INSTRUCTION MANUAL SERVA HPE™ ScreenPicker Semi-manual spot picking system (Cat. No. HPE-SP1) SERVA Electrophoresis GmbH Carl-Benz-Str. 7 D-69115 Heidelberg Phone +49-6221-138400, Fax +49-6221-1384010 e-mail: [email protected] http://www.serva.de Contents 1. Introduction 2 2. Features of the Screen Picker 3 2.1. Specifications 3 2.2 Description of the ScreenPicker 4 2.3. Packing List 5 3. Installation 6 4. Operation 6 4.1. Image Viewer software manual 4.1.1. Automatic mode 4.1.2. Manual mode 4.1.3. Mixed mode (automated with additional manual spots) 7 13 15 5. Software re-installation 18 5.1. General comment on software protection 18 5.2. Installation from Boot menu 19 5.3. Installation from USB backup key 19 5.4. License key installation 20 6. Care and maintenance 20 7. Picker.ini configuration** 20 8. Closing and Shutdown procedure 21 9. Important comments 21 10. Legal Information 21 11. Order Information 22 Vers. 11/12 1 8 1. Introduction Manually picking from a fluorescent 2D electrophoresis gel is a challenge without an automated picking robot. It requires to post stain the gel in order to visualize the spots. Many classical staining methods interfere with mass spectrometry or if not, have lower sensitivity than fluorescent staining. The SERVA HPE™ ScreenPicker is a device to semi-manually pick fluorescent 2D gels (DIGE, LavaPurple, Sypro Ruby, Flamingo…) without the need of post staining and/or sophisticated robot and software. It consists of an embedded screen connected to a small computer to display the scanned image of the gel, an XY carriage to guide the picker accurately over the spot position and a software which can read images and picking lists from most evaluation packages. The ScreenPicker is adapted for gels on 3 mm glass plates (fluorescent markers placed on 3 corners before scanning) and HPE™ gels on non-fluorescent plastic backing (punched holes performed on 3 corners before scanning). The scanned image is projected under the gel. The provided software allows performing a fine-tuning between the size of the displayed image with a mouse and the size of the real gel with the help of markers or punched holes used as landmarks. An XY carriage is used to guide the manual picker over the target to be excised and to prevent parallax errors. The ScreenPicker guides visually the user to place the picked plug in the right well of the microtiter plate using the file generated by the evaluation software. At the end of the process a file containing spot numbers and microtiter plate plug positions is generated for use by digester robot. Because it clearly marks the spots to pick on the image, the ScreenPicker can also be used for Coomassie® blue or silver stained gels, increasing the picking accuracy and making the process entirely error free. Another interesting application is the picking of protein spots from 2D gels after the immunological visualization of specific spots by western blotting: The scanned image of the blot membrane is projected under the gel, which contains still a great portion of the proteins after the electro-transfer. The files from the evaluation software are transferred to the Netbook PC via an USB stick. The special Crunchbang (Debian) Linux installation on the Netbook PC is minimalistic. The Netbook PC setup is not designed for any other purpose than picking spots. 2 Benefits • Space saving: easy to store on lab bench; dimensions length, width, height compared to huge automatic picker. • Time saving, accuracy, easy to use, no need for specific training, no need for manual corrections of the gaps always encountered with automatic pickers. Full workflow integration with generation of picking-digester list. Ready within seconds. • Costs saving: no need for expensive instrument or maintenance contract. • Error free picking with help of pick list and plate well identifier. • Flexibility: initialization file defining visual appearance and requirements of digestion robot like input file and reserved positions on plate. Reads any image file format or picking list. • Safety: no transilluminator or dangerous UV sources. • Security: Linux avoids transfer of viruses by USB memory keys. PC and Software protected. 