I I I R T acquisition screen Extended OD Ext OD Off FrameGrabber 31 ImageQuant 400 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 Image frames images you want for the sequence Setup gt Preview There 1s a maximum of 50 frames images and a minimum of 1 frame that can be captured with Noise Reduction each sequence The FRAME selection is used for setting up the ee l E a a 4 Seconds a o o o conditions for each frame image For example if three 3 images are to be captured you would ia 104 choose FRAME 1 and setup all of the desired lighting and filter requirements You can then T Auto Expose __Auto Expose Setup 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 ne High Mediurn all lighting is off and the filter wheel is positioned F ace igh Lowy for the chemiluminescence position for all frames Thus the only variable that is changing FrameGrabber _ Super Se
15. 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 51 ImageQuant 400 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 will 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 o
16. 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 LUE Util i il Original Image 3 D Image Level 5 amp lower right 3 D Image Level 5 amp upper shadow middle shadow Sharpen Noise resect Gane Sharpen Noise resect M sane Sharpen Noise EN disa 2 s 4 s o r e 9 1 pes alza slzlsls H Tee tfallefe sizjejoj t gt _ wore Original filter TE Lower right shadow Upper middle SNAJA 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
17. 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 with 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 t
18. 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 OKR 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 DAD2 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 99 ImageQuant 400 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
19. 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 Passyvord AddRemove Users OK Cancel 101 ImageQuant 400 User Manual Each time a new name is used it is added to the master user list Since this list requires a password in case the system is later changed to a closed password protected system ImageQuant 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 M User Logging Active Closed System Auto Logout 5 minutes 10 minutes 20 minutes Change Master Passyword AddRemove Users OK Cancel 102 ImageQuant 400 User Manual Option 4 Closed System with User Name Log In and
20. image only To save the entire sequence save Select the save icon on the FrameGrabber tab in the tool box 1 6 13 Loading Saving an Entire Sequence 8 BR You can load or save an entire sequence by using the load and save icons in apen gave 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 33 ImageQuant 400 User Manual dialog box will appear prompting you to select the location and name for the sequence that you would like to save the images as 1 6 14 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 Ed oj xi Cabinet Controls aj m Frame Option 1 gt Disconnected Normal Sequence Stack Frames Frame a Pf ransillumination I Noise Reduction l zi r Delay rs w o Is White Exposure Time Ed jE gt F gt Copy to Next whe a a Minates Seconds iiliSecs o lif o l iw 104 iv Copy to End af Auto Expose Auto Expose Setup Reflective Display Sensitivity Resolution hemi Displ Bon Normal High MV Show Saturation Medium Medium Auto Contrast High Medium i Reverse enue arm Super Sensitivity DE sa Load Setup gt Zoom ROI
21. 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 black pixel values At an edge 89 ImageQuant 400 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 resu
22. insert the camera fastening knob with washer from the other side of the post then turn the knob clockwise until the camera is secured at the top end of the post Important Note The camera must be fastened at the extreme top end of the upper post to prevent damage to the filter wheel when the lens is fully extended see illustration below 4 Place the foam rubber camera stand gasket in the cabinet over the location where the camera stand will be placed 5 Carefully lower 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 When lowering the camera stand assembly into the cabinet make sure that the lens does not damage the filter wheel when it pokes through the aperture 6 Fasten the camera post to the top shelf of the cabinet using the screws that are provided Camera stand camera and View showing correct Completed camera camera stand fastening knob laid out on table location of camera At top assembly which then gets placed into end of post the cabinet and fastened down ImageQuant 400 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 l At the bottom of the cabinet there is an extendable metal tray that is held in place by a single screw this
23. 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 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 85 ImageQuant 400 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 details and 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 eee rte rrr rere etree rere rer reer rre errr errr rier r ttre ree
24. r m UV o x White Exposure Time Ei Ea x gt gt Copy to Next Whe ac o jmil o l 10 xj Coppte endl eg ff Auto Expose Auto Expose Setup ux Reflective m Display Sensitivity Resolution I Chemi Display Normal High M Show Saturation Medium Medium Auto Contrast High Medium Reverse High Low f i Super Sensitivity 7 i Fes of ppe Mowe Se Filter Wheel Position Cancel Zoom ROI sss Load Setup and L p Seset Save Setup buttons ert Wey Uh Ge De TD Taye Door CLOSED 4 9 1 Frame Stacking At the top of the Camera Setup and Preview 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 2 3 4 5 sec 95 ImageQuant 400 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
25. the appropriate checkbox to show text with or without a background An opaque background is 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 8 COLOR LINE ENDS R TRANSPARENT BACK PEN WIDTH 2 TEXT STYLE Set Forit O is 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 Font O OOOO 2 x Font Font style Size arial R equilar fi z Cancel Arial Black Italic O Arial Narrow Bold Aral Rounded MT Bok Bold Italic O Arial Unicode MS Baskerville Old Face Batang Effects Sample Strikeout M Underline Color Lo white Script wester Font Selection Window Note To choose Greek symbols such as a B A 1 and 0 choose the Symbol font abcdefghijklmnopqrstuvwxyz apyoedvOyntgnKrAUvVOTOPGOtv DoaAEWC 79 ImageQuant 400 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 e N olo x a 8 cotor LINE ENDS FJ CLOCKWISE ANGLE PEN WIDTH TEXT STYLE PJ aNTICLOCKWISE fo gt FEN STYLE TEXT O
