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Typhoon 8600 Phosphoimager User Manual

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1. 6 11 6 4 7 Entering user comments optional 6 12 Typhoon User s Guide v Table of Contents vi Typhoon User s Guide 6 5 Starting a scan and checking the progress n saana aaan 6 5 1 Startingascan ocarina sior unine aaa ee 6 5 2 Monitoring the scan progress saaana aaa 6 5 3 Viewing the preview image for saturated data 6 5 4 Completing the scan sanasana eee 6 6 Cleaning up after the scan cece cece eee eens 6 7 Storing the storage phosphor screen 0 cc cece eee eee 6 8 Analyzing or preprocessing the scanned image 0055 Part three Scanning in the fluorescence mode Chapter 7 About fluorescence scanning 7 1 How fluorescence works 10 0 0 c ccc tees 7 2 Advantages of direct fluorescence 0 0 0 c eee eee 7 3 Emission filters 2 5 3 22k SoA ALA at eh Re ee 7 4 Beamsplitters icing ian eee eee doesn dita E E eons 7 5 Multilabel multichannel experiments 0000 0 ee 7 6 Common fluorochromes 0 ccc cee eee eee eee Chapter 8 Setting up for fluorescence scanning 8 1 Preparing the sample 000s 8 1 1 General guidelines 2 2 eee 8 1 2 Label guidelines 0 0 c eee eee 8 1 3 Low fluorescence sample support guidelines 8 2 Cleaning the glass platen and samplelid 0 000 8 2 1 Cleaning the glass platen 0 0 c cece eee 8 2 2 Cleaning the sample lid 0 0 eee eee eee 8
2. 2 Using a Phillips screwdriver remove the two screws that hold the clip support and diffuser screen in place 3 Lift off the clip support and diffuser screen 4 Using a 7 64 inch hex wrench or a slotted screwdriver loosen the screw holding the bulb clamp around the bulb and fixture and then remove the clamp 5 Unscrew the bulb and fixture from the receptacle and replace the bulb and fixture with a new bulb and fixture figure 14 2 Screws Image Eraser Clip support Grasp here and unscrew Figure 14 2 Removing a bulb from the Image Eraser Typhoon User s Guide p14 3 Part five Maintaining the Typhoon system hardware Warning 6 Replace the bulb clamp on the new bulb and fixture and use a 7 64 inch hex wrench or a slotted screwdriver to tighten the screw until the clamp is secure 7 Repeat steps 4 through 6 to replace the three remaining bulbs and fixtures 8 Replace the front diffuser screen and clip support and reinsert the two screws 9 Reconnect the power cord and turn on the Image Eraser figure 14 1 14 3 Changing the fuses in the Image Eraser If the fuses must be replaced repeatedly the Image Eraser may have an electrical problem Do not use the Image Eraser Contact MD Scanner Technical Support See Assistance in the preface for contact information You can use the Image Eraser with either a 220 volt to 240 volt 230V power source or a 10
3. Chapter 2 Safety When using the Image Eraser follow the precautions below Always plug the power cord into a grounded outlet Before cleaning the Image Eraser always turn off the power and unplug the eraser Follow the instructions in section 14 1 e Before changing a bulb always turn off the power and unplug the eraser Follow the instructions in section 14 2 e When changing a bulb do not remove covers other than the front diffuser panel There are no user serviceable parts inside e Before changing a fuse always turn off the power and unplug the eraser Follow the instructions in section 14 3 e If the covers become damaged do not use the eraser Contact M D Scanner Technical Support for repair See Assistance in the preface for contact information e If fuses must be replaced repeatedly do not use the eraser Contact M D Scanner Technical Support for repair Computer and monitor See the manufacturer s computer and monitor precautions before opening the computer or monitor 2 3 Laser light safety Using controls making adjustments or performing procedures other than those specified herein may result in hazardous laser light exposure The Typhoon instrument is a Class laser instrument that houses two Class IIIB lasers and under the specified operating procedures does not allow operator exposure to laser light The lasers with power from 10 mW to 20 mW are accessible in the interior of the instrument
4. For samples that require a higher resolution choose the 100 microns setting The 100 micron pixel size produces 100 data points per centimeter and 200 data lines per grid square e For samples that require very high resolution choose the 50 microns setting The 50 micron pixel size produces 200 data points per centimeter and 400 data lines per grid square After you change the pixel size Scanner Control updates the image size the scan time and the number of data lines per channel in the Scanning Information area figure 9 1 9 1 7 Selecting the focal plane The Focal Plane area allows you to adjust the focal point of the laser Two settings are available e Platen Use this setting if you placed a thin less than 2 mm or surface sample such as a gel or membrane directly on the glass platen of the Typhoon instrument Scanner Control adjusts the focal point of the laser beam to the area just above the glass platen of the Typhoon instrument p9 6 Typhoon User s Guide Important Chapter 9 Scanning a fluorescent sample 3 mm Use this setting if you placed a microplate or a sandwich gel on the glass platen of the Typhoon instrument Scanner Control adjusts the focal point of the laser beam 3 millimeters higher than the glass platen of the Typhoon instrument If you used different thicknesses of electrophoresis glass plates make sure you place the 3 mm electrophoresis glass plate of the sandwich gel on the glass plate
5. Part five Maintaining the Typhoon system hardware Important Important 1 Close the filter door and tighten the fingernut Make sure you tighten the fingernut The Typhoon instrument will not scan if the fingernut is loose 2 Close the access door and use a 9 64 inch hex wrench to tighten the screw 3 Align the pins on the instrument frame with the holes on the front cover and gently push the cover back into place 4 Usea 9 64 inch hex wrench to tighten the two socket head screws at the bottom of the front cover 13 3 6 Registering a new optical filter in Scanner Control Every time you start the Scanner Control software Scanner Control checks the current optical emission filters installed in the instrument If Scanner Control detects a filter with a new bar code Scanner Control displays the Define the Filter window so that you can define the filter 1 Start the Scanner Control software The Define the Filter window appears figure 13 9 Define the Filter x An unregistered filter has been detected in the Typhoon Please provide additional information below Bar Code Type Name Peak WL BW 11 Band Pass 7 cv3 555 20 Don t Register Now Register Filter Figure 13 9 The Define the Filter window If you installed more than one filter make sure you enter the correct filter information for the bar code number displayed in the window 2 Inthe Type list select the type of filter that you installed
6. Sample placement in the Screen placement on the For face up sample For face down sample exposure cassette glass platen choose choose E A1 corner A1 corner Al corner poe sac ich Gepost Al corner Al corner p weeperoperryerey Al corner Figure 6 6b Determining which orientation button to use SESE p6 10 Typhoon User s Guide Chapter 6 Scanning a storage phosphor screen To display the orientation buttons place the pointer on the button next to Orientation in the O ptions area and hold down the mouse button The orientation buttons appear Using figure 6 6 as a guide select the orientation button that you want to use and then release the mouse button During the scan the software maps the pixels to permit display of the sample image face up and top end up in ImageQuant 6 4 5 Selecting the pixel size Pixel size refers to the size of each individual picture element that is recorded and together with the thousands of other pixels forms the image You choose the pixel size from the Pixel Size list in the Setup area of the Scanner Control window figure 6 4 For most standard electrophoresis samp
7. 12 1 6 Selecting the pixel size Pixel size refers to the size of each individual picture element that is recorded and together with the thousands of other pixels forms the image You choose the pixel size from the Pixel Size list in the Setup area of the Scanner Control window figure 12 1 e For most samples choose the 200 microns setting which provides the fastest scan time and the smallest image file size The 200 micron pixel size produces 50 data points per centimeter and 100 data lines per grid square For samples that require a higher resolution choose the 100 microns setting The 100 micron pixel size produces 100 data points per centimeter and 200 data lines per grid square For samples that require very high resolution choose the 50 microns setting The 50 micron pixel size produces 200 data points per centimeter and 400 data lines per grid square After you change the pixel size Scanner Control updates the image size the scan time and the number of data lines per channel in the Scanning Information area figure 12 1 p12 6 Typhoon User s Guide Important Chapter 12 Scanning a chemiluminescent sample 12 1 7 Selecting the focal plane The Focal Plane area allows you to adjust the focal point of the laser Two settings are available Platen Use this setting if you placed a thin less than 2 mm membrane directly on the glass platen of the Typhoon instrument Scanner Control adjusts the focal po
8. Access door open Filter door Access door open Prongs Filter door open Figure 13 7 Removing the front cover and opening the access and filter doors p13 10 Typhoon User s Guide Warning A Caution Chapter 13 Maintaining the Typhoon instrument 13 3 3 Inserting a new optical filter You must leave the Typhoon instrument turned on when you change the optical filter selection After you remove the front cover of the Typhoon instrument and open the access and filter doors make sure you follow the procedures in this section to avoid injury Before you insert the optical filter into the filter changer make sure the filter is clean If the filter is not clean e Remove dust using puffs from a dust free blower e Remove water spots using a soft cloth e Remove fingerprints using a lens tissue and a few drops of spectroscopic grade isopropyl alcohol Before using the Typhoon Direct Instrument Access Web page make sure you close the Scanner Control software Leaving Scanner Control running while you use the Web page can cause unpredictable problems in Scanner Control You use the Insert a N ew Optical Filter into the Filter Changer Web page to insert a new filter If you do not see this page return to section 13 3 1 1 Makesurea bar code label is attached squarely to the top of the filter holder as shown in figure 13 8 Bar code za S Notch gt 37 xq Figure 13 8 The optical fil
9. Displays the order in which the scans will be performed by the Typhoon instrument Emission Filter list Allows you to select the emission filters to use for each scan After you select the emission filter Scanner Control determines the most appropriate laser to use with the selected filter PMT Voltage list Allows you to select the PMT voltage to use for each scan Values range from 0 to 1 000 and are in 5 volt increments Laser list Allows you to change the laser that Scanner Control selected as the most appropriate laser to use with the emission filter You choose either Green 532 or Red 633 Sensitivity list Allows you to select the sensitivity limit of detection to use for each scan Normal collects one data point from each pixel Medium collects four data points from each pixel and averages the results High collects eight data points from each pixel and averages the results Beamsplitters list After you link scans displays the beamsplitter you should use for the linked scan Allows you to change the selected beamsplitter You can choose either 560 or 580 Link With area Scan buttons Allows you to select the scans you want to link Scanner Control displays brackets that connect the Use boxes of the linked scans Link With area Auto Link button Allows you to link the scans automatically Scanner Control links the optimal scans displays brackets connecting the Use boxes of the