2. Features of the Screen Picker 2.1. Specifications The system is mainly designed for film-backed HPE™ gels and gels on 3 mm glass plates. Screen box Dimensions Weight Features 55 x 36 x 8 cm 7 kg Visible light for safe usage Leakage-free Flat screen protected by glass platen Maintenance-free Precise XY carriage Robust default picker head 1.5 mm diameter Voltage Power Consumption AC 90V to 264V; 60 Hz Power rating 1.2 A at start (protected by fuse in switch ) 37 W nominal 10° C to 40° C Relative Humidity: 20% to 85% Operating Temperature: Environment 3 Computer hardware Netbook PC 10.1“, 1024 RAM, 250 GB HD, AC 90V to 260V; 50-60 Hz. 30 W maximum. Linux-based software environment, virus-safe Connection to screen box via monitor cable Cable free mouse USB stick for data transfer ImageViewer software Flexible: reads any image or pick list format Variable configuration for plug disposal in X or Y direction; reserved positions in MTP; spot cross hair size on screen. Manual spot selection saved to file. Function icons for easy handling Adjustable image contrast “SYSTEM BACKUP” USB stick Kolourpaint tool Paint program for marking and documenting manually selected spots when working without image analysis software. 2.2 Description of the ScreenPicker Fig. 1: The complete Screen Picker system 4 Fig. 2: Screen box setup 2.3. Packing List • • • • • • Screen picker box Y- rail and carriage for picking head One Touch 2D gel spotpicker 1.5 mm (Cat. No. P2-D15) Power cord VGA Monitor cable Netbook PC with power supply and power cord • Cordless mouse • USB key for data transfer • USB key for system backup • • Dust cover (Cat. No. HPE-SP02) 450 Fluorescent stickers (5 mm diameter, Cat. No. HPE-FS05) • Waterproof tape • Instruction manual 5 3. Installation 1. Unpack the ScreenPicker and place it on a flat surface with the label facing you. 2. Unpack the Y rail and insert it from the left or right of the two parallel X rails, with the double guiding insert facing you.. 3. Insert the carriage with picker support on the Y rail. You can decide to have the picker head on the left or on the right of the Y rail. 4. Connect the provided VGA (15 pins sub-D) cable on the computer and the mains cable to the unit. Connect computer to mains via power supply according to computer installation guide. 5. Start the computer, the software running under Linux will load immediately after security check. 6. Use USB keys or drive to transfer the image and picklist of the gel to pick. Format allowed are Tiff, .gel, mel, img, jpg, png, bmp, gif, 8, 12, 16 bits or more... Conversion is fully automated. 7. Pick list can be imported from major evaluation software packages; format must be ASCII comma delimited. 8. The system is delivered fully configured but can be customized at installation by editing the picker.ini file located in ../param folder (see section 4.1.1.). 4. Operation The ImageViewer software delivered on the Netbook computer is running under a special version of Linux (CrunchBang Linux). . Fully charge the computer before first use following computer installation guide. On start up, a user name and a password must be provided to access the safe environment. Unless it has been changed during installation, the username is “picker” and password is “spot”; both are case sensitive. In case of software failure, follow procedure in chapter 5, “Software re-installation”. Important before Scanning the Gels: • Remember to put fluorescent markers (adhesive white dots on the glass plate on 3 edges before casting the gel in case of traditional vertical gels. (Fig. 3) • While running HPE gels, either use small scissors to make V cuts on 3 edges (Fig. 3) or the fluorescent adhesive dots. 6 Fig. 3: Marker positions on plastic-backed gels and glass plate gels. Switch on the device using the button on the left side (Fig. 2). Then switch on the Netbook computer The booting process takes approximately 30 sec. 4.1. Image Viewer software manual ImageViewer software is automatically loaded after boot. If the program has been closed, a right click with the mouse opens a dialog box, click on first menu item <ImageViewer>. An array of icons (figure 4) makes handling easy: Fig. 4: Imageviewer icons on the screen. Please notice that the direction of filling double arrow is just an indicator, you can change the direction via picker.ini file in chapter 7. 7 4.1.1. Automatic mode In automatic mode, the loaded picking list is entirely driving the process by highlighting one spot at a time by a cross hair indication and the corresponding well position to release the picked plug. 1. Choose the file name of the gel to pick from the browser window popping up as per Fig. 5, and click <Open> It is also possible to choose an image file using camera icon on menu bar (The USB key used to transfer files is labelled . “xGb filesystem “ on the folder list ). 