26. 630nm Hoechst Blue 460nm Note Each Filter has an approximate bandwidth of 40nm to allow for use with other fluorescence stains as they are developed 22 ImageQuant 400 User Manual 1 6 4 Sensitivity Resolution Settings The ImageQuant System 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 to generate 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 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 4X High Sensitivity Medium Resolution Recommend for Chemiluminescence This mode captures the image performing a 2x2 pixel bin with a gain setting of 3 to further decrease exposure times and still maintain a publication quality 1024x1024 pixel resolution image The High Medium sensitivity selection will decrease the exposure time needed by about 2X over the Medium Medium selection High Sensitivity L
27. AT ses escteicceeadcsaeaeainesusaacseaa ieee sasaki 44 Ds A VON ey ON asetccstae cheba aemaceee E sian seeuosaah ones wee se ean ore AA 47 2 ALS PSA aen a T a a ea a vane Guha ede 47 Chapter 3 Pull Doyn Men s oxscesiiesavust soxetesscasedenenantoasesadesewonastbadevacedsetaaaesedebanedsesanaesasevasersetance 48 JEPET Ue MNU seati O a 48 JALAT OD Mess a errnet ements n erates entre nner eee 49 Shoe Wile OV Cidy cae teal asada teil h ie tapct E saan ele tea adn ts 50 JES NG LOSE inks Societies ee nastnnita easteiccacactsc A EE eta duets teed 50 AAEE S IVC AUN AVG A Sie sin wets a E E E E eiacoastntaniones 51 AES PNET SE UD ean ee nee 53 PEOP a EAE E OTE E A E 54 TA E O E EEE E E A A ENEA ETE E E EEE A EET E E E 54 Diodes Oe ERSE SAVE UDC O Merria E O O EO 54 Fene 2 dE MeN seine E mer et On nae EN I ae ate eT 55 PIM FRESS andiC lEI pCR eee ma N EE BRR SUD a ERA ee ee 56 Jo Tne MACS Memrise E E S 57 FOL EQUI Z eenen e E A a FILAMENT te sach axsdeedess casey A axsaeneas maaan exeaontaeies a7 Io CONV CUS LOM eet speedy a aad santa heath alec aad eatebee taney alee aad entabees 58 IIA PIA Feld C a NDA oa RR ee eC E N eR oe ee 59 Jo limage RES ZO nee A a E a En acon EAEE TOE 60 DOs MINA M Onera A N 60 zA WS STU Py VS IE ana a E N E NER 6l SAT Dele PI EES e ea eee Oa 6l IA DAVE DETU eraa A tan A a 62 IAS Load Deilt onie ernn E E OS 62 IAA SCUTT oar n e E E E E 63 FES Pant I Oea eee 63 FOP TL OG a A TTE E or ausaaeeteasdeorede 64 IATP DAE eae A
28. CTION 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 1s 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 E 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 4 8 2 Loading Saving an Entire Sequence 6 BR You can load or save an entire sequence by using the load and save icons in 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 94 ImageQuant 400 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 7 zj o x Cabinet Controls Total frames 1 Disconnected Frame Option Ej Normal Sequence Stack Frames Frame af P l l 1 E Tr nsillumination Noise Reduction
29. Chemiluminescence Save Setup Load Setup and ee gg ee Save Setup buttons gt Door CLOSED 1 6 15 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 2 3 4 5 sec 34 ImageQuant 400 User Manual 1 6 16 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 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 35 ImageQuant 400 User Manual Chapter
30. EE OT EN 64 Or E Ce SEEE O E E E A E as 64 33 LING Ova Me iarere a aE A A AEE A 67 Jok SaV naan OVT la V e a a a a 67 392 Loading al OVerla y aree E A 68 50rd MS UNGES MENU oireina aea E T E E T 69 FOLEI ON Poeren n E NE E taeda led cade ad eee toes 69 IGL NOEP IU err E A EEE TE E AAA 69 IE TACEN ENOM aaea T AR 70 Dol ZOOMIE UM CUOMS aa a a a a a eee aes 71 IL OROW ANOA ON S oiai n T T O E TE 71 1S Loe olel em M e iea a E Rae een Ir eta 12 BS Oi ATC IN ONG aaaea T AE E AS TS 72 De NO E EEE A EE aie cereal GE A PRTA E E ear ET T2 Chapter 4 Fhe Tool BOX ywsssssceesiesescieciasscustesedsssiaaseenscedscasasiassdeegedsaseatetubavscedsasiasssecsnsdelsasestaseetes 73 dek TBE OO TMA LOO Perera O S N N 73 4 2 TNE ROME FIO TOO rriena ii E E N TTE S 74 Ao EAS LOO T a ee clase daeu sain amie caare nace ae cana wees eee 75 A A Amoma MATIC CMA C IN ainina aT E ETA 76 AyD ATMO CAMO S aa A owed en eae eee en Ti ADA OBI CCU AU OU Ue irr n aE A TO val AD 2 MIMO TAO CO OFS erinra a E A OEE EA E 71 ASS Ene MMIC KIE SSi rasa heetahusaaecaue ta cea aula soaeunte aauanewiaatetoaseaaueesoaeaee 78 BD A AIG VY DCS e tan cted ial ect seen a ded ist lais ine taetaa 78 ASS ATrOws ands Stal One LANCS conien e E E E E 78 A D0 TEX BACK Or OU lI OU 555655 slat ca ied a E daniel tal ouaiaubadieetedeacesates 79 MIT LR ANE ES aE E AET E TE E ES E AE 80 AaS No Drawno TOO Beien A ANA 80 AD OT Ne Gite TORni a a a 82 REPS ONG EATE E ace eect alte tative irate taal cei ae
31. GE Healthcare ImageQuant 400 Capture User Manual www amershambiosciences com Waste Electrical and Electronic Equipment WEEE ENG H FRA F GER a 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 rappresentante
32. I Extended OD Est OD Off 20 Auto Exposure Setup options Click this button to view and Edit Auto Exposure settings ImageQuant 400 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 1 gin 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 In Preview mode the software will continue exposing 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 in Preview mode tt does not start the calculation over from scratch S
33. Manual 3 Turn the power on to the camera by pushing the switch to the on position Note The camera should be turned off at the end of every day with all of the equipment by pushing the switch into the off position 0 or by turning off the power strip 1 3 4 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 prong power cable on the back of the printer and into the power 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 12 ImageQuant 400 User Manual 1 3 5 Connecting the Cabinet The cabinet connects to the back of the computer via an RS232 cable Connector on the back of the cabinet Connector on the back of the computer S e i T i Cas a T E E i Sr ts Ports on the back of the cabinet and computer respectively Power on the
34. RIENT Text Orientation 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 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 typi
35. aboratory Practices files glpl Paint Brush files pcx Color Targa files tga Headerless files pic JPEG format ja Mac Tiff Files rac 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 screen To dismiss the dialog box without loading an image click on the Cancel button 49 ImageQuant 400 User Manual 3 1 2 File Overlay D 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 if you have a saved gra
36. aches the desired size o 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 Release the left mouse button when the circle reaches the desired size 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 81 ImageQuant 400 User Manual Annotated image showing freehand drawings lines with various characteristics 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