10. Index cleaning 5 4 condensation 5 8 enhancers 2 9 5 2 erasing 5 4 exposing 5 8 exposure guidelines 5 6 exposure temperature 5 8 exposure time 4 2 5 11 function 4 1 GP description 4 3 LE description 4 4 light leak A 4 linear dynamic range 4 2 loading in instrument 6 5 mounted 4 3 placing in cassette 5 8 5 11 protecting 5 3 removing from exposure cassette 6 4 saturated data 6 15 scintillants 2 9 5 2 sensitivity 4 2 solvents 5 8 storing 6 17 TR description 4 4 units 5 2 unmounted 4 3 using plastic wrap 5 2 substrate 10 2 11 1 surge protector 2 4 T template creating 3 8 Delete Template s window 3 12 deleting 3 12 modifying 3 11 Save As Template window 3 11 selecting 3 11 selecting default 3 12 selecting for chemiluminescence 12 2 selecting for fluorescence 9 2 Typhoon User s Guide Index 7 Index selecting for storage phosphor 6 7 selecting options illustrated 3 9 selecting setup options illustrated 3 10 terminology computer xii timer on eraser 5 5 TLC plate exposing 5 6 guidelines for using 5 1 orientation on glass platen 8 6 precaution 2 9 TR screen see also storage phosphor screen caution 5 8 cleaning 5 4 description 4 4 erasing 5 4 using with wet gel 5 2 trained operator definition xi transparency support 8 4 troubleshooting A 1 Typhoon Direct Instrument Access Web page 13 1 to 13 2 Typhoon instrument changing fuse 13 16 contamination 6 1 electrical safety 2 4 fuse location 13 16 f
11. e Clean the storage phosphor screen section 5 2 3 e Erase the storage phosphor screen section 5 2 4 e Check the Image Eraser for contamination and clean the eraser section 14 1 e Check the glass platen and sample lid for contamination and clean the glass platen and sample lid of the Typhoon instrument section 6 1 e Check the exposure cassette for contamination and clean the exposure cassette section 14 4 p6 16 Typhoon User s Guide Chapter 6 Scanning a storage phosphor screen 6 7 Storing the storage phosphor screen Before storing the storage phosphor screen you should clean decontaminate if necessary and erase the screen Then store the screen e Atroom temperature Away from sources of radiation such as strong beta or gamma emitters or x ray machines e Ina protective box or in a clean exposure cassette Damaged screens are unusable and must be replaced 6 8 Analyzing or preprocessing the scanned image You should display the image of the sample to determine the quality of the scan If you selected image analysis software in the Scanner Control window the software opens and displays the image Refer to the applicable user s guide or online H elp for information on how to use the selected software Typhoon User s Guide p6 17 Part three Scanning in the fluorescence mode Chapter 7 About fluorescence scanning This chapter describes the theory behind fluorescence The topics in this
12. e Selecting a laser shut off time section 13 2 e Changing the optical filter selection section 13 3 e Changing the definition of an optical filter section 13 4 e Changing the fuses section 13 5 e Moving the Typhoon instrument section 13 6 13 1 Starting the Typhoon Direct Instrument Access Web page Several maintenance procedures use the Typhoon Direct Instrument A ccess Web page to control the instrument or change settings that affect the instrument To use the Typhoon Direct Instrument Access Web page you must have Internet Explorer installed on the computer controlling the Typhoon instrument If you use a proxy server to access the Internet you must modify the Internet options to exclude the address 192 168 1 1 or the IP number you are using with the Typhoon instrument from the proxy server For more information on proxy servers see the Molecular Dynamics Web page www mdyn com or your browser software documentation To display the Typhoon Direct Instrument Access Web page 1 Close Scanner Control Before using the Typhoon Direct Instrument Access Web page make sure you close the Scanner Control software Leaving Scanner Control running while you use the Web page can cause unpredictable problems in Scanner Control 2 Start the browser software Typhoon User s Guide p13 1 Part five Maintaining the Typhoon system hardware ei 3 Type the following in the Address box http 192 168 1 1 or the IP addr
13. ma Nant oes Cherri EE Figure 9 2 Selecting the scan area Typhoon User s Guide p9 3 Part three Scanning in the fluorescence mode oo 9 1 4 Selecting the sample orientation You choose the orientation from the Setup area in the Scanner Control window figure 9 1 The orientation buttons allow you to change how the scanned image will be displayed in ImageQuant To do this select the icon that represents how you aligned the upper left corner of the sample with the grid markings on the glass platen Figure 9 3 describes each sample orientation option If you placed the sample on the glass platen so that the top of the sample was aligned with the numbered rows you can use the orientation buttons to display the top of the sample at the top of the mage window in ImageQ uant To display the orientation buttons place the pointer on the button next to Orientation in the O ptions area and hold down the mouse button The orientation buttons appear M ove the pointer to the button that represents how you aligned the sample on the glass platen and release the mouse button During the scan the software maps the pixels to permit display of the sample image face up and top end up in ImageQ uant p9 4 Typhoon User s Guide Chapter 9 Scanning a fluorescent sample Orientation buttons Upan miin A R H ce Pua JE Sample placement on the For face up sample For face down sample glass platen choose choose
14. The safety interlocks figure 2 2 in the Typhoon instrument are designed to prevent you from being exposed to the laser beams If you open the sample lid while the scanner is in operation the laser shutter closes and blocks the light from the lasers Do not attempt to defeat the safety interlocks on the sample lid or try to gain access to the interior of the instrument through any other opening Exposure to laser light can cause injury For example viewing the laser light directly can cause blindness Typhoon User s Guide p2 7 Part one Introduction 2 3 1 Laser light warning labels The labels shown in figure 2 6 warn of exposure to laser light The exact locations of the labels are shown in figure 2 2 CLASS 1 LASER PRODUCT Per IEC 825 CAUTION DANGER Laser radiation when open and interlock defeated AVOID DIRECT EXPOSURE TO BEAM VORSICHT GEFAHR Laserstrahlung wenn offen und Verriegelung ausgeschaltet DIREKTE BESTRAHLUNG VERMEIDEN CAUTION Laser Radiation when open AVOID EXPOSURE TO BEAM ATTENTION DANGER L ouverture du couvercle entraine une exposition au rayonnement laser si le dispositif de verrouillage a t mis hors service EVITER TOUTE EXPOSITION DIRECTE AU FAISCEAU International compliance Eye exposure Direct exposure Figure 2 6 Laser light warning labels Caution Vorsicht Attention Danger Laser radiation when open and interlock defeate
15. Thescan area you selected appears in white The Scanning Information area displays the image file size the approximate scan time and the number of lines per channel Co a e Uram leben fix pple im IFEN Lead hija TI Taim Fi i HHHH EPCLELCLLT TET CTILC A A D u AHHH HH po A A HH A AGH HG LH VL L E j ipea a a a e a u HHHH HHH HH feo HIH amp em T O E b m o mn CrO A1 corner Sea Ay 12d RR PEP HNMR BTA Oe Ms Fama fps e Sham Fe i i Tmi liae ere H imge imh ows Career 5 E Figure 12 2 Selecting the scan area Typhoon User s Guide p12 3 Part four Scanning in the chemiluminescence mode 12 1 4 Selecting the sample orientation You choose the orientation from the Setup area in the Scanner Control window figure 12 1 The orientation buttons allow you to change how the scanned image will be displayed in ImageQ uant To choose an orientation select the icon that represents how you aligned the upper left corner of the sample with the grid markings on the glass platen Figure 12 3 describes each orientation option N ote If you placed the sample on the glass platen so that the top of the sample was aligned with the numbered rows you can use the orientation buttons to display the top of the sample at the top of the Image window in ImageQ uant To display the orientation buttons place the pointer on the button next to Orientation in the O ptions area and hold down the mouse button The
16. and monitor are high voltage electronics Typhoon instrument In general you do not need to access the interior of the Typhoon instrument H owever to install or remove an optical emission filter you must remove the front cover while the instrument is turned on If you need to install or remove emission filters make sure you follow the procedures in section 13 3 Do not remove the front cover of the instrument unless you are installing or removing optical emission filters There are no other user serviceable components inside When you change the optical filter selection you must leave the Typhoon instrument turned on After you remove the front cover of the Typhoon instrument and open the access and filter doors make sure you follow the procedures in section 13 3 to avoid injury Image Eraser Because the Image Eraser uses high voltage you must disconnect the power cord from the eraser before performing any maintenance task Figure 2 5 displays the high voltage hazard label on the Image Eraser See figure 2 3 for the exact location of the label CAUTION DISCONNECT POWER BEFORE SERVICING ATTENTION COUPER LE COURANT AVANT L ENTRETIEN WARNUNG VOR DER WARTUNG VOM NETZ TRENNEN Figure 2 5 High voltage hazard label on the Image Eraser Disconnect power before servicing Couper le courant avant l entretien Vor der wartung vom netz trennen p2 6 Typhoon User s Guide Warning A Warning A
17. and then lift up the opposite edge of the screen 3 Protect the screen from direct light and proceed immediately to the next section p6 4 Typhoon User s Guide Important Chapter 6 Scanning a storage phosphor screen 6 3 Loading the storage phosphor screen into the instrument To load the storage phosphor screen into the Typhoon instrument 1 If the sample lid is not already open push up on the lid release under the center front of the sample lid until the lid opens figure 6 1 2 Makesure the white phosphor side of the screen is face down 3 Usethe grid markings that surround the glass platen to align the Al corner of the screen with the Al corner of the glass platen figure 6 3 If you place a small screen so that the J1 corner of the screen aligns with the A1 corner of the glass platen make sure you use the orientation buttons section 6 4 4 in the Scanner Control window to rotate the scanned image Top view Back of the Typhoon instrument Glass platen Small screen as viewed from above Label Al corner Front of the Typhoon instrument Figure 6 3 Load the screen onto the glass platen of the Typhoon instrument Typhoon User s Guide p6 5 Part two Scanning in the storage phosphor mode E 4 Gently lower the screen on the glass platen N ote T he unmounted screen may not lie completely flat against the glass at this time When you initiate the scan the instrument lowers the in
18. anual m 63002831 Typhoon User s Guide for Microsoft Windows NT and Apple Mac OS molecular dynamics part of Amersham Pharmacia Biotech Amplify Cy ECF Fluorlmager FluorSep ImageQuant Molecular Dynamics Typhoon Vistra Green and Wonder Wedge are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia amp Upjohn Inc Apple and Mac are trademarks of Apple Computer Inc BODIPY NanoOrange PicoGreen RiboGreen SYBR SYPRO Texas Red and TOTO are trademarks of Molecular Probes Inc Coomassie is a trademark of Imperial Chemical Industries Ltd Ethernet is a trademark of Xerox Corporation FAM HEX ROX TAMRA and TET are trademarks of the Perkin Elmer Corporation Kapton and Mylar are trademarks of DuPont Corporation Kodak is a trademark of Eastman Kodak Company Microsoft and Windows NT are trademarks of Microsoft Corporation SeaKem is a trademark of FMC Corporation Tween is a trademark of ICI Americas Inc Whatman is a trademark of Whatman International Ltd The Typhoon instrument is covered by one or more of the following U S patents 5 528 050 5 578 818 and foreign equivalents The Typhoon system is for research purposes only It is not intended or approved for diagnosis of disease in humans or animals All goods and services are sold subject to the terms and conditions of sa
19. beamsplitter If you select emission filters and a beamsplitter that are separated by less than 5 nm Scanner Control disables the Auto Link button To activate the Auto Link button change the emission filters to more optimal pairs After you click the Auto Link button Scanner Control links the scans that display the colored dots Brackets appear connecting the Use check boxes and the Scan numbers change to indicate which scans Scanner Control combined into one scan figure 9 5 Manually linking scans If you do not want to use the Auto Link feature you can manually link two scans using the link buttons in the Link With area For each scan that can be linked link buttons 1 2 3 4 appear in the Link With area An optimal link displays a colored button above the link button N onoptimal links do not display a colored button You can create both optimal and nonoptimal links In the Link With area for one of the scans you want to combine click the link button that corresponds to the scan you want to combine with this scan After you select the link button brackets appear connecting the Use check boxes for the linked scans In addition the Scan numbers change to indicate which scans you combined into one scan For example figure 9 5 a shows scan 1 can be linked to either scan 2 or 3 The colored dot above the link button for scan 3 shows that the optimal link is between scan 1 and scan 3 To manually link scan 1 and scan 3 you clic