2. A second dialog box opens to choose the corresponding picking list . Select the file with the mouse and press <Open> (Fig. 6) or press <Cancel> to pick without pick list (see Manual mode 3.1.2) It is also possible to choose picking file using printer icon on menu bar. The folder containing image and picking list files will be saved automatically at the end for the next run. Choose “Ettan™ picker” as picking robot in Imaging software to generate the picking list Fig. 5: Choosing image from gel to pick Fig. 6: Choosing corresponding picking list As the last position used in the microtiter plate is saved at the end of the run , before starting, you have to choose between filling the next empty position on an existing plate or restart at the default position on a new plate (the default position is defined in the initialization file). This feature allows the user to pick several gels using the same microtiter plate. (Fig. 7.) Fig. 7: Continue to fill existing plate or choose a new one 8 3. Lay your gel on the picker window, gel side up. Start by positioning one V cut or marker on the corresponding image position. In general, it is not necessary to fix the gel but if needed, use the tape provided. 4. Using mouse, enlarge the image by dragging right and bottom sides to get the 2 other markers properly coincide with the real gel (holes or markers); Use the XY carriage without the picker to avoid any parallax distortion. Lock the image position and size by pressing locker icon; icon becomes red. 5. The 96-wells microtiter plate used to collect the polyacrylamide plugs is placed on the bottom right corner of the screen picker. There is only one way to position the plate due to the special design (Fig. 8). Fig. 8: Positioning the microtiter plate 17. Image contrast is adjusted using icons on menu bar (Fig. 4) in section 4.1. This icon opens dialog box enabling the choice of contrast steps: This icon increases contrast by number of steps defined above This icon decreases contrast by number of steps defined above 6. ImageViewer is running in Auto mode (icon A of Fig. 4), the software will display the first spot to pick with a crosshair on the image and it will also highlight the well position to use by means of the plate coordinates and a red spot visible underneath the plate at the correct well position. (Fig. 9,10). 9 7. Move the carriage to the pick position like on Fig. 11; move Y rail by moving left-right only the X carriage located on front side (Fig. 12, 13). Immobilize the XY carriages by rotating clockwise 1/4 turn the black knurled thumb screw knobs, introduce the picker into the 2 holes of the picker head (Fig. 14). Fig. 9: Plug position highlighted below the Microtiter plate and indicated by arrows (for black MTPs). Fig. 10: Positions in black are reserved for calibration, the display shows the plate Nr 3 position D4 to put spot 1830 Fig. 11: Crosshair generated on spot to pick Fig. 12: Moving X carriage from front side 10 Fig. 13: Align the carriage over the spot using XY lines and the spot crosshair. Fig. 14: Use black knurled thumb screw knobs to lock carriages. 8. Pick the spot by pushing downwards the black tube of the picker to close the jaws. Rotate the picker one full turn to detach the plug from glass or plastic (Fig. 15). 9. Remove the picker from the carriage and dispense the plug into the microtiter plate at the indicated / highlighted position (Fig. 16). Carefully rinse your picker after each spot to avoid cross-contamination. Fig. 16: Placing the spot plug into plate ready for digestion. Fig. 15: Picking the spot. 10. Use arrows keys on the menu bar to continue with next spot. 11 11. When you reach the last position on the plate a message invites you to put a new plate : The microtiter plate number will be incremented on screen and in file. 12. At the end of the process, a window pops up saying “You are on the last spot”; click “OK” and ... 13. At that stage you can switch to manual mode to define new spots to pick or not; If you select “Yes”, refer to 3.1.2 step 7 If you answer “No”, continue with step 15. 14. Click the green arrow icon on the menu bar to save the picking list as a file for digester; The created file will contain spot numbers, spot positions, microtiter plate number (default=1) and the plate well position for each spot picked (e.g.: 2C11= plate 2 raw C column 11). The software automatically identifies the positioning markers (IR1 & IR2) used by Ettan™ picker (GE Healthcare) and remove these from picking process and saved file. The file name and folder are entered via the dialog box. By default, the file will be saved in the folder used to load image and pick list but you can save it in any other folder by selecting <Browse for other folders> (Fig. 17). An example of the saved list is visible on Fig. 18. 12 Fig. 17: Browsing between folders. 15. At that stage, you can switch to the following gel to pick by pressing the Camera button on menu bar. You are now jumping again at step 1 of this chapter. If you didn’t save the file for the digester yet, you will get a reminder ... If you do not save the pick list, the last well position used is not saved! 