37. ake 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 38 ImageQuant 400 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 39 ImageQuant 400 User Manual 2 1 6 The Reverse Button The Reverse button inverts the gray levels of the displayed image converting a positive 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 i
38. ance Exposure Time E Ey a i E Annotate False Color Filters FrameGrabber Houra Hours a Minutes Minutes Seconds Seconds MiliSecs MiliSecs al l 0 0 h 104 T Auto Enpose _ Auto Expose Setup _ a ale f gt a ai 2l REY REC STOP PAUSEPLAY FWD DEL OPEN SAVE Display Chemi Display if Show Saturation Auto Contrast Reverse FrameGrabber Setup in the Tool Box Sensitivity Aesalution Normal High Medium Medium High Medium High Low Super Sensitryvit FrameGrabber button in the camera setup acquisition screen FrameGrabber oom AOI Extended OD Est OD Off 92 ImageQuant 400 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 is 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 FRAME 2 and repeat the above Or you can click on COPY TO NEXT to help speed up the Frame Option a Normal S
39. 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 Italy T 02 27322 1 F 02 27302 212 Japan T 81 3 5331 9336 F 81 3 5331 9370 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
40. cabinet after the Windows operating system has fully loaded The ImageQuant Imaging System is now ready for use 1 3 6 Starting ImageQuant Capture To start ImageQuant Capture from Windows double click the ImageQuant icon on the windows desktop ImageQuant 400 13 ImageQuant 400 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 ijj After the computer has booted up completely to the Windows Ed desktop turn on the power to the cabinet ImageQuant Activate ImageQuant Capture by double clicking on the desktop 2E icon In the Tool Bar select the Acquire icon to activate the image a Acquire acquisition software features has Setup In the Image Acquisition window select the as button Open the door to the cabinet and position your sample on the preferred illumination source 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
41. ck 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 Utilities View Help Load Defaults Save Defaults Adjust input level Security ie Print Mode t ow Bottom Print Date GFF 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 63 ImageQuant 400 User Manual 3 4 6 Print Mode ImageQuant Capture provides custom printing options File Edit Image Setup Overlay Ukilities wiew Help Load Defaults Save Defaults Adjust input level Security Prink Info Print Mode a 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 sele
42. ction 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 application 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 64 ImageQuant 400 User Manual There are four tabs in the Preferences 1 General Configure prompts and file saving opening formats 2 Image Acquire Inverts the image seen by the camera and adjusts the ROI values Preferences Normal High Medium f Medium High Lowy Super sensitivity 65 ImageQuant 400 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 7 8 1 Filters Selection Jeter Colorimetric ore to 4 Auto Enhancements Used to customize the Auto Enhance Levels located in the tool box General Image Acquire Cabinet Settings Auto Enhancements Enhance Tonal 4djustment selection Black Level 0 00 white Level 0 35 Gamma Lewel 1 00 Device Corrected Negative V Enable Auto Levels Negative Change ca
43. d 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 ee Enhancement Tools Annotate False Color Filters FrameGrabber Histogram Enhance Zoom Rotate Flip ROTATE o UNDO FLIP 90 H 90 Ki al 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 Also 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 l sia ie Rotate Flip box at 12 degrees Rotate Flip box at 12 degrees Rotate Flip box at 12 degrees and vertical flip button pressed 74 ImageQuant 400 User Manual 4 3 Histogram The histogram is a graphical display of the proportion of pixels assigned to each o
44. d 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 a 50 millisecond exposure is sufficient with final adjustments of the aperture for best image quality e For UV fluorescence applications usually a 250 millisecond to 4 seconds is sufficient and the aperture should be adjusted fully open The show saturation 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 ACQUIRE 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 24 ImageQuant 400 User Manual 1 6 6 Optimizing the Gray Scale for Saturation and Co
45. 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 is 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 25 ImageQuant 400 User Manual 1 6 7 Extended OD Acquisition Feature Extended OD is a process that delivers Full Dynamic Range Performance for digital imaging applications Extended OD acquisition accomplishes this by extending the range of different light or brightness levels from the sample that can be viewed as a single image beyond that of a standard image Extended OD acquisition can produce an image that represents more than 100 fold extension of the brightness range In the TOOLBAR window click on the Camera Acquire _ icon Acquire The Camera Setup and Preview window appears Camera Setup amp Preview i 2 215 xi Image Cabinet Controls l Noise Reduction r UV White Exposure Time i ak Ea t gt gt ers es oe o lw o r oO awl 104 v I Auto Expose Auto Expose
46. 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 84 ImageQuant 400 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 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 0 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
47. ecomes progressively darker This is because all pixels at the specified gray level and lower are shown on the screen as black pixels Ifthe slider is set to 0 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 37 ImageQuant 400 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 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 m