20. chapter are How fluorescence works section 7 1 e Advantages of direct fluorescence section 7 2 e Emission filters section 7 3 Beamsplitters section 7 4 e Multilabel multichannel experiments section 7 5 Common fluorochromes section 7 6 7 1 How fluorescence works The absorption of light energy by a fluorochrome boosts an electron to a higher energy shell excited state The characteristic light absorption pattern of a fluorochrome is shown by its absorption spectrum figure 7 1 The excited state has a very short half life on the order of a few nanoseconds During the brief lifetime of the excited state the excited electron generally decays toward a lower energy level within the excited state When the electron falls to the ground state the fluorochrome emits light of specific wavelengths The distribution of fluorescent emissions among different wavelengths is plotted in the emission spectrum figure 7 1 The fluorescent emission spectrum is always shifted toward a longer wavelength lower energy relative to the excitation spectrum The difference in wavelength between the maxima of the absorption and the emission spectra is called the Stokes shift Because of this shift optical filtration can be used to separate excitation light from the longer wavelength emitted light For more information on how fluorescence is generated see M olecular Dynamics Technical N ote 57 Understanding Fluorescence See app
21. collected by either one or two PM Ts Scanner Control creates an image file and automatically adds the correct file extension which is either Data file gel Creates a single image file Dataset file ds Creates the set of files and folders required for a multichannel image If you are scanning the sample using a beamsplitter Scanner Control adds this extension N ote If you select or type the wrong file extension Scanner Control changes the file type back to the correct extension p12 10 Typhoon User s Guide Chapter 12 Scanning a chemiluminescent sample 12 3 1 Starting a scan To start a scan 1 Make sure that the sample is in place the sample lid is shut and all the settings are correct in the Scanner Control and Chemiluminescence Setup windows 2 Inthe Scanner Control window click the Scan button The Save As window appears figure 12 5 ave ae remp ora tie Windows NT Mac OS Figure 12 5 The Save As window Caution 3 In the Save As window type a name in the Windows NT File name box e Mac OS Saveas box If you type a name and extension that has already been used a message appears and asks you if you want to replace the existing file Before you click Yes make sure you want to replace the existing image file Clicking Yes deletes the image and all the associated auxiliary files If you have analyzed the image using ImageQuant all the data will be dele
22. double the erasure time If a fluorescent bulb in the Image Eraser needs to be replaced you should replace all four bulbs before using the eraser To replace the bulbs see section 14 2 Front view Side view Image Eraser Clip Storage phosphor screen white side toward eraser Time Erase button Power switch ae Eee ope Bs Figure 5 1 Erasing the storage phosphor screen Typhoon User s Guide p5 5 Parttwo Scanning in the storage phosphor mode 5 3 Guidelines for exposing storage phosphor screens Use the following guidelines when exposing the storage phosphor screen Use an exposure cassette to expose the screen to the sample The exposure cassette seals out light and keeps the sample flat against the storage phosphor screen during exposure To expose multiple samples on the same screen make sure the samples are the same thickness If the samples are not the same thickness the thinner sample will not contact the screen uniformly which can result in a bad exposure poor image and poor quantitation results Some samples such as wet gels or TLC plates are too thick to fit in an exposure cassette For thick samples expose the screen in a light tight drawer or other light tight enclosure Place the screen on the sample correctly the first time Caution Adjusting the position of the screen following initial placement can result in a double image If you must adjust
23. e Lowering the inner lid of the Typhoon instrument to hold the sample flat against the glass platen 12 3 2 Monitoring the scan progress After you start the scan the ImageQ uant Preview window figure 12 6 appears and the green Scan indicator light on the top of the instrument blinks As the Typhoon instrument scans the sample Scanner Control displays the image in the ImageQ uant Preview window In addition Scanner Control displays the number of data lines scanned and the total scan time remaining excluding initialization Scanner messages may also appear as the scan progresses p12 12 Typhoon User s Guide Chapter 12 Scanning a chemiluminescent sample Leen Serd E Total Scan Tea Heeg fa Joi SrH Mt le TT lt Figure 12 6 The ImageQuant Preview window showing a chemiluminescent sample A More Info button appears in the ImageQ uant Preview window when the Typhoon reports errors Click the M ore Info button to view the error message If the error isa nonfatal error the instrument continues to scan and Scanner Control saves the file when the scan is finished You should check the image in ImageQ uant to make sure the data are not corrupted Ifthe error is a fatal error the instrument aborts the scan and Scanner Control saves the data collected before the fatal error occurred You should check the image in ImageQ uant Depending on where the fatal error occurred in thescan you might be able to use the da
24. lid opens figure 6 1 2 Optional Check the glass for chemicals 3 Clean the glass with distilled water and a clean lint free cloth or paper If visible spots remain clean the glass first with 75 ethanol and then with distilled water Caution Because window cleaners use ingredients that can fluoresce do not use window cleaners to clean the glass platen In addition the use of acetone or the excessive use of ethanol can shorten the lifetime of the instrument Protect the glass from scratches Scratches interfere with accurate imaging and quantitation p6 2 Typhoon User s Guide Chapter 6 Scanning a storage phosphor screen Pushupon the lid release Sample lid open DONO ATTAIN LATTA Figure 6 1 Opening the sample lid 6 1 3 Cleaning the sample lid Under normal circumstances the surface of the inner lid should not come in contact with contaminants H owever it is good laboratory practice to check the surface periodically for contamination For example you can perform a wipe test or use the procedure that you used to check the glass platen section 6 1 1 except place the screen in the instrument with the phosphor side up The surface of the inner lid should be clean and free of contamination from radioactive samples If necessary clean the surface with a damp not saturated cloth moistened with a small amount of distilled water If visible spots remain clean the surface first with 75 et
25. 2 wet gel see also sample caution 5 8 exposing 5 2 guidelines for using 5 2 window cleaners using 8 5 11 2 windows Chemiluminescence Setup description 12 8 Chemiluminescence Setup quick reference B 5 Define the Filter 13 14 13 16 Delete Template s 3 12 Fluorescence Setup description 9 8 Fluorescence Setup quick reference B 4 ImageQuant Preview 6 14 9 17 12 13 Save As 6 13 9 15 12 11 Save As Template 3 11 Scanner Control chemiluminescence 12 2 fluorescence 9 2 quick reference B 2 storage phosphor 6 6 workflow chemiluminescence 3 6 fluorescence 3 5 storage phosphor 3 3 Typhoon User s Guide Index Index 9 Index Index 10 Typhoon User s Guide
26. 810 UNV 100 120 220 240V 1 5A 50 60Hz 62 SERIAL en torn IARAA Ge MANUFACTURED AA AA AAA AAAA a T 1 5A 250V B1 Figure 2 1 The serial number certification labels p2 2 Typhoon User s Guide Chapter 2 Safety ooo Frontview gt 1 Serial number E3 i as i molecular dynamics sunnyvale CA 94086 IOAW an NE CE i 4 6 srar e a eee This product conforme to he applicable equremonta of TOF subchapter J at th dave fm 2 Laser light eye exposure warning Right side view 4 A CETE EEANN a Top view with sample lid open _ J TSA 250V 5 General warning 6 High voltage Back view 7 Laser light international compliance CLASS 1 Per IEC 825 1 f f 1 f f 1 f f i f l LASER PRODUCT f f 1 f f 1 f f 1 f f 1 Figure 2 2 Location of important labels on the Typhoon instrument Typhoon User s Guide p2 3 Part one Introduction Right side view 2 High voltage warning CAUTION DISCONNECT POWER BEFORE SERVICING ATTENTION COUPER LE COURANT AVANT L ENTRETIEN WARNUNG VOR DER WARTUNG VOM NETZ TRENNEN 3 Serial number A 928 E Arques Ave aaRolecular dynamics Sunnyvale CA 94086 MODEL 810 UNV 100 120 220 240V 1 5A 50 60Hz C SERIAL E C mnano AAAA No SIISII m Figure 2 3 Locat
27. Al corner Al corner Al corner Figure 9 3 Determining which orientation button to use Typhoon User s Guide p9 5 Part three Scanning in the fluorescence mode Important Caution 9 1 5 Selecting the Press Sample option The Press Sample option is not saved as part of a template You must select this option each time you scan If you are scanning a sample such as a dry membrane or filter paper that does not lie flat against the glass platen of the instrument you should select the Press Sample check box When you scan the sample the inner lid of the Typhoon instrument lowers to press the sample flat If you do not select Press Sample the inner lid remains retracted during the scan Do not select Press Sample if you are scanning wet membranes wet gels or soft samples Pressing wet or soft samples can damage the instrument In addition pressing a soft sample can distort the sample and the image 9 1 6 Selecting the pixel size Pixel size refers to the size of each individual picture element that is recorded and together with the thousands of other pixels forms the image You choose the pixel size from the Pixel Size list in the Setup area of the Scanner Control window figure 9 1 e For most samples choose the 200 microns setting which provides the fastest scan time and the smallest image file size The 200 micron pixel size produces 50 data points per centimeter and 100 data lines per grid square
28. Chemiluminescence Setup window without saving any changes OK button Saves the changes and closes the Chemiluminescence Setup window Typhoon User s Guide pB 5 Appendix C Literature references The following references are cited in this manual Fluorescence Imaging Applications Guide Molecular D ynamics Publication 7500 9806 Sonada M M Takano J M ayahara H Kato 1983 Computed radiography utilizing scanning laser stimulated luminescence Radiology 148 833 838 Understanding Fluorescence M olecular D ynamics Technical N ote 57 Using Dyes and Filters in the Fluorlmager System M olecular D ynamics Technical N ote 58 Typhoon User s Guide pC 1 Glossary ds the file extension for a multichannel dataset See also datasets gel the file extension of a single channel image absorption the energy transfer from a photon of light to a dye molecule acquisition mode the type of scan Choices are storage phosphor fluorescence and chemiluminescence bandpass filter an optical filter that allows light of wavelengths between two specified wavelength cutoffs to pass through The filter rejects most of the light with wavelengths shorter than the first cutoff or longer than the second cutoff channel one image in a set of images that make up a multichannel image For a chemiluminescent sample two channels are created using a beamsplitter For a fluorescent sample two three or fou