16. Before removing the USB key, press the USB icon on the title bar to close files and eject key or drive; remove your key only when you see this message: 4.1.2. Manual mode The manual mode gives the scientist the freedom to decide which spot he wants to pick. The spot on the gel will be identified later by his XY coordinates in the image and will be reported as such in the digester file. There is no limit to the number of manual spots to pick. ImageViewer allows working in mixed mode: automatic mode, switching to manual and back to automatic (See section 4.1.3.). We know by experience that researchers are sometimes interested to pick additional spots for instance in order to identify a train of different glycosylations on a gel near a spot identified by the imaging software as a statistically relevant difference. In case of large spots, it could also be a good idea to pick more than once; this may help to identify multiple proteins present in that spot. Most imaging software give the possibility to only pick one time per spot. With automated picker, it is also not so easy to add more positions to the picking list or to change the picker head for a larger one. Screen Picker gives you all these features. If you have a faint spot, why not choosing a picker of 3 mm instead of 1.5 mm diameter or picking more than once in the spot! 13 1. Choose the file name of the gel to pick from the browser window popping up as per Fig. 5, and click <Open> It is also possible to choose an image file using camera icon on menu bar. (The USB key used to transfer files is labelled . “xGb filesystem “ on the folder list) 2. A second dialog box opens to choose the corresponding picking list . Press <Cancel> to pick without pick list. The folder containing image and picking list output files will be saved automatically at the end for the next run. As the last position used in the microtiter plate is saved at the end of the run , before starting, you have to choose between filling the next empty position on an existing plate or restart at the default position on a new plate (the default position is defined in the initialization file). This feature allows the user to pick several gels using the same microtiter plate (Fig. 7). 3. Lay your gel on the picker window, gel side up. Start by positioning one V cut or marker on the corresponding image position. In general, it is not necessary to fix the gel but if needed, use the tape provided. 4. Using the mouse, enlarge the image by dragging right and bottom sides to get the 2 other markers properly coincide with the real gel (holes or markers); Use the XY carriage without the picker to avoid any parallax distortion. Lock the image position and size by pressing locker icon; icon becomes red. 5. The 96 wells microtiter plate used to collect the polyacrylamide plugs is placed on the bottom right corner of the screen picker. There is only one way to position the plate due to the special design (Fig. 8). 6. Image contrast is adjusted using icons on menu bar (Fig. 4 in section 4.1) 7. Click on the image at the position you plan to pick a spot, a dialog box will pop up: click “No”, to reselect another position; click “Yes” will validate the spot to pick. 8. The spot number will be incremented to Mxx (starting with M1) and displayed on the image, Move the carriage to the pick position like on Fig. 13; move Y rail by moving left-right only the X carriage located on front side (Fig. 12). Immobilize the XY carriages, introduce the picker into the 2 holes of the picker head. 9. Pick the spot by pushing downwards the black tube of the picker to close the jaws. Rotate the picker one full turn to 14 detach the plug from glass or plastic (Fig. 15). 10. Remove the picker from the carriage and dispense the plug into the microtiter plate at the highlighted position (Fig. 16). Carefully rinse your picker after each spot to avoid cross-contamination. 11. Continue with step 7 for the next manual spot 12. Terminate the picking process by clicking the green arrow icon on the menu bar to save the picking list as a file for digester; The created file will contain spot numbers (M1 to Mx), spot X,Y positions, microtiter plate number (default=1) and the plate well position for each spot picked (e.g. 2C11= plate 2 raw C column 11). The file name and folder are entered via the dialog box. By default, the file will be saved in the folder used to load image but you can save it in any other folder by selecting <Browse for other folders> (Fig. 17). 13. At that stage, you can switch to the following gel to pick by pressing the Camera button on menu bar. You are now jumping again at step 1 of this chapter. If you did not save the file for the digester yet, you will get a reminder ... If you do not save the pick list, the last well position used is not saved! 