48. elweutanay 8 1 3 Computer Camera and Peripheral Installation ce eeccccccccceceeseeeeeeecceeeeeaaeeeees 9 1 3 1 Setting up the Power Strip Surge Protector c cc ceesssseessseeeececeeeeeeeeeeeeeaaaeas 9 1 3 2 Setting up the Computer Monitor Mouse and Keyboard eeeeees 10 Ezo Gamera Msala hoM oe secs occ 9 ssen a a E emus 11 1A COMME CUMS MEPAMG arrarena n a E saw ltadeiweweasendiae oa aserase 12 1 3 5 Connecting the C ait Eesnime E 13 L3 OStarmas Imat c Ouant Cape eano a a Ta R 13 1 4 ImageQuant System Quick Guide ou eeccccccccccccceeeeeeeeeeceeseseeeseeeeeeeeeeeeeeeeeeeaaaaaas 14 TAC aDIMe EC ONTO S ernen a ala liaavasouiaonesoees 16 Es System Mmao N snese O 17 LO Acg imne an Mate enana e E O EE 18 126 A OGTISII Oi AI Mma e a N 19 1 6 2 Auto Exposure and Auto Exposure Setup ccccccccsssssseececceeeeeeasseeeeeeeeeeeas 20 1 6 3 Cabinet Controls Activating the Light Source and Selecting a Filter 22 16 4 Sensitivity Resolution SCMINGS serine a EE E ee keane 23 1 6 5 Use Preview to Set Exposure TIME menreeiinna n a in 24 1 6 6 Optimizing the Gray Scale for Saturation and Contrast Displays 5 25 16 7 Extended OD ACquisilion F Catuire sicceincntccinnss iei e EE TEE 26 1 6 8 Using the Zoom ROT TOO scesencscadcscedeactcnssdcdauwouliseandesstonsededesuiedecdedeousdaedvdaqiuebes 29 1 6 9 Acquiring a Standard Image Without Extended OD eee 30 1 6 10 Acquire and Transfer the Ima
49. ently 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 Capture icon on the windows desktop ImageQuant 400 ImageQuant 400 User Manual 1 2 ImageQuant Imaging System Setup 1 2 1 System Components The ImageQuant System includes High performance high resolution CCD camera Zoom lens Computer with keyboard mouse and monitor Windows operating system preinstalled ImageQuant Capture image processing software preinstalled and calibrated with computer and hardware system ImageQuant light cabinet with UV transilluminator and white light fold down transilluminator and interference filter optional wide angle fast lens optional epi illuminating UV lights op
50. equence Stack Frames Fame Noise Reduction Delay ma 0 E Copy to Nest Copy to End Ripe Pee Exposure Time Hours EE EN Minutes 4 Seconds Seconds a MiliSecs MiliSecs al 0 104 Auto Expose Setup Auto Expose Display Chemi Display e Show Saturation Auto Contrast Reverse Sensitivity A esolution Normal High Medium Mediurni High Medium High Low Go Super Sensitivity setup process COPY TO NEXT copies all settings from the previous frame to the current frame Load Setup oom AOI 1 0 Save Setup wt I I I I I 1 I 1 I Fat 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 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 is consistent for the entire sequence then once you set up the first frame you can select the COPY TO END selection to
51. esentations 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 Height li O24 Cancel iY 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 width 1136 Image Height ggz 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 af Modified no Exposure Time OhOmOs 18ms Sensitivity Factor 1 0 Binning ixl Gain 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 60 ImageQuant 400 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 Uilitie Load Defaults Save Defaults Adjust input level Security Print Info Print Mode d Print Date Preferences Setup pull down menu 3 4 1 Default Parameters Default
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53. ettes 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 ere rrr rere reer errr reer rier errr terrier rere errr rrr etree treet eter irre reir erie rrr Enhancement Tools oom Rotate Flip Histograrn Enhance Annotate False Color Filters Frame rabber GRAY PALETTE SATURATION MEST 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 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
54. f the 4 095 gray levels This tool is found in the Tool Box Tool Box errr rte errr rrr rere rer rere rire rrr rier errr errrrre rrr ttre r irr r rir r rer rrr e errr errr e errr etree rrt errr rrr rrr Enhancement Tools Annotate False Color Filters FrameGrabber Zoom Rotate Flip Histogram Enhance Ed Linear Scale Log Scale Jix 4x lix 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 white 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 Lineal Scale Log Scale 1x de eee 1 Eine Eide 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 h
55. for the set It is 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 57 ImageQuant 400 User Manual most common application for quotient 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 publication
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57. ghborhood is different from the neighborhood average either greater or lesser by a threshold a multiple of the standard deviation it 1s 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 outliers 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 88 ImageQuant 400 User Manual In addition 3 D
58. haat dee ad ilu t adalat 95 4 9 2 Capturing a Color Image Using the FrameGrabber Function 0066 96 Chapter 5S Analyzing IMA OS a doucsasdescutecsceovsaaiavussacsteeis 97 Appendix A Opening ImageQuant Capture Files in Other Software Programs 98 Appendix B Security Fedt ES sissisotaa a 100 ImageQuant 400 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 ImageQuant Capture Annotations are superimposed on the image when a hard copy 1s printed and can either be saved as a template file or as part of the image All of these features a
59. he total acquisition time for this acquisition will be 10 milliseconds x 544 5440 milliseconds or 5 44 seconds Initial Exposure Time for Extended OD Acquisition Whenever Extended OD 1s activated the acquisition interface disables the exposure time input fields and switches to Auto Exposure mode The initial exposure time used for Extended OD is determined by a proprietary Auto Exposure algorithm Clicking the Preview button in Extended OD mode will calculate and display the initial exposure time Clicking the Acquire button in Extended OD mode will start the Extended OD acquisition process which first determines the initial exposure value and then performs the Extended OD acquisition process automatically Acquiring Extended OD Images Slide the Extended OD slider bar to the desired Extended OD factor Click the Acquire button When the process is complete the resultant Extended OD image will be displayed in the ImageQuant Capture main window 27 ImageQuant 400 User Manual Canceling Stopping the Extended OD Acquisition Process During the Extended OD Acquisition you may want to cancel the process or stop the process and save the resultant image Disconnected Click the Cancel Button to cancel the process and return to the acquisition window Otherwise select the Stop and Save button The resultant image will be displayed as it appears at that point in the acquisition process Acquiring image Cored stoend
60. his 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 45 ImageQuant 400 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 46 ImageQuant 400 User Manual 2 3 Tool Box The Tool Box window contains an intuitive interface for performing all image enhancement functions Toots oom Rotate Flip Histogram Enhance Annotate False Color Filters FrameGrabber 4 dale n gt tai REY REC STOP PAUSEPLAY FWO DOEL OPEN SAVE Tool Box Display Window It contains 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 positio