29. Place the storage Place the screen on the sample To avoid a 5 6 phosphor screen double image of the sample do not reposition on the sample the screen after it has touched the sample 6 Expose the storage Leave the screen in the exposure cassette or 5 7 phosphor screen to _ light tight drawer until the screen is exposed the sample To determine the length of exposure consider a 2 hour exposure to the screen to be equal to an overnight exposure to basic x ray film Typhoon User s Guide p3 3 Part one Introduction Table 3 1 Storage phosphor scanning workflow continued Task Description Section Scanning the storage phosphor screen 1 Prepare the Decontaminate optional and clean the 6 1 Typhoon instrument glass platen and sample lid of the Typhoon instrument 2 Place the storage In subdued light remove the storage phosphor 6 2 phosphor screen screen from the exposure cassette or light tight 6 3 in the Typhoon drawer Keep the screen face down while you instrument move it from the cassette to the instrument With the screen still face down position the screen on the glass platen so that the Al corner of the screen aligns with the Al corner of the glass platen Close the sample lid 3 Select scan settings In the Scanner Control window select an 6 4 using the Scanner existing template that contains the scan settings Control software you want to use or manually select the settings To manua
30. Reflects light of wavelengths shorter than 580 nm and passes light longer than 580 nm The instrument uses the beamsplitter you select in the Chemiluminescence Setup window of the Scanner Control software section 12 2 3 to create two images from a single scan This optimizes scanning of samples containing two chemiluminescent substrates by reducing the scan time The beamsplitter separates the emissions from the substrates so that one PM T collects the longer wavelength emissions while the other PM T collects the shorter wavelength emissions figure 10 2 Typhoon User s Guide p10 3 Part four Scanning in the chemiluminescence mode C O Emission filter Mirror Al Short wavelength Emitted light Long wavelength Beamspiitter Emission filter Figure 10 2 How the beamsplitter separates short and long wavelength emissions E p10 4 Typhoon User s Guide Important Chapter 11 Setting up for chemiluminescence scanning This chapter describes how to prepare a sample for chemiluminescence scanning and how to place the sample in the Typhoon instrument The topics in this chapter are e Preparing the sample section 11 1 e Cleaning the glass platen and sample lid section 11 2 e Loading the sample section 11 3 11 1 Preparing the sample You should follow the manufacturer s guidelines for preparing membranes or microplates for chemiluminescence scanning To avoid compromising the chemistry o
31. Starting a scan and checking the progress 0 0 00 9 14 9 3 1 Starting a Scan noaua aaa 9 15 9 3 2 Monitoring the scan progress saana aaa 9 17 9 3 3 Viewing the preview image for saturated data 9 18 9 3 4 Completing the scan cece eee 9 19 9 4 Removing the sample from the Typhoon instrument 9 19 9 5 Cleaning the glass platen and sample lid naana 9 19 9 6 Analyzing or preprocessing the scanned image 005 9 19 E Typhoon User s Guide vii Table of Contents viii Typhoon User s Guide Part four Scanning in the chemiluminescence mode Chapter 10 About chemiluminescence scanning 10 1 How chemiluminescence works 0 0000 e eee eee 10 1 10 2 Advantages of chemiluminescence 0 cece eee ee eee ee 10 1 10 3 How to optimize the chemiluminescent experiment 10 2 10 4 Beamsplitters i siarsio euio a a ote waa ean 10 3 Chapter 11 Setting up for chemiluminescence scanning 11 1 Preparing the sample aaau 11 1 11 2 Cleaning the glass platen and sample lid aaa aaa 11 1 11 2 1 Cleaning the glass platen oaan naaa 11 1 11 2 2 Cleaning the sample lid 0 00 a 11 3 11 3 Loading the sample anaana 11 3 11 3 1 Determining the sample orientation 11 3 11 3 2 Determining the sample placement 11 3 11 3 3 Placing a sample on the glass platen 11 4 Chapter 12 Scanning a chemiluminescent
32. TR screen Store the screen in the exposure cassette or other dark place 3 4 2 Workflow for fluorescence scanning Table 3 2 describes the workflow for fluorescence scanning and lists the sections in this user s guide that contain the procedural steps for each task Table 3 2 Fluorescence scanning workflow Task Description Section Preparing for fluorescence scanning 1 Prepare the sample Prepare the sample using fluorescent dyes that 8 1 are compatible with the green 532 nm or the red 633 nm excitation mode 2 Prepare the Typhoon Decontaminate optional and clean the glass 8 2 instrument platen and sample lid of the Typhoon instrument Scanning the fluorescent sample 1 Place the sample In subdued light position the sample on the 8 3 in the Typhoon glass platen Make a note of the grid coordinates instrument on the glass platen Close the sample lid 2 Select scan settings In the Scanner Control window select an 9 1 using the Scanner existing template that contains the scan settings 9 2 Control software you want to use or manually select the settings To manually select the settings make sure Fluorescence is the selected scan acquisition mode Then select the grid area pixel size sample orientation press sample optional focal plane and image analysis software from the Scanner Control window Then click Setup and select the emission filter PMT voltage laser and sensitivity for
33. The Typhoon Typhoon User s Guide p4 1 Parttwo Scanning in the storage phosphor mode instrument uses a bandpass filter which allows the light near the peak emissions of the screen to pass through to the detector The detector collects and measures the light The emitted light intensity is proportional to the radioactivity in the sample Emission Absorption 390 600 Excitation eat 633 t t t 300 400 500 600 700 800 Wavelength nm Figure 4 1 Emission and absorption spectra of the activated storage phosphor screen The wavelength with maximum stimulation or emission is shown above the curves These curves are approximations based on data presented in an article by Sonoda et al 1983 See appendix C for a reference list 4 2 Advantages of storage phosphor screen autoradiography Storage phosphor screen autoradiography offers many advantages over traditional film autoradiography p4 2 Typhoon User s Guide Exposure time is approximately one tenth that of traditional autoradiography using x ray film Sensitivity is 10 to 100 times that of film depending on the isotope used and the sample type The linear dynamic rangeis 1 to 100 000 5 orders of magnitude Film has a linear dynamic range of only 1 to 500 2 5 orders of magnitude The linear dynamic range of the storage phosphor screen allows both weak and strong signals to be visualized and quantitated in a singl
34. The choices are long pass short pass and bandpass 3 Inthe Name box typea name for the filter For example use the name of a fluorescent dye that you will scan using this filter 4 In the Peak WL box type the wavelength of the filter For example type 555 for a 555 nm filter p13 14 Typhoon User s Guide 5 Chapter 13 Maintaining the Typhoon instrument If the filter type you selected in step 2 is bandpass the BW box is active Type the bandwidth of the bandpass filter For example type 20 if the bandwidth is 20 nm Click Register Filter If you click Don t Register N ow you can use the Typhoon instrument for scanning but you will not be able to use the new filter O ptional If you installed more than one filter the D efine the Filter window appears again and displays the bar code number for the next filter you inserted Repeat steps 2 through 6 to register the additional filter s You can now use the new optical filter s for scanning 13 4 Changing the definition of an optical filter After you install an optical emission filter in the Typhoon instrument and define the filter in Scanner Control you can change the definitions for the filter To do this 1 In the Scanner Control window choose Fluorescence as the scan acquisition mode and then click the Setup button The Fluorescence Setup window appears Select the emission filter you want to redefine from the Emission Filter list To display
35. Typhoon system works 0 0 e cece eee eee 1 3 1 3 Typhoon scan acquisition modes cc eee ees 1 4 1 4 Before you begin 6 tee 1 4 Chapter 2 Safety 2 1 General safety precautions 0 cee 2 1 2 2 Electrical safety 0 0 0 cece 2 4 2 2 1 Electrical connections 0 2 0 00 c eee 2 4 22 2 BUSES Renia wee edn di den Ta wa eek BG 2 5 2 2 3 High voltage hazard and precautions 2 6 Typhoon User s Guide iii Table of Contents iv Typhoon User s Guide 2 3 Laserlightsafety 0 cee 2 7 2 3 1 Laser light warning labels 0000 cee eee ee 2 8 2 3 2 Safety precautions ccc ee eee eens 2 8 2 4 Hazardous materials precautions 0c cece eee ees 2 9 Chapter 3 Getting started 3 1 Turning on and warming up the Typhoon instrument 3 1 3 2 Turning on the computer 00 cece eee 3 2 3 3 Opening the Scanner Control software saasa eee 3 2 3 4 Workflows forscanning 00 0 0 eects 3 2 3 4 1 Workflow for storage phosphor scanning 3 3 3 4 2 Workflow for fluorescence scanning 000 3 5 3 4 3 Workflow for chemiluminescence scanning 3 6 3 5 Using templates 02 nace abe a dada begeee de ee Reed 3 8 3 5 1 Creating a new template 0 c eee eee 3 8 3 5 2 Selecting a template 0 cee eee eee 3 11 3 5 3 Modifyingatemplate 0 eee eee eee 3 11 3 5 4 Selecting a tem
36. aintenance information for the Typhoon instrument and accessories Warnings A Chapter 2 Safety The Typhoon instrument and accessories have been designed for safe operation It is imperative that you follow the precautions in this chapter The topics in this chapter are General safety precautions section 2 1 e Electrical safety section 2 2 e Laser light safety section 2 3 Hazardous materials precautions section 2 4 2 1 General safety precautions The operator of the Typhoon system is assumed to be trained in the correct operation of the instrument and the safety issues Throughout the Typhoon User s Guide the word you refers to this trained operator Using controls making adjustments or performing procedures other than those specified in the Typhoon User s Guide may result in hazardous exposure to laser light high voltage or moving parts Exposure to these hazards can cause severe injury or death Please observe the following precautions If the cover or doors of the Typhoon instrument become damaged do not continue to use the instrument e If the Image Eraser becomes damaged do not continue to use the eraser If you add or remove optical emission filters make sure you follow the procedures in section 13 3 Because the instrument is turned on during the procedures you may be exposed to moving parts in the instrument To protect your warranty the Typhoon instrument and Image Eras
37. and click 12 3 Save Windows NT or Open Mac OS The ImageQuant Preview window appears While the Typhoon instrument scans the sample an image of each scan appears in the window 4 Evaluate the results During scanning check the image in the 12 3 3 ImageQuant Preview window for saturation Saturated pixels appear in red If the image appears saturated you may need to repeat the scan If the image appears usable continue with the next scan If you are scanning another sample make sure you Clean the glass platen and sample lid before you place the next sample in the instrument 5 Clean up after the scan Remove the sample from the Typhoon 12 4 instrument and dispose of the sample using 12 5 the appropriate laboratory procedures Clean the Typhoon instrument Typhoon User s Guide p3 7 Part one Introduction Important 3 5 Using templates The Scanner Control software allows you to save the scanner settings you use frequently as a uniquely named template You use the template name to retrieve the settings and display them in the Scanner Control and Setup windows When you create a template for the fluorescence acquisition mode you select an emission filter or filters from the filters installed in the Typhoon instrument If you remove an emission filter from the instrument Scanner Control removes all the templates from the template list that used the filter If you reinstall the filter the templa
38. blots using fluorescently labeled probes such as Cy 5 labeled DN A probes or antibodies Pixel by pixel fluorescent excitation eliminates fluorescent blooming which is caused by constant excitation in traditional systems and provides better resolution of closely spaced bands Quantitation is simplified because unlike instant film the Typhoon system provides a linear response to the fluorescent signal intensities Chapter 7 About fluorescence scanning 7 3 Emission filters The Typhoon instrument contains 6 standard emission filters and can accommodate a total of 17 emission filters The instrument uses emission filters to reject reflected and scattered excitation light and background fluorescence while allowing the emitted light from the sample to pass through to the detector The instrument contains the following standard set of filters 555 nm bandpass filter 555 DF 20 H as a transmission peak centered at 555 nm and transmits light between 545 nm and 565 nm Use this filter with fluorochromes such asR6G and HEX 580 nm bandpass filter 580 D F 30 H as a transmission peak centered at 580 nm and transmits light between 565 nm and 595 nm Use this filter with fluorochromes such as TAM RA and Cy3 e 610 nm bandpass filter 610 DF 30 H as a transmission peak centered at 610 nm and transmits light between 595 nm and 625 nm Use this filter with fluorochromes such as ROX and EtBr e 670 nm bandpass