14. Before removing the USB key, press the USB icon on the title bar to close files and eject key or drive; remove your key only when you see this message : 4.1.3. Mixed mode (automated with additional manual spots) In mixed mode, the loaded picking list is entirely driving the process by highlighting one spot at a time and the corresponding well position to release the picked plug. At any time, the user can decide to switch to manual for choosing additional spots to pick; the automated process will continue and the manual spots chosen will be added at the end of the process. The advantage of this mode is for the user to make multiple picks into one large spot or to pick adjacent spots which seems to be related to spots chosen by the evaluation software. 1. Choose the file name of the gel to pick from the browser window popping up as per Fig. 5, and click <Open> It is also possible to choose an image file using camera icon on menu bar. (The USB key used to transfer files is labelled . “xGb filesystem “ on the folder list). 15 2. A second dialog box opens to choose the corresponding picking list. Select the file with the mouse and press <Open> (Fig. 6) or press <Cancel> to pick without pick list (see Manual mode 4.1.2) It is also possible to choose picking file using printer icon on menu bar. - The folder containing image and picking list files will be saved automatically at the end for the next run. - Choose “Ettan™ picker” as picking robot in Imaging software to generate the picking list - As the last position used in the microtiter plate is saved at the end of the run, before starting, you have to choose between filling the next empty position on an existing plate or restart at the default position on a new plate (the default position is defined in the initialization file). This feature allows the user to pick several gels using the same microtiter plate. (Fig. 7). 3. Lay your gel on the picker window, gel side up. Start by positioning one V cut or marker on the corresponding image position. In general, it is not necessary to fix the gel but if needed, use the tape provided. 4. Using mouse, enlarge the image by dragging right and bottom sides to get the 2 other markers properly coincide with the real gel (holes or markers); Use the XY carriage without the picker to avoid any parallax distortion. Lock the image position and size by pressing locker icon; icon becomes red. 5. The 96 wells microtiter plate used to collect the polyacrylamide plugs is placed on the bottom right corner of the screen picker. There is only one way to position the plate due to the special design (Fig. 6). 6. Image contrast is adjusted using icons on menu bar (Fig. 4 in section 4.1 ) 7. ImageViewer is running in Auto mode (icon A of Fig. 12), the software will display the first spot to pick with a crosshair on the image and it will also highlight the well position to use by means of the plate coordinates and a red spot visible underneath the plate at the correct well position (Fig. 9,10). 8. Move the carriage to the pick position like on Fig. 13; move Y rail by moving leftright only the X carriage located on front side (Fig. 12). Immobilize the XY carriages , introduce the picker into the 2 holes of the picker head. 9. Pick the spot by pushing downwards the black tube of the picker to close the jaws. Rotate the picker one full turn to detach the plug from glass or plastic (Fig. 15). 10. Remove the picker from the carriage and dispense the plug into the microtiter plate at the highlighted position (Fig. 16). Carefully rinse your picker after each spot to avoid cross-contamination. 11. At any time, switch to manual mode by depressing icon A on menu bar, the icon will turn white in manual mode and the message bar at the bottom of the image window will display: 16 12. Click on the image at the position you plan to pick a spot, a dialog box will pop up: click No, to reselect another position; click Yes will validate the spot to pick. Perform this step as many times you want to pick new manual spots. The software will remember the manual coordinates of new spots and will guide you at the end of the automatic process. 13. Use arrows keys on the menu bar to continue with next spot. ImageViewer is now jumping to next automatic spot to pick; at the end of the spots from the picking list, the plate display will change to show the manual spots starting with M1 until Mxx. The process can be interrupted as many times as you want by switching back again to step 9. 14. When you reach the last position on the plate a message invites you to put a new plate: The microtiter plate number will be incremented on screen and in file. 15. At the end of the process, a window pops up saying “You are on the last spot”; click OK and ... 16. At that stage you can still add manual spots (see step 10). After each manual spot added, you will get the message as in step 13. 