61. ht Auto Expose l Select the Momal High resolution setting 14 h ImageQuant 400 User Manual Once the image in the preview does not contain any saturation red false color palette for white bands green for dark bands select Sa 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 oe SI9 ho 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 Mf Show Saturation Select Mf Chemi Display Select hf Auto Expose Select the Supe Sensitivity resolution setting 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 is still present in the image Select Medium Medium resolution Capture Select eee 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
62. ibration 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 the Preview option 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 1f the UV transilluminator was used place the paper onto the UV transilluminator if 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 transilluminator 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 59 ImageQuant 400 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 pr
63. illumination 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 is currently being used 1 No filter use for colorimetric gels and film 2 Ethidium Bromide 3 5 Optional filters 16 ImageQuant 400 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 ImageQuant Capture ImageQuant CAPTURE Version 1 0 0 serial 0 GE Healthcare Copyright 1993 2005 All Rights Reserved 4 GE Healthcare ABOUT Pop Up Dialog x 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 clic
64. imilarly if a different sensitivity binning mode is selected after the exposure calculation has been calculated 1t will do a quick conversion and does not have to start the calculation from scratch 21 ImageQuant 400 User Manual 1 6 3 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 Transillumination UV Reflective White Reflective UV optional No Light Selected For protein gels autorads film plates flasks For fluorescent gels such EtBr SYPRO Red etc For colorimetric blots and membranes For SYBR green TLC plates and Chemifluorescence For chemiluminescence You can also select your FILTER to correspond with your sample staining The options include Filter Position 1 Filter Position 2 Filter Position 3 optional Filter Position 4 optional Filter Position 5 optional optional Chemiluminescence samples no filter Ethidium Bromide colorimetric stains film SYPRO Orange 595nm SYBR green 557nm Fluorescein SYBR Gold 520nm SYPRO Red Texas Red
65. 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 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 2 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 gm o Close the cabinet door Choose the appropriate optical 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 Kf Show Saturation Select
66. 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 With the tray fully extended re insert the screw This ensures that you will have the screw if you ever have to ship the unit and also prevent light coming through the threaded hole that seats the screw 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 Plug the power cord at the bottom of the cabinet into the socket at the back of the UV transilluminator 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 gn the tray making sure that Note that there is a screw that original position This will its rubber feet are firmly holds the tray in place for prevent light from entering into seated in the holes on the shipping Remove this screw the cabinet through the tray and then plug the to allow the tray to slide freely threaded hole cabinet s power cord into the back of the transilluminator ImageQuant 400 User Manual Unfastening the Fold Down White Light Tray The cabinet contains a fold down light tray that 1s locked for shipping see illustration below Take the following steps to unfasten the light tray
67. isplay 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 71 ImageQuant 400 User Manual 3 8 The Help Menu Help On Line Mote Abot Help pull down menu 3 8 1 On Line Note On line help is available in the ON LINE NOTE section of the help menu Common tips are included for the image enhancement tools detailed in Chapter 4 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 software version number Use this information when contacting technical support upgrading software etc ImageQuant Capture x ImageQuant CAPTURE Version 1 0 0 serial 0 GE Healthcare Copyright 21995 2005 All Rights Reserved GE Healthcare ImageQuant Capture About Help Dialog Box To close the box click on the OK button 72 ImageQuant 400 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 Colo
68. istogram 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 15 ImageQuant 400 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 darker Each increasing button click will lighten up the image until button 9 1s pressed which will make the image the lightest possible Tool Box Ce Enhancement Tools Annotate False Color Filters FrameGrabber Zoom Aotate Flip Histogram Enhance Foto Enhance Level EEEE 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 Hpo0000ORE Jodee Pees Original Auto Enhance Toolbox Auto Enhance Toolbox with level 2 Auto Enhance Toolb
69. k on the OK button 17 ImageQuant 400 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 REVIEW 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 FrameGrabber setup is controlled and activated from this window This window will open with the Setup control mode activated left button which provides a near real time readout to allow for easy sample positioning and optics adjustments Camera Setup amp Preview 2 101 x Cabinet Controls Image M Noise Reduction Disconnected Exposure Time i Ea Ea x gt 0 x 0 x 0 104 x I Auto Expose Auto Expose Setup Display Sensitivity Resolution Chemi Di M oisi Normal High M Show Saturation Medium Medium Auto Contrast High Medium Reverse eman eS FrameGrabber Super Sensitivity Filter Wheel Position Chemiluminescence mm Door CLOSED m Zoom AOI We yo ies tes 15x Extended OD Ext OD Off 18 ImageQuant 400 User Manual 1 6 1 Focusing on an Image Focusing on an image in the ImageQuant Capture is eas