39. close the Scanner Control software See section 13 3 for more information on changing an emission filter Use the following instructions to close the Scanner Control software Windows NT Choose Exit from the File menu or click the Close button K Mac OS Choose Quit from the File menu or click the Close box E To scan again open the Scanner Control software section 3 3 3 7 Turning off the Typhoon instrument To turn off the Typhoon instrument press the on off switch on the lower right side of the Typhoon instrument figure 3 1 to the off position You can continue to use the computer after you turn off the Typhoon instrument 3 8 Turning off the computer Before you turn off the computer you should save and close any open files and then close any open applications Use the computer manufacturer s instructions for turning off the computer Typhoon User s Guide p3 13 Part two Scanning in the storage phosphor mode Chapter 4 About storage phosphor screen autoradiography This chapter describes storage phosphor screen autoradiography The topics in this chapter are How Typhoon generates an image section 4 1 e Advantages of storage phosphor screen autoradiography section 4 2 e Types of storage phosphor screens section 4 3 4 1 How Typhoon generates an image Generating an image from a storage phosphor screen is a two step process First you expose an erased storage phosphor scr
40. each scan Optionally you can select scans that can be linked Type comments you want saved with the image Typhoon User s Guide p3 5 Part one Introduction E Table 3 2 Fluorescence scanning workflow continued Task Description Section 3 Start the scan Click Scan Type a file name and click 9 3 Save Windows NT or Open Mac OS The ImageQuant Preview window appears While the Typhoon instrument scans the sample an image of each scan appears in the window 4 Evaluate the results During scanning check the image in the 9 3 3 ImageQuant Preview window for saturation Saturated pixels appear in red If the image appears saturated you may need to repeat the scan If the image appears usable continue with the next scan If you are scanning another sample make sure you Clean the glass platen and sample lid before you place the next sample in the instrument 5 Clean up after the Remove the sample from the Typhoon 9 4 scan instrument and dispose of the sample using 9 5 the appropriate laboratory procedures Clean the Typhoon instrument 3 4 3 Workflow for chemiluminescence scanning Table 3 3 describes the workflow for chemiluminescence scanning and lists the sections in this user s guide that contain the procedural steps for each task Table 3 3 Chemiluminescence scanning workflow Task Description Section Preparing for chemiluminescence scanning 1 Prepare the sample Prepare the
41. for detailed information p3 2 Typhoon User s Guide Chapter 3 Getting started 3 4 1 Workflow for storage phosphor scanning Table 3 1 describes the workflow for storage phosphor scanning and lists the sections in this user s guide that contain the procedural steps for each task Table 3 1 Storage phosphor scanning workflow Task Description Section Preparing for storage phosphor screen autoradiography 1 Prepare the sample Prepare a sample using a radioactive isotope 5 1 that can be collected by the general purpose GP low energy LE or tritium TR storage phosphor screen Use the laboratory procedures established for the type of sample you are preparing If you are preparing a wet gel wrap the gel in plastic wrap or polyester film before exposing it to the screen N Prepare the storage Decontaminate optional clean and erase the 5 2 phosphor screen GP or LE screen Erase the TR screen Protect the Image Eraser from contamination when erasing the TR screen Ww Prepare the Decontaminate optional and clean the 14 4 exposure cassette exposure cassette 4 Place the sample Place the sample in the exposure cassette and 5 5 in the exposure note the grid coordinates where you placed the cassette sample If the sample is thick or wet do not use the exposure cassette Instead expose the sample to the screen in a light tight environment such as a light tight drawer 5
42. in the exposure cassette again pA 6 Typhoon User s Guide Appendix A Troubleshooting e Thesample may have shifted when the screen was inserted into the cassette Erase the screen and re expose it to the sample Do not tip the cassette upright during exposure The image created from a fluorescent sample contains a double image Thesample may have moved after the initial placement on the glass platen of the instrument If fluorescent material has come off onto the glass platen carefully remove the sample and clean the glass Then place the sample on the glass platen again and do not readjust the placement e Remove excess liquid from gels so that the gels do not move on the glass platen The image created from a chemiluminescent sample contains a double image e Thesample may have moved after the initial placement on the glass platen of the instrument If chemiluminescent material has come off onto the glass platen carefully remove the sample and clean the glass Then place the sample on the glass platen again and do not readjust the placement Typhoon User s Guide pA 7 Appendix B Quick reference for menus and windows The following is a quick reference for the Scanner Control software The topics in this appendix are e Menus section B 1 e Windows section B 2 B 1 Menus The following menus and commands are used in the Scanner Control software File menu Command Description Name Dataset Dis
43. intense samples pixel intensities in your scanned image should be within a specific range To determine the correct PM T voltage scan a test sample and display the image in ImageQ uant Use the Gray Color Adjust feature to display the intensity values of the image See the mageQ uant User s Guide or online H elp for more information If the values are more than 1 count and less than 100 000 counts all spots in your image are within the dynamic range of the instrument If some of the values are equal to or greater than 100 000 counts part of your image is at or near saturation Lower the PMT voltage rescan and confirm that the image is not saturated e For intense samples that saturate the system decrease the PM T voltage This brings high intensity signals into the linear range of the instrument You may lose the sensitivity and the accuracy of the quantitation at the lower end of the signal range If you decrease the voltage below 400 you may lose linearity Increasing the PMT voltage can cause the signal to noise ratio to deteriorate and lower the sensitivity For the best sensitivity use the High sensitivity setting with a lower PMT voltage If you change your sample matrix you may need to test several PM T settings between 400 volts and 1000 volts to determine which setting gives an acceptable background As a suggested starting point use 600 volts Typhoon User s Guide p12 9 Part four Scanning in the chemilumine
44. lock it into place Figure 5 4 Closing the exposure cassette for mounted screens 5 6 2 Positioning an unmounted screen To position an unmounted screen in the exposure cassette 1 Remove the screen from the Image Eraser 2 Hold the white side of the screen facing down toward the sample The top of the label on the screen should be toward the top end of the cassette 3 Gently lower the screen into place over the grid area in the cassette The screen should be centered over the grid area and should lie straight and flat in the cassette figure 5 5 Caution Do not reposition the screen after it has touched the sample Because the screen is extremely sensitive adjusting the position of the screen can result in a double image 4 Close the exposure cassette and press the lid shut until the lock s click p5 10 Typhoon User s Guide Exposure cassette 1 Place the screen in the exposure cassette 2 Press the lid shut until both locks click Chapter 5 Preparing for storage phosphor screen autoradiography Inside lid Outside lid Locks w x a a Se et i ina E ll nc Pa Gal hoa Phosphor screen white side down Figure 5 5 Loading a large unmounted screen into the exposure cassette 5 7 Exposing the storage phosphor screen The storage phosphor screen requires approximately one tenth the exposure time of normal x ray film When determining the exposure time consider a 2 hour exposur
45. multichannel image A multichannel image is a set of images channels that can be viewed individually or as a composite overlaid To provide accurate detection and separation of the signals you label the sample with two three or four fluorochromes that can be collected and distinguished from each other using the available filters You include a spectral separation standard for each fluorochrome in the sample to assist in reducing cross contamination between the fluorescent emissions After the scan you use the FluorSep software to remove the cross contamination and create a new image for each fluorochrome in the sample The multichannel image can be viewed in ImageQ uant In ImageQuant you can view the set of images channels overlaid in different colors or you can view the channels side by sidein gray scale For information on removing cross contamination using FluorSep see the FluorSep online H elp or user documentation For viewing images in ImageQ uant see the ImageQ uant online H elp or user documentation 7 6 Common fluorochromes Table 7 1 lists the absorption and emission maxima of some common fluorochromes The chemistry of the experiment may alter the absorption and emission spectra slightly For more information on selecting fluorochromes see the M olecular Dynamics publication Fluorescence maging Applications G uide See appendix C for a list of references Typhoon User s Guide p 5 Part three Scanning in t
46. sample using chemiluminescent 11 1 enzyme substrate systems To create two images with a single scan use substrates that can be collected and separated using a beamsplitter 2 Prepare the Decontaminate optional and clean the 11 2 Typhoon instrument glass platen and sample lid of the Typhoon instrument p3 6 Typhoon User s Guide Chapter 3 Getting started Table 3 3 Chemiluminescence scanning workflow continued Task Description Section Scanning the chemiluminescent sample 1 Place the sample in the Typhoon instrument In subdued light position the sample on the 11 3 glass platen Make a note of the grid coordinates on the glass platen Close the sample lid N Select scan settings using the Scanner Control software In the Scanner Control window select an 12 1 existing template that contains the scan settings 12 2 you want to use or manually select the settings To manually select the settings make sure Chemiluminescence is the selected scan acquisition mode Then select the grid area pixel size sample orientation press sample optional focal plane and image analysis software from the Scanner Control window Then click Setup and select the sensitivity and PMT voltage for the scan To create two images with a single scan select a beamsplitter and a second PMT voltage Type comments you want saved with the image 3 Start the scan Click Scan Type a file name
47. sandwich gel M ake sure the sandwich gel does not leak Make sure the electrophoresis glass plate you place on the glass platen is 3 millimeters thick The 3 mm focal plane setting is designed for 3 mm thick glass Make sure both the electrophoresis glass plates of a sandwich gel have a low fluorescence background M embranes You should scan a test piece of each type of membrane you plan to use and check that the background is low enough for your purposes M icroplates Because the bottoms of the wells in most microplates will raise the sample at least 2 mm above the glass platen you should use the 3 mm focal plane setting in the Scanner Control window section 9 1 7 You might want to scan a test microplate and check the results in ImageQ uant to make sure the selected focal plane provides the best limit of detection and spatial resolution In addition check the background fluorescence of the microplate to make sure it is within an acceptable range To prevent the microplate from moving during the scan use the Press Sample option M icroplate strips Scan a test microplate strip using the Platen focal plane section 9 1 7 Check the results in ImageQ uant to make sure the selected focal plane provides the best limit of detection and spatial resolution In addition check the background fluorescence of the microplate strip to make sure it is within an acceptable range Typhoon User s Guide p8 3 Part three Scanning in t