17. Terminate the picking process by clicking the green arrow icon on the menu bar to save the picking list as a file for digester; The created file will contain spot numbers (automated and manual at the end), spot positions, microtiter plate number (default=1) and the plate well position for each spot picked (e.g. 2C11= plate 2 raw C column 11). The software automatically identify the positioning markers (IR1 & IR2) used by Ettan™ picker and remove these from the picking process and from the saved file. The file name and folder are entered via the dialog box. By default, the file will be saved in same folder than the folder used to load image and picklist, but you can save it in any other folder by selecting <Browse for other folders> (Fig. 13). An example of the list saved is visible in Fig .18. 17 Fig. 18: Example of pick list saved for the digester. 18. At that stage, you can switch to the following gel to pick by pressing the Camera button on menu bar. You are jumping again at step 1 of this chapter. If you didn’t save the file for the digester yet, you will get a reminder ... If you do not save the pick list, the last well position used is not saved! 19. Before removing the USB key, press the USB icon on the title bar to close file and eject key or drive; re-move your key only when you see this message: 5. Software re-installation 5.1. General comment on software protection ImageViewer application is protected by a dual level security encrypting key. You cannot install the package on another machine than the computer which is delivered with the system. We sell additional pre-installed computers in case of need. Please ask your distributor for a quotation. There are two ways of re-installing the software on the computer. 18 5.2. Installation from Boot menu • Always attach the computer to mains supply before continuing. • At boot, the GRUB Linux loader is visible. If you do not do anything, the computer will automatically boot and load the software after 20 sec. Pressing the “Enter” key forces the system to boot immediately. • In case of boot failure identified by a message on screen, switch off the computer by pressing the Power on switch for a few seconds until computer power goes off. • Power computer on again and when you see the GRUB Linux loader, move down using arrow keys to the last line labelled : “Software installation (use only in case of boot failure)” • A dialog will ask you to confirm your choice, answering “No” will start the process again, “Yes”, will initiate the complete re-installation at factory defaults. • Follow instructions on screen until end of the process.. DO NOT POWER OFF COMPUTER DURING INSTALL PROCESS. 5.3. Installation from USB backup key A special USB key containing a full backup of operating system and application is included with the package in case the software must be reinstalled due to computer crash. This key is labelled “SYSTEM BACKUP” In case you cannot find back the USB key included in the original package, please ask your distributor for a copy provided at production cost price. • Always attach the computer to mains supply before continuing. • If the process detailed in 5.2 has failed, please switch off your computer by pressing power on switch for a few seconds until computer power goes off. • Attach your “System Backup” USB key to any USB port and switch computer on again. Follow all instructions on screen until you get the final message inviting you to remove the USB key and reboot. The system must be up and running again at factory defaults. DO NOT POWER OFF COMPUTER DURING INSTALL PROCESS 19 5.4. License key installation In case the license key must be re-installed, use contact details (phone, email) provided on screen to fix the issue on your system. 6. Care and maintenance Immediately after use, wipe the glass plate with a clean tissue and water (can use 0.1% SDS). Never use any other solvent than methanol or 50:50 ethanol. Always make sure that the rails are clean; eventually clean them with distilled water; never use grease, oil or any lubricant. For smooth travelling of Y rail, use only X carriage located on front side (Fig. 8 bis). Rinse properly OneTouch spot picker between each plug to avoid crosscontamination. 7. Picker.ini configuration** There is a configuration file located in /home/picker/imageViewer/param. We do not recommend to change the X and Y offset for the 96 wells plate, size and gap of the spot crosshair and plateW, plateH (this has been optimized for each machine). You can change the default start, the reserved positions on the microtiter plate. Some users like to keep wells for standards or calibration. It is also possible to adjust the contrast changing speed, the plate geometry and the way of filling the plate. Procedure: - Double click on file picker.ini file to open it. - Enter the position to start in the line “start= “ - Enter positions to skip in line “reserve” without using space between values. - Contrast is the default contrast change speed (0.5 steps) - Way is the plate filling direction; V means A1,A2,A3..., H means A1,B1,C1,D1... (An orange double arrow indicating the direction, is visible on image window title bar (Fig. 12)). - PlateR is the plate geometry; 0 is normal, 1 is inverted - Save the file after edition by clicking “Save” in menu bar.