70. k 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 S 1Qcapture def Notepad a i l0 x Eile Edit Format Help File Type AppDets Version 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 ExposureCompLimits 4 0 4 0 Notepad Display Window 69 ImageQuant 400 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 w Contrast Window wW Tool Box Default tools position zoom In Ctrl zoom Cut Ctrl zoom 1x Fit In Screen wv Show Annotation View pull down menu Four main tools exist within ImageQuant Capture Contrast Adjustment Tool Bar Status Bar and Tool Box x mi j Acquire Overlay Ch AAR Ch oom Out oor Iri Contrast Adjustment Gamma Linear Log Equal Low o j gt i Hich J gt l Gamma 0 55 gt Reverse Auto Contrast
71. lts of this filtering process 4 7 10 Custom Filter This function also allows the user to customize filters Using the weighting factors of the other filters as a frame of reference it s possible to 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 90 ImageQuant 400 User Manual 4 7 12 Examples of Filter Results SS e ss Original Image SSS ee ee ee 3 D Contour Level 5 Horizontal Edge Vertical Edge 91 ImageQuant 400 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 the Tool Box FrameGrabber setup 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 the acquisition screen Image ae N m C srh ra Setup Freview Laoture Noise Reduction Tool Box oo Enhancement Tools Pr rere rrrr rr rrrrirrrrrrrtirrrttriritr irri rttr ttre try Zoom SHE Flip Histogram Enh
72. 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 82 ImageQuant 400 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 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 B 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 83 ImageQuant 400 User Manual 4 6 False Color These tools consist of eleven pre defined color pal
73. n the image zoom factor and the grayscale intensity at the mouse cursor x y position p 89 2 Zoom 200 Intensity 416 47 ImageQuant 400 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 ii ImageQuant Capture File Edit Image Setup Overlay Utilities wiew 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 close an image log off of the system or exit the system OEM Overlay Close Save Save Modified Save As Princ Printer Setup 1 Cf Images Sample tif 2 Cf Images Testpatn tif 3 CfImages coomassiel 6 tif Exit Alt F4 Logoff ImageQuant Capture File pull down menu 48 ImageQuant 400 User Manual a 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 Fie Edit Image Setup Overlay Utilities View Help Look ir kl Blwhi tif w coomassiel g tif w Sample tif w Testpatn tif lk Testpatt tif File name Blwhi tt Cancel Good L
74. n 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 O8BLACK OVR lane labels in white black IOWHITE OVR IOBLACK OVR 10 lane labels in white black I2WHITE 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 HINDIIILOVR Hindi label The objects in these overlays can be repositioned resized re colored copied or 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 68 ImageQuant 400 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 blan
75. nd the Contrast Adjustment tools are configured for color image adjustments Contrast Adjustment 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 43 ImageQuant 400 User Manual 2 2 Tool Bar The Tool Bar window provides intuitive buttons for the most common functions in ImageQuant Capture ToolBar Es Tool Bar 0 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 oon Ir 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 LLI Sat
76. ng 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 52 ImageQuant 400 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 4x Printer Name SAVIN 2560 PCL amp Properties Shatus Ready Type SAVIN 2560 PCL 6 Wher 216 132 94 4002560 Comment Network Cancel Source Auto Tray Select Margins inches i Portrait Right f Top Bottom f Cancel Printer Printer Setup Dialog Box Printer Dialog Box Lett r C Landscape For more information on using the Print menu see the Windows manual 53 ImageQuant 400 User Manual fi 3 1 6 Print aint 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 54
77. ng 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 s 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 80 ImageQuant 400 User Manual 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 5 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 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 Release the left mouse button when it re
78. nly 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 data 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 If an image is saved as a Modified file it permanently retains any changes made to the Black level White level and Gamma setti
79. nnect 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 the monitor s video cable to the video card connector Connect the monitor s power cable to the power strip d z TE a port printers The mouse and keyboard connectors 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 for printers ront of the unit j f and other devices 10 ImageQuant 400 User Manual 1 3 3 Camera Installation Make sure that the power supply is set to the off o position before beginning the camera installation 1 Connect the data cable and power supply barrel connector to the top of the camera Power Supply SCIE Barrel Connector W a t Data Cable j connects here j k i 7 at Complete the camera connection by attaching the other end of the Data Cable to the computer interface card that came preinstalled in the computer system Connect the Data Cable to the D shaped connector on the computer interface card Data Cable _ from camera fee Computer a interface card t O ul 7 2 Connect the standard three prong end to the power strip 11 ImageQuant 400 User
80. nsitivity from one frame to the next is the exposure time In this situation COPY TO NEXT is a useful Zoom AOI tool to save time in the setup process 1 0 u l I I I I I I I I z 7 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 Display Chemi Display IY Show Saturation Auto Contrast Reverse Sensitivity Aesolution Normal High Medium Wedium Extended OD Ext OD Off Once the sequence is set up to the desired configuration click on the GO button The software will then begin the process of 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 32 ImageQuant 400 User Manual Tool Box Peretti etree errr e
81. ntrast 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 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
82. or Selection Tools T7 ImageQuant 400 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 jo Lloje 4 a l coroR LINE ENDS T _ TEST STYLE P PEN STYLE JTEXT 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 All annotations subsequently entered will appear in that style Note these pen styles only work with a thin line see Line Thickness above ae el TENER COLOR LINE ENDS PEN WIDTH TEXT STYLE Bless lt 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 e N ojo x a l COLOR LINE ENDS PEN WIDTH TEXT STYLE gt PEN STYLE TEXT ORIENT _ Line Ends Selection Tools 78 ImageQuant 400 User Manual 4 5 6 Text Background and Font The TEXT STYLE menu specifies the style of text Click on