48. scans the area just above the glass platen 6 4 1 Selecting a template If a template exists with the settings you want to use you can select the template review the settings add user comments section 6 4 7 and then start the scan section 6 5 For more information on creating and using templates see section 3 5 To select a template choose Load from the Templates menu and then select the template name from the list If a template does not exist select the settings using the instructions in sections 6 4 2 through 6 4 7 6 4 2 Selecting the storage phosphor scan acquisition mode The storage phosphor scan acquisition mode is the default mode when you first start the Scanner Control software If the Typhoon instrument has been used for fluorescence or chemiluminescence scanning select Storage Phosphor from the Acquisition M ode list in the Scanner Control window figure 6 4 6 4 3 Selecting the scan area The grid in the Scanner Control window allows you to select the area to scan The letter and number markings correspond to the markings in the exposure cassette and on the glass platen of the Typhoon instrument The white rectangle on the grid designates the area you want to scan You can either use the current scan area or select a new one To select a new scan area 1 Place the pointer in the grid square corresponding to the lowest lettered and numbered grid square of the area you want to scan For example if you
49. the Define the Filter window e Windows NT Hold down the right mouse button and click the selected emission filter in the Emission Filter list Mac OS Choose Redefine Filter from the Emission Filter list The Define the Filter window appears and displays the current definition of the filter you selected in step 2 M odify any of the following e Choose a new filter type from the Type list e Typea new filter name in the N ame box Typea new peak wavelength in the Peak WL box e If the filter is a bandpass filter type a new bandwidth in the BW box Click Register Filter to redefine the filter and close the window If you do not want to redefine the filter click Don t Register N ow to close the window Typhoon User s Guide p13 15 Part five Maintaining the Typhoon system hardware Warning A Define the Filter x Please correctly redefine the selected filter Bar Code Type Name Peak WL BW 11 Band Pass 7 es 555 20 Don t Register Now Figure 13 10 The Define the Filter window 13 5 Changing the fuses If fuses must be replaced repeatedly the Typhoon instrument may have an electrical problem Do not use the instrument Contact MD Scanner Technical Support See Assistance in the Preface for contact information You can use the Typhoon instrument with either a 220 volt to 240 volt 230V power source or a 100 volt to 120 volt 115V power source The power supply in the instrument switche
50. the Typhoon instrument except to install or remove optical emission filters There are no other user serviceable components inside and you can be exposed to laser light Do not defeat the safety interlocks of the sample lid figure 2 2 The safety interlocks are designed to protect you from exposure to the laser light If you lift the sample lid during a scan the safety interlocks signal the laser shutters to close and block the light from the lasers Do not continue to use the instrument if a cover or the sample lid becomes damaged and the instrument is no longer light tight Contact M D Scanner Technical Support immediately to arrange for repair See Assistance in the preface for contact information To protect your warranty your Typhoon instrument should be serviced only by an authorized M D Scanner Technical Support representative If the instrument is not working correctly please contact M D Scanner Technical Support e If fuses must be replaced repeatedly do not use the instrument Contact M D Scanner Technical Support 2 4 Hazardous materials precautions Some materials used to label samples can be hazardous Use good laboratory procedures and follow the manufacturer s precautions when working with these materials Before using the Typhoon system become familiar with your laboratory s hazardous materials procedures Do not expose the Typhoon instrument and storage phosphor screen to volatile organic solvents such
51. the longest edge of the sample along the scan head path minimizes the scan time p8 6 Typhoon User s Guide Chapter 8 Setting up for fluorescence scanning To minimize the image file size scan only the number of grid squares covered by the sample If you do not want to scan the entire sample scan the squares that contain the part of the sample you are interested in analyzing To record multiple samples gels membranes and so on during the same scan place the samples close together M ake sure the samples are the same thickness If the samples are not the same thickness the thinner sample may not contact the glass platen uniformly which can result in a bad scan poor image and poor quantitation results Align the lanes and rows of the sample with the edges of the grid on the glass platen so that the scanned image will be straight Top view Back of the Typhoon instrument Coordinates of this grid square L10 Coordinates of this grid square Al Front of the Typhoon instrument Figure 8 2 Sample placement on the glass platen of the Typhoon instrument Typhoon User s Guide p8 7 Part three Scanning in the fluorescence mode Important Important 8 3 3 Placing the sample on the glass platen You can place the sample directly on the glass platen H owever for gels and membranes you can avoid contaminating the glass platen and possible double images if you place the gel on a low fluorescence el
52. the position remove the screen and erase it Then place the screen on the sample again 5 4 Guidelines for placing the sample in the exposure cassette Use the following guidelines when placing the sample in the exposure cassette p5 6 Typhoon User s Guide In general place the sample in the cassette in the orientation you want to view the scanned image face up on the grid and with the top of the sample toward the top end of the cassette To avoid possible edge effects on the image you should place the sample at least 1 cm from the edge of the screen For example use the area B2 through J 13 for the small screens and the area B2 through Q21 for the large screens as shown in figure 5 2 To minimize the scan time Place the sample near the upper left corner of the grid for example start in grid square B2 Position a rectangular sample so that the shortest edge is along the numbered side of the exposure cassette as shown in figure 5 2 Because each pass of the scan head moves the entire length of the glass platen from front to back placing the longest edge of the sample along the scan head path minimizes the scan time Chapter 5 Preparing for storage phosphor screen autoradiography Coordinates of this grid square B2 o 1 1 o A 5 i c D f F G H 1 J K L M N o P a 3 Sampl
53. the standards should bein a matrix similar to the final sample For example if the sample isa dried 10 polyacrylamide gel the standards should be madein a 10 polyacrylamide gel and dried as well Standards are commercially available from a number of companies that sell radioactive products Contact these companies for assistance in choosing an appropriate set of standards for your application Alternatively you can make your own standards 5 2 Preparing the storage phosphor screen for exposure Before exposing the sample to the storage phosphor screen you should clean erase and if necessary decontaminate the screen 5 2 1 Protecting the storage phosphor screen The storage phosphor screens consist of a relatively soft matrix To protect the screen e Handle the screen on the edges or back only Do not touch the white phosphor surface e Do not fold roll or gouge an unmounted screen 5 2 2 Verifying that the storage phosphor screen is clean Between exposures or if the screen has not been used for several days verify that the storage phosphor screen is clean using the following procedure 1 Clean and erase the screen using the procedures in sections 5 2 3 and 5 2 4 Before you erase the screen check the Image Eraser for contamination from radioactive samples To clean the eraser see section 14 1 2 Store the screen in a clean light tight box 3 Provide enough time to register the contamination as an image on
54. visible spots still remain clean the surface first with 75 ethanol and then with distilled water Caution Do not allow liquid to seep into the exposure cassette Use a damp cloth only Do not pour or spray liquid in the cassette p14 6 Typhoon User s Guide Part six Appendixes Appendix A Troubleshooting If you are having problems with the Typhoon instrument or the Scanner Control software use the troubleshooting sections below to locate the description that matches your problem If you cannot find a solution call M D Scanner Technical Support See Assistance in the preface for contact information Problems in bold print are followed by possible causes and solutions The topics in this appendix are Power and communication section A 1 e Scanning section A 2 e Image section A 3 A 1 Power and communication The power indicator light on the Typhoon instrument will not turn on e Theinstrument may not be plugged in or the surge protector switch may be turned off Plug in the instrument or turn on the surge protector e Thewall outlet may be faulty Test the outlet or try another one e The fuse s may have blown To change the fuse s see section 13 5 The Typhoon instrument is on but cannot communicate with the computer You may be using the wrong type of Ethernet cable or the cable may be bad The Typhoon instrument requires a special crossover Ethernet cable or an Ethernet hub If you are using the cabl
55. your application scan a test gel to make sure the background is low enough for your purposes M ake sure the agarose is completely dissolved and well mixed before pouring the gel Also avoid generating bubbles when mixing and pouring Uneven agarose concentration and bubbles affect light scatter and can cause artifacts and interfere with quantitation If you poured the gel into a plastic tray remove the gel from the tray before placing the gel on the glass platen of the Typhoon instrument Important Chapter 8 Setting up for fluorescence scanning If the agarose gel containing the sample is more than 4 mm thick scan the sample using the 3 mm focal plane setting in the Scanner Control window section 9 1 7 If the agarose gel is less than 4 mm thick scan the sample using the Platen focal plane setting Check the results in ImageQ uant to make sure the selected focal plane provides the best limit of detection and spatial resolution Polyacrylamide Polyacrylamide gels are usually clear and thin less than 1 mm The background contribution from the gel material is generally very low M ake sure your electrophoresis glass plates are absolutely clean before you pour the gel Grease and fingerprint oils from the plates can stick to the gel surface and attract dust and fluorescent dyes Electrophoresis glass plates Use glass that has a low fluorescence background M ake sure the electrophoresis glass plate is clean before preparing a
56. 0 volt to 120 volt 115V power source The selected operating voltage of the Image Eraser appears in the fuse window on the right side of the eraser next to the power switch figure 14 1 The fuse holder of the Image Eraser takes two 1 5A 250V T1 5A 250V slow acting fuses The fuse holder is designed to accept both 0 5 inch x 1 5 inch English fuses designated 3AG fuses and 5 mm x 20 mm metric fuses To change a fuse 1 Turn off the Image Eraser and disconnect the power cord figure 14 1 2 Note the voltage 115V or 230V showing in the fuse window figure 14 3 3 Look for the small groove above the fuse window 4 Place the end of a small flathead screwdriver in the grooveand pry open the fuse box figure 14 3 5 Place the screwdriver in the groove on the upper edge of the fuse holder and pry out the fuse holder When the holder is loose pull it out of the fuse box 6 Oneor both fuses may be blown Replace the blown fuse s with new fuse s of the same type and rating Fuse specifications are listed on the serial number label located to the left of the fuse box 7 Insert the fuse holder into the fuse box making sure the correct voltage rating for your power supply is right side up p14 4 Typhoon User s Guide Chapter 14 Maintaining the Image Eraser and exposure cassettes Pry here Pry here to open the to remove the fuse cover fuse holder Groove Hf Fuse holder r Fuse cover o