** **: ask help from your IT department or Proteomics Consult if questions 20 8. Closing and Shutdown procedure • Close ImageViewer by pressing the <Close> icon on image title bar (Fig. 4). • Press Computer power button once to initiate shutdown procedure or right mouse click and choose “Shutdown” • Shutdown screen picker using Power button on the left (Fig. 2). 9. Important comments The special Crunchbang (Debian) Linux installation is minimalistic. The system is not designed for any other purpose than picking spots. In case of network installation, please communicate network details before installation and inform IT people in advance to help configuring the system on the network at the install. We do not recommend using file transfer via network to avoid support problems. In case of network link, we truly recommend to subscribe a maintenance contract with the support team. Please ask your distributor for a quotation. ImageViewer is fully protected from changes or copy via dual-level encrypting license key. The delivered package on an USB key cannot be installed on another computer. Please contact your distributor 10. Legal Information Screen Picker is covered by Intellectual Property rights assigned to Proteomics Consult for the design and the hardware, and to Alltux for the software (http://www.alltux.be ) 21 11. Order Information Product HPE™ ScreenPicker Plexiglass protection shield, 50 cm wide OneTouch 2D gel spot picker, 1.5 mm diameter OneTouch 2D gel spot picker, 3.0 mm diameter Screen Picker Protection Cap Fluorescent Stickers (450 pcs., Ø 5 mm) Cat. No. HPE-SP1 HPE-PS50 P2-D15 P2-D30 HPE-SP02 HPE-FS05 Important Specifications, software bundles, and accessories are subject to change without notice. Not responsible for typographic errors. Related products Product Trypsin NB Premium Grade, MS approved Trypsin NB Sequencing Grade, modified Endoproteinase Glu-C (V8), MS approved 22 4 x 25 µg 4 x 25 µg 4 x 25 µg Cat. No. 37384.01 37283.01 20986.01 Appendix A When spots are picked from a gel without the use of a picking list, there is a tool to annotate the image in advance with the spots to pick. This feature is useful if you do not have an imaging software application, or if the number of gels is anyway too small to be analyzed by such a software. 1. Scan the gel with the markers as described in chapter 3. 2. Load your image on the USB key and insert the key into the picker computer. The file manager will pop up to display the folders on the USB key. 3. Double click on the image to modify. The application Kolourpaint is loaded with the image. 4. You can zoom in and out or modify contrast using the buttons “Zoom in” “Zoom out” and “Enhance Image” (Fig. A.1) Fig. A.1: Kolourpaint Zoom and contrast buttons Fig. A.2: Kolourpaint painting buttons 5. From the left toolbox, choose the ellipse object, the line width and from the bottom, you can select the colour. Click and drag on the gel around the spot you will select for later picking. (Fig. A.2) 6. It is possible to write a label annotation near the spot for easy identification. The font type & size are selected via the buttons on top (Fig. A.1.) The font colour is selected via the colours on the bottom. Select the text tool via the left toolbox; you can now see a new icon to choose between transparent background or solid. Choose transparent, click and drag near the spot and write the annotation. (Fig A.3) 23 7. Continue at step 5 again for additional spots. 8. When the selection is finished, save the file via the menu bar : File->Save Image As ; Image format can be chosen via Filter, but according to original format, the software will propose the closest possible. See Fig. A.4. Fig. A.3: Kolourpaint writing tools Fig. A.4: Kolourpaint Saving image at the end 9. The modified image can now be imported into the ImageViewer software for picking. Please notice that the palette colour of the image is modified to show the same colour like the one you used during image annotation. A good advice will be to pick the spots in manual mode in the same order as the spots annotations. The spots in the final saved digester file will correspond to the image (Fig. A.5 and A.6). 24 Fig. A.5: Kolourpaint image at saving Fig. A.6: ImageViewer image during picking of spot 1 25
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