83. or 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 ImageQuant 400 User Manual 1 3 Computer Camera and Peripheral Installation 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 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 400 User Manual 1 3 2 Setting up the Computer Monitor Mouse and Keyboard The computer will need to have the monitor mouse and keyboard Keyboard Mouse connected to it in the correct ports m amp CONNECNOND ACONI ae coded purple and see the descriptions below Also t a 3 prong standard power cable should be plugged into the back of i the computer and the power strip aes COMI port for DE eee 500 Light Cabinet Co
84. ow Resolution Recommend for Chemiluminescence This mode captures the image performing a 3x3 pixel bin with a gain setting of 3 This will decrease the exposure time needed by approximately 2X over the High Medium setting but yield a lower resolution image containing 682x682 pixels This mode is ideal for high throughput screen applications for verification of absence or presence of target DNA or Protein Super Sensitivity Low Resolution Recommend for Chemiluminescence This mode captures the image performing an 8x8 pixel bin with a gain setting of 3 This will significantly decrease the exposure time required for acquiring a long chemiluminescent exposure by about 2X over the High Low setting or 8X over the Medium Medium setting The resolution using this mode is 256x256 pixels and is not recommended for publications This mode is ideal for previewing when using auto expose to determine the signal level in the chemiluminescent blot It is also ideal for high throughput screen applications for verification of absence or presence of target DNA or Protein 23 ImageQuant 400 User Manual 1 6 5 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 1s turned on Also make sure the cabinet door indicator in the Cabinet Control software interface indicates CLOSED Click on the blue PREVIEW button an
85. ox with level 9 76 ImageQuant 400 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 Cr COLOR _ LINE ENDS FEN WIDTH TEXT STYLE PEN STYLE TEXT ORIENT Annotation 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 Annotations 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 alelsiolo m E FEN STYLE TEXT ORIENT Pen Col
86. 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 IQcapture 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 61 ImageQuant 400 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 can be loaded later and applied to any image lll Save in E ImageQuant 400 ee EE 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
87. r 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 14x 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 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 73 ImageQuant 400 User Manual 4 2 The Rotate Flip Tool The Rotate Flip tools are foun
88. r errr errr etree rrr errr err rrr rrr Enhancement Tools oom Rotate Flip Histograrn Enhance Annotate False Color Filters FrameGrabber 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 tab 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 applied to a single image Tool Box Enhancement Tools oom Rotate Flip Histograrn Enhance Annotate False Color Filters FrameGrabber Sharpen Moise Despeckle 3 D UNO Low High ilalal slelzlels 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 86 ImageQuant 400 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
89. re 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 400 User Manual 1 1 1 Mouse Functions The mouse supplied with the ImageQuant System has two buttons The left button 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 rec
90. re interface i inegal kani apt ure Lo Peni an Files ae 1 AO Jlenage commana 1a 2 yr Zoomi imema 5075 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 56 ImageQuant 400 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 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
91. roet_ 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 66 ImageQuant 400 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 image 1s 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 DAautorad 1 6bit tif 1280x1024x16 1 s 3 4 5 G Uie S 9 10 ow ot oe eee ow ot oe ae kperstd120 Save As 21x Save in E Image TE c amp i Demotutr a 12white ovr fa O8black ovr fa 15black ovr a O8white ove ia 1 Swhite ovr
92. rrr 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 become displayed in the Tool Box The buttons on this tab perform the following tasks REC Opens the Camera Setup and Preview window 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 Moves forward in 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 1 6 12 Saving an Individual Image from a Sequence After you load play and stop a sequence at the desired image it 1s 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 B Note This will save the current
93. s 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 File Edit Image Setup Overlay Utilities View Help Equalize Sample tiff Arithmetic Conversion 68 bit grayscale Flat Field Calibrate 16 bit grayscale Resize Image Info Image Conversion dialog box 58 ImageQuant 400 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 use 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 Cal
94. s 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 40 ImageQuant 400 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 41 ImageQuant 400 User Manual 2 1 8 Making Linear Log or en Adjustments yutersnd i tut tit 1 Patel Gi Original image of film with default BWG settings l rires Ajus Imer Gama Log Eaa 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 film 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 42 ImageQuant 400 User Manual 2 1 9 Using the Contrast Adjustment Tools for Color Images ImageQuant Capture can also import RGB color images The software automatically detects this process a
95. scribed 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 1 Introduction and Setup ssssssssececcccsssscccceccossssccececcosssssseccecoossssscececosssssseceeesssssssseee 1 1 1 ImageQuant Capture Imaging System c ccc ccccccccccccecceceeeeeeeeeessseeeceeeeeeeeeeeeeaaaaas l Td cdl IMIOUSE FUNCIONS ioan cara vieeuateouteeseamceneres 2 LEZA DOUC iis Manta lcron aiT ET TAAT 2 bal otarine Imas cQuant CAPUT ena a E TE associ 2 t2 Ima ceQuant Imas me Sy Stem SetU ep EA E 3 F2 l System OM PONGHIS serea eria oa nanei anasa ae eE ANENE EER ONEN 3 EOE Ste a HAC CHIC E E EET E EEE E E ETS 3 1 2 Ca bme ASSEMDIY oiee a E aia du dasuiady daa eee iee esau aadunauieds 4 2A Cable 2 OMNE CONS ics wstasinccmenetnontacainesnenntnonisecle ning niwousuyuteanindaleeaiveneanie