57. 3 Loading thesample 0 0 0 cece eee ee 8 3 1 Determining the sample orientation 8 3 2 Determining the sample placement 8 3 3 Placing the sample on the glass platen Table of Contents SSS Chapter9 Scanning a fluorescent sample 9 1 Selecting the scan parameters in the Scanner Control window 9 1 9 1 1 Selectingatemplate 0 cece ee 9 2 9 1 2 Selecting the fluorescence scan acquisition mode 9 2 9 1 3 Selecting the scan area 1 kee 9 3 9 1 4 Selecting the sample orientation 0 000 9 4 9 1 5 Selecting the Press Sample option 000 9 6 9 1 6 Selecting the pixel size nana 9 6 9 1 7 Selecting the focal plane cee eee eee 9 6 9 1 8 Selecting the image analysis software 9 7 9 1 9 Entering user comments optional 0 000 9 7 9 2 Using the Setup window to select the fluorescence parameters 9 7 9 2 1 Displaying the Fluorescence Setup window 4 9 8 9 2 2 Selecting the number of scans ccc cece eee 9 8 9 2 3 Selecting the emission filter 0 0 eee eee 9 8 9 2 4 Setting the PMT voltage 0 cece eee 9 9 9 2 5 Selecting the laser 0 cece cee 9 10 9 2 6 Setting the limit of detection 0 0 00s 9 10 9 2 7 INKING SCANS Accu 7 ake eas fear ia a aes 9 11 9 2 8 Closing the Fluorescence Setup window 9 14 9 3
58. 8 6 9 4 11 3 12 4 placement guidelines 5 7 to 5 8 placing in exposure cassette 5 8 placing on glass platen 8 6 to 8 7 11 3 precautions 2 9 preparation guidelines 5 1 removing from instrument 9 19 scanning several at once 8 7 11 4 scintillants 2 9 5 2 solvents 2 9 weak 9 9 wet 5 8 wet gel guidelines 5 2 sample lid cleaning 6 3 8 5 11 3 closing 6 6 8 8 8 10 11 5 opening 6 5 8 7 11 4 sample orientation buttons illustrated 6 10 9 5 12 5 Scanner Control window 6 8 9 4 12 4 sample orientation in exposure cassette 5 8 sample support low fluorescence 8 2 sandwich gel assembling 8 9 placing 8 6 placing on glass platen 8 9 to 8 10 saturated data 6 15 9 18 12 14 Save As Template window 3 11 Index 6 Typhoon User s Guide Save As window 6 13 9 15 12 11 scan acquisition mode chemiluminescence 1 4 12 2 fluorescence 1 4 9 2 storage phosphor 1 4 6 7 scan area glass platen 8 7 11 4 selecting 6 7 9 3 12 3 scan coordinates 8 8 8 9 11 5 scan errors 6 14 9 17 12 13 scan head securing for move 13 18 Scanner Control window closing 3 13 commands B 2 creating template 3 8 deleting template 3 12 grid 6 7 9 3 12 3 Image Analysis Software box 6 11 9 7 12 7 modifying template 3 11 opening 3 2 Pixel Size box 6 11 9 6 12 6 Press Sample option 9 6 12 6 registering new optical filter 13 14 sample orientation buttons 6 8 9 4 12 4 selecting default template 3 12 selecting focal plane 9 6 12 7 selecting
59. Amersham Pharmacia Biotech K K Sanken Bldg 3 25 1 H yakunincho Shinjuku ku Tokyo Japan 169 0073 Telephone 81 3 5331 9344 Fax 81 3 5331 9368 Australia and N ew Z ealand Amersham Pharmacia Biotech Unit 38 5 7 Anella Avenue Castle H ills N ew South Wales 2154 Australia Telephone 612 9894 5188 Latin America Amersham Pharmacia Biotech do Brasil Ltda Rua Brigadeiro Galvao 288 Barra Funda Sao Paulo SP 01151 001 Brazil Telephone 55 11 3667 5700 Fax 55 11 3667 5899 Web Site http www mdyn com xi Typhoon User s Guide Part one Introduction Chapter 1 Introduction to the Typhoon system The Typhoon system consists of an instrument instrument control software and a variety of accessories The Typhoon instrument is a variable mode imager that produces digital images of radioactive fluorescent or chemiluminescent samples This chapter introduces you to the Typhoon system and contains the following topics Typhoon system hardware components section 1 1 e How the Typhoon system works section 1 2 e Typhoon scan acquisition modes section 1 3 Before you begin section 1 4 1 1 Typhoon system hardware components The Typhoon system includes the following hardware components figure 1 1 Typhoon instrument Scans exposed storage phosphor screens fluorescent samples and chemiluminescent samples Image Eraser Erases storage phosphor screens for reuse Storage phosphor scr
60. Before the Typhoon instrument begins the scan an initialization process occurs Depending on the state of the instrument and the scan parameters you chose in Scanner Control the initialization can take as long as 3 minutes The initialization procedures include e Moving the scan head into position e Changing the filters to match the scan parameters e Stabilizing the laser if the laser has been idle shut down or recently turned on e Lowering the inner lid of the Typhoon instrument to hold the sample flat against the glass platen p9 16 Typhoon User s Guide Chapter 9 Scanning a fluorescent sample 9 3 2 Monitoring the scan progress After you start the scan the ImageQ uant Preview window figure 9 7 appears and the green Scan indicator light on the top of the instrument blinks As the Typhoon instrument scans the sample Scanner Control displays the image in the ImageQ uant Preview window In addition Scanner Control displays the number of data lines scanned and the total scan time remaining excluding initialization Scanner messages may also appear as the scan progresses A More Info button appears in the ImageQ uant Preview window when the Typhoon reports errors Click the M ore Info button to view the error message If the error is a nonfatal error the instrument continues to scan and Scanner Control saves the file when the scan is finished You should check the image in ImageQuant to make sure the data are not corrup
61. Control window click the Scan button The Save As window appears figure 6 7 3 In the Save As window type a name in the e Windows NT File name box Mac OS SaveAs box p6 12 Typhoon User s Guide Chapter 6 Scanning a storage phosphor screen SSS ae Caution If you type a name and extension that have already been used a message appears and asks you if you want to replace the existing file Before you click Yes make sure you do not want to keep the existing image file Clicking Yes deletes the image and all the associated auxiliary files If you have analyzed the image using ImageQuant all the data will be deleted ieee a3 ajae Teo ine e Tied o Windows NT Mac OS ave ae mp ore fe Figure 6 7 The Save As window Caution 4 Optional Change to a different folder O therwise the software saves the image file in the Data folder Using a network to save the scanned data to a folder on a remote computer may cause a loss of data For the best results choose a folder on the local hard drive After you scan the screen move the image file to the remote computer 5 To start the scan Windows NT Click Save e Mac OS Click Open Before the Typhoon instrument begins the scan an initialization process occurs Depending on the state of the instrument and the scan parameters you chose in Scanner Control the initialization can take as long as 3 minutes Typhoon User s Guide p6 13
62. E screen low energy storage phosphor screen link the process of creating two images with one scan Uses emission filters and beamsplitters to separate and collect light of two different wavelengths long pass filter an optical filter that allows light of wavelengths longer than a specified cutoff to pass through The filter rejects most of the light with wavelengths shorter than the cutoff multichannel image a set of images that can be viewed as a composite overlaid or viewed as individual images side by side A chemiluminescent sample can be used to create a multichannel image consisting of two images A fluorescent sample can be used to create multichannel images made up of two three or four images Each image is called a channel See also channel photomultiplier tube PM T an electro optic device in the Typhoon instrument that converts light energy into electrical current and amplifies the current quantitation the process in which signal intensities of the sample and other values are calculated sample lid the under side of the cover of the Typhoon instrument short pass filter an optical filter that allows light of wavelengths shorter than a specified cutoff to pass through The filter rejects most of the light with wavelengths longer than the cutoff Stokes shift the difference in wavelength between the apex of the excitation spectrum shorter wavelength higher energy and the apex of the emi
63. Figure 8 1 Opening the sample lid 2 Optional Check the glass periodically for chemicals 3 Clean the glass with distilled water and a clean lint free cloth or paper If visible spots remain clean the glass first with 75 ethanol and then with distilled water Caution Because window cleaners use ingredients that can fluoresce do not use window cleaners to clean the glass platen In addition the use of acetone or the excessive use of ethanol can shorten the lifetime of the instrument Protect the glass from scratches Scratches interfere with accurate imaging and quantitation 8 2 2 Cleaning the sample lid The surface of the inner lid should be clean and free of fluorescent contamination If necessary clean the surface with a damp not saturated cloth moistened with a small amount of distilled water If visible spots remain clean the surface first with 75 ethanol and then with distilled water To prevent liquid from seeping inside the lid or rolling down onto the glass do e not spray liquid on the lid Typhoon User s Guide p8 5 Part three Scanning in the fluorescence mode Caution 8 3 Loading the sample Before you load the sample determine how to position it on the glass platen If you place the sample directly on the glass platen make sure you place the sample in the correct position when you first set it down Although unlikely any fluorescent material left on the glass after you move a sample can result in a d
64. Guide p9 15 Part three Scanning in the fluorescence mode Sa 3 In the Save As window type a name in the Windows NT File name box e Mac OS Saveas box Caution If you type a name and extension that have already been used a message appears and asks you if you want to replace the existing file Before you click Yes make sure you do not want to keep the existing image file Clicking Yes deletes the image and all the associated auxiliary files If you have analyzed the image using ImageQuant all the data will be deleted If you type a name and select the gel extension and that name has already been used with the ds file extension a message appears and asks you to change the file name 4 Optional Change to a different folder O therwise the software saves the image in the Data folder Caution Using a network to save the scanned data to a folder located on a remote computer can cause a loss of data For the best results choose a folder on the local hard drive After you scan the sample move the image file s from the folder on the local hard drive to a folder on the remote computer For dataset image files make sure you move both the ds file and the dir directory into the new folder 5 To start the scan Windows NT Click Save e Mac OS Click Open If you selected Press Sample in section 9 1 5 the inner lid of the Typhoon instrument lowers to hold the sample flat against the glass platen of the instrument
65. IUOGUUUGUULOGNUNGGULOGUUNGGHD lt the fuse holder Fuse soer Fuse cover open T E E E E E E E A A EEE E AEN E E Figure 13 12 Opening the fuse box Typhoon User s Guide p13 17 Part five Maintaining the Typhoon system hardware Warning A Caution 13 6 Moving the Typhoon instrument If you need to move the instrument contact M D Scanner Technical Support See Assistance in the preface for contact information Use this section to determine a new location for the instrument and to prepare for the move The Typhoon instrument weighs approximately 160 kg 350 Ib Only personnel authorized by Amersham Pharmacia Biotech or Molecular Dynamics should move the instrument using the proper lifting equipment Injury to personnel and damage to the instrument can result from attempting to lift the instrument without authorized personnel and proper equipment If you use unauthorized personnel to move the Typhoon instrument Amersham Pharmacia Biotech and Molecular Dynamics are not responsible for damage to the instrument or injury to personnel Damaging the instrument can void the warranty and may affect the accuracy of the collected data 13 6 1 Determining a new location for the instrument If you need to move the Typhoon instrument make sure the new location for the instrument is away from strong air currents The airflow can carry dust or dirt particles into sensitive parts of the instrument In a