96. setting instead Alternatively if the exposure time is short enough omal High may be selected for a full resolution 4 million pixels chemiluminescent image 5 Save the original image d 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 window 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 d 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 digital filters found in the Tool Box under ENHANCEMENT and 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 window under ENHANCEMENT and ANNOTATE 7 Print the image using the large PRINT button in the Tool Bar window 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 15 ImageQuant 400 User Manual 1 4 1 Cabinet Controls J Sg UV Override UV Trans
97. seve 28 ImageQuant 400 User Manual 1 6 8 Using the Zoom ROI Tool Zoom ROL 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 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 1s now possible to adjust the region of interest of the acquired image using the ImageQuant Capture 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 the Preview and Capture buttons to acquire the final image This eliminates a post acquisition step of cropping the image down to the appropriate size Camera Setup amp Preview 2i 15 xj Image Cabinet Controls l Noise Reduction Disconnected Exposure Time ay Es gt z gt gt o j o J o 104 wf n Td I Auto Expose Auto Expose Setup Reflective m Display
98. t before the image is transferred from ImageQuant Capture to the appropriate ImageQuant TL module 97 ImageQuant 400 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 100AV Results may vary for different software versions and or hardware configurations Adobe Photoshop 2 51 LE Mac Open Open As TIFF Adobe Photoshop 3 0 Mac no Canvas 3 5 Mac use No ResEdit ResEdit Microsoft Word 6 0 Mac Insert Insert Insert no Picture Picture Picture Microsoft Word 6 0a Win Insert Insert Insert Insert Picture Picture Picture Picture Microsoft Excel 5 0 Mac no no no no Microsoft Excel 5 0c Win Insert Insert Insert Insert Picture Picture Picture Picture NIH Image 1 59 Mac Open 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 98 ImageQuant 400 User Manual Using ResEdit l 2 Save image in IS 1000 500 as a TIFF or MACTIFF Obtain a copy of the freeware ResEdit by downloading from Apple Computers through the Internet Open ResEdit An
99. tional 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 ImageQuant 400 User Manual 1 2 3 Cabinet Assembly When you remove the light cabinet from its shipping carton it 1s 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 1 Remove the camera and lens from the packaging and attach the lens to the camera Attach the lens to the camera 2 Place the camera stand on its side 3 Holding the camera in your left hand position it against the camera post so that the data and power sockets are next to the post With your right hand
100. to an ImageQuant TL module for analysis then you will be prompted to save as part of the transfer process 30 ImageQuant 400 User Manual 1 6 11 Capturing Images Using 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 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 the acquisition screen Image E A fe ee Preview Lapture Hose Reduch PELELLELETETLLLLETETEELELLETETLELLELELLTELELELLLELELELELLLLLELELELELELELLLELLELETEELLLLETETEETELLETETEELLLLTTETELELELELELELELELETEEELELLTETEEELELLLLELETLELLLLETETELLLETETEELELLETI Enhancement Tools OOM Rotate Flip Histograrn Enhance Exposure Time J f e Ber ae PST an Houra Minutes Seconds Milisecs notate False Color Filters rameGrabber 0 0 0 104 Image 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 esalutiorn Normal High l FrameGrabber Setup in the Tool Box Medium Medium High Medium High Low
101. uration The Saturation button allows for a quick image display of saturation Completely saturated black regions gray scale 0 will turn green and 44 Rue Img Drag ImageQuant 400 User Manual saturated white regions i e gray scale 255 4095 65 535 will turn red This is a useful tool to check for linearity of an image before analysis occurs Saturation is a feature that 1s 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 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 t
102. y with the real time display Simply adjust the parameters on the manual lens or if the lens 1s controlled via the software click on the appropriate software control button 1 To open the aperture turn the lens s aperture ring counter clockwise for more light meen Conversely if the image is too Al Hil HAH IMMA Tee Eae salonbobed se i f turning it clockwise until an i image is visible on the monitor ES 2 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 3 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 a zoom ring 19 ImageQuant 400 User Manual 1 6 2 Auto Exposure and Auto Exposure Setup The acquisition window for the ImageQuant Capture includes a check box and setup button for Auto Exposure mage Noise Reduction Exposure Time i E Ei J gt Hours Hours Minutes blinutes a Seconds Seconds a MiliSecs MiliSecs al T l T 104 Auto Expose Auto Expose Setup Display Chemi Display IY Show Saturation Auto Contrast Reverse Sensitivity Resolution Normal High Medium Medium High Medium High Low n EERE Super Sensitivity oom AL
103. 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 necessary x Closed System Passwords ute Logout f 5 minutes 10 minutes f 20 minutes Change Master Password Add Remove Users OK Cancel 100 ImageQuant 400 User Manual Security Features Dialog Box 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 1s required and anyone can use the system SS t RE P dosed System Passvvords Aute Logout f 5 minutes 10 minutes 20 minutes Change Master Passyword 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
104. ype 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 87 ImageQuant 400 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 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 nei
105. yscale 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 50 ImageQuant 400 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 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 nave in Jlmages si amp kl Blyhi tif a coomassie1 6 tif a Sample tif a Testpatn tif lal Testpatt tif File name ti nave as type Tit Files tif Cancel Tiff Files tif Windows bitmaps bmp Good Laboratory Practices files glp Paint Brush files pcx Color Targa files tga File Formats tif bmp glp pcx tga pic jpg mac Headerless files pic JPEG format p Mac Tiff Files mac All image files Enter

Chapter 1 - GE Healthcare Life Sciences

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