66. Parttwo Scanning in the storage phosphor mode SSS The initialization procedures include e Moving the scan head into position e Changing the filters to match the scan parameters e Stabilizing the laser if the laser has been idle shut down or recently turned on e Lowering the inner lid of the Typhoon instrument to hold the screen flat against the glass platen 6 5 2 Monitoring the scan progress After you start the scan the mageQ uant Preview window figure 6 8 appears and the green Scan indicator light on the top of the instrument blinks As the Typhoon instrument scans the storage phosphor screen Scanner C ontrol displays an image in the mageQ uant Preview window In addition Scanner Control displays the number of data lines scanned and the total scan time remaining excluding initialization Scanner messages may also appear as the scan progresses A More Info button appears in the ImageQ uant Preview window when the Typhoon reports errors Click the M ore Info button to view the error message If the error isa nonfatal error the instrument continues to scan and Scanner Control saves the file when the scan is finished You should check the image in ImageQ uant to make sure the data are not corrupted e If the error is a fatal error the instrument aborts the scan and Scanner Control saves the data collected before the fatal error occurred You should check the image in ImageQ uant Depending on when the fatal error occu
67. Q uant U ser s G uide If the Scanner Control window is not open see section 3 3 Typhoon User s Guide p9 1 Part three Scanning in the fluorescence mode Tee ek rin EI Bie J pii Gets berm et evn Haii Papeete ike TELE Spel barter EHE Tad Fewer i pea T PREP EPR ANH EDMAN ice Eia ine bT ent Da File Lom Eedi PRE Spe ni Sage Fe brs F hae MPA leim Ae EO 2 Ln fuh z irs Geto Hea BY image ha pa re 100 ell Figure 9 1 The Scanner Control window set for a fluorescence scan 9 1 1 Selecting a template If a template exists with the settings you want to use you can select the template review the settings add user comments section 9 1 9 and then start the scan section 9 3 For more information on creating and using templates see section 3 5 To select a template choose Load from the Templates menu and then select the template name from the list If a template does not exist select the settings using the instructions in sections 9 1 2 through 9 2 8 9 1 2 Selecting the fluorescence scan acquisition mode Storage phosphor is the default scan acquisition mode when you first start the Scanner Control software To change to the fluorescence mode select Fluorescence from the Acquisition M ode list The selections for the fluorescence scan acquisition mode appear in the Scanner Control window figure 9 1 and in the additional Fluorescence Setup window figure 9 4 p9 2 Typhoon User s Gui
68. ack of inherent background Typhoon User s Guide p10 1 Part four Scanning in the chemiluminescence mode Se and an extended output of the light signal caused by enzyme catalysis contribute to sensitive detection over a wide dynamic range A variety of chemiluminescent substrates are commercially available for different enzymes The substrates are optimized for either solution or solid phase assays You can use the Typhoon system for membrane based chemiluminescent detection of protein and nucleic acid blots In addition you can use the Typhoon system to scan microplate assays 10 3 How to optimize the chemiluminescent experiment When you design the chemiluminescent experiment you need to evaluate how to prepare the sample so that the image you create using the Typhoon instrument can be used for quantitation Several factors affect the quality of the experiment For accurate quantitation and limit of detection time is a critical factor in the chemiluminescent experiment You should make sure you understand the effect the following have on the quality of the scan e Substrate You should test several substrates and evaluate which substrate emits light for the length of time required for the Typhoon instrument to scan the sample If the signal intensity of the substrate diminishes significantly before the instrument completes the scan you may not be able to perform accurate quantitation on the last part of the sample scanned by
69. al numbers 9 2 8 Closing the Fluorescence Setup window After you finish selecting the fluorescence settings click OK to save the settings and close the Fluorescence Setup window Scanner Control warns you if you have selected a nonoptimal link laser or beamsplitter If you do not want to save the settings click Cancel to return to the Scanner Control window The Scanning Information area in the Scanner Control window displays the settings you selected in the Fluorescence Setup window including the linking information In the main Scanner Control window the scans are labeled by channel number not scan number If you linked scans the channel numbers may not match the order of the scan number in the Fluorescence Setup window Scanner Control reorders the scans so that linked scans appear as contiguous channel numbers For example if you linked scans 1 and 3 they become channels 1 and 2 in the Scanning Information area In addition the scans are arranged so that the scan with the longest wavelength is listed first in the channel order ImageQuant uses the channel number to display multichannel images See the ImageQuant User s Guide or online Help for more information 9 3 Starting a scan and checking the progress Do not attempt to defeat the safety interlocks on the sample lid These interlocks are designed to protect you from laser light exposure Never turn off the Typhoon instrument or disconnect the Ethernet cable while sc
70. aning the storage phosphor screen 000 5 4 5 2 4 Erasing the storage phosphor screen 0 0005 5 4 5 3 Guidelines for exposing storage phosphor screens 5 6 5 4 Guidelines for placing the sample in the exposure cassette 5 6 5 5 Placing the sample in the exposure cassette 0 05 5 8 5 6 Placing the screen in the exposure cassette 00 0002 ae 5 8 5 6 1 Placing amounted screen 1 0 0 cee eee 5 8 5 6 2 Positioning an unmounted screen ese e eee ee 5 10 5 7 Exposing the storage phosphor screen 0 0 cece eee eee 5 11 Chapter6 Scanning a storage phosphor screen 6 1 Verifying the Typhoon instrument is clean 00 00 ec eee eee 6 1 6 1 1 Checking for radioactive contamination 0 00 6 1 6 1 2 Cleaning the glass platen 0 0 0 eee eee 6 2 6 1 3 Cleaning thesamplelid 0 0 0 2 e eee eee 6 3 6 2 Removing the exposed screen from the exposure cassette 6 4 6 3 Loading the storage phosphor screen into the insttument 6 5 6 4 Selecting the scan parameters in the Scanner Control window 6 6 6 4 1 Selecting a template eee 6 7 6 4 2 Selecting the storage phosphor scan acquisition mode 6 7 6 4 3 Selecting the scan area 1 eee ees 6 7 6 4 4 Selecting the sample orientation 00005 6 8 6 4 5 Selecting the pixel size 2 2 ee eee 6 11 6 4 6 Selecting the image analysis software
71. anning You can severely damage the internal mechanism of the instrument Keep the sample lid closed during the scan Opening the lid causes the instrument to close the laser shutter and block the laser light Then Scanner Control aborts the scan and saves the data already collected During the scanning process light illuminates each position on the sample only while the corresponding pixel is being recorded The position is minimally exposed during the rest of the scan p9 14 Typhoon User s Guide Chapter 9 Scanning a fluorescent sample SSS Scanner Control creates an image file and automatically adds the correct file extension which is either Data file gel Creates a single image file Dataset file ds Creates the set of files and folders required for a multichannel image If you are scanning the sample more than once or you linked scans Scanner Control adds this extension N ote If you select or type the wrong file extension Scanner Control changes the file type back to the correct extension 9 3 1 Starting a scan To start a scan 1 Make sure that the sample is in place the sample lid is shut and all the settings are correct in the Scanner Control and Fluorescence Setup windows 2 In the Scanner Control window click the Scan button The Save As window appears figure 9 6 p p a F e Eo aoo a TT Mac OS Windows NT Figure 9 6 The Save As window Typhoon User s
72. are Scan a test sample to make sure the instrument is working properly If the instrument is not working properly contact M D Scanner Technical Support See Assistance in the preface for contact information p13 20 Typhoon User s Guide Warning A Caution Chapter 14 Maintaining the Image Eraser and exposure cassettes This chapter provides information on the care and maintenance of the Image Eraser and the exposure cassettes The topics in this chapter are e Cleaning the Image Eraser section 14 1 e Changing the bulbs in the Image Eraser section 14 2 e Changing the fuses in the Image Eraser section 14 3 e Cleaning and protecting the exposure cassettes section 14 4 14 1 Cleaning the Image Eraser The surface of the Image Eraser must be clean and free of contamination from radioactive samples when you erase screens O therwise the screens will be exposed to radioactive contamination during the erasure process Because the Image Eraser uses high voltage always turn off and unplug the eraser before cleaning the surface Do not allow liquid to seep into the Image Eraser Use a damp cloth only Do not pour or spray liquid over the surface Make sure the instrument is totally dry before you reconnect the power cord 1 Turn off the Image Eraser and disconnect the power cord figure 14 1 2 Clean the surface of the eraser with a damp cloth moistened with a small amount of distilled water If visible spots rema
73. aring sample 11 1 removing sample 12 14 saturated data 12 14 scan acquisition mode 1 4 12 2 selecting a template 12 2 workflow 3 6 Chemiluminescence Setup window closing 12 10 displaying 12 8 PMT Voltage box 12 9 quick reference B 5 selecting beamsplitter 12 9 Sensitivity setting 12 8 cleaning exposure cassette 14 6 glass platen 6 2 8 4 11 1 Image Eraser 14 1 sample lid 6 3 8 5 11 3 computer connecting new 13 20 terms xii troubleshooting A 1 turning off 3 13 turning on 3 2 condensation on storage phosphor screen 5 8 contamination checking for 5 3 chemiluminescent 11 1 11 3 cross contamination 7 5 dust 5 4 6 2 8 1 8 4 8 5 11 1 11 3 fingerprint oil 5 4 6 2 8 4 8 5 11 1 11 3 fluorescent 8 4 8 5 glass platen 6 1 powder 8 1 Index 2 Typhoon User s Guide radioactive sample 5 3 5 4 6 1 6 3 storage phosphor screen 5 3 Typhoon instrument 6 1 coordinates see also scan area exposure cassette 5 8 glass platen 8 7 11 4 Scanner Control window 6 7 9 3 12 3 counts Typhoon system 5 2 cross contamination in multichannel experiments 7 5 D data file gel 6 12 9 15 12 10 dataset file ds 9 15 12 10 default template see template Define the Filter window 13 14 13 16 Delete Template s window 3 12 detergent powdered 5 4 14 1 documentation xi double image 5 8 8 6 dust 5 4 6 2 8 1 8 4 11 1 electrophoresis glass plate 8 3 emission chemiluminescence 10 2 fluorescence 7 1 to 7 2 stora
74. armup time The instrument may be damaged and is no longer light tight If so do not continue to use the instrument Contact M D Scanner Technical Support to arrange for repair See Assistance in the preface for contact information Dust fingerprints or other dirt may be on the screen sample or glass platen Clean the glass platen If necessary for fluorescence scans filter the liquid samples reagents and components used to make the gels For more information on removing contaminants see section 5 2 storage phosphor 8 2 fluorescence or 11 2 chemiluminescence The storage phosphor screen may not have been erased immediately before you exposed it to the sample or the residual image from an intense sample may not have been erased completely The sample support may have high autofluorescence Use a low fluorescence material section 8 1 3 The wrong emission filters may have been used for a fluorescent sample section 9 2 3 The image is incomplete The selected scan area in the Scanner Control window may be incorrect See section 6 4 3 storage phosphor 9 1 3 fluorescence or 12 1 3 chemiluminescence The signal emitted by a chemiluminescent sample may have diminished before the instrument finished scanning The edges of the image created from a storage phosphor screen show a loss of signal A light leak may have occurred during exposure M ake sure you expose screens in the exposure cassette or with wet g
75. as D efault Template check box N ote You can select the original default template system supplied with the instrument from the Templates menu 6 Click OK The template name appears above the grid area in the Scanner Control window 3 5 2 Selecting a template If you want to use a different template choose Load from the Templates menu and then select the template name from the list 3 5 3 Modifying a template You can modify a template and use the modified settings to scan a sample After you change a setting an asterisk appears next to the template name to show that the settings have been changed If you want to use the original unmodified settings select the template from the Load menu The original settings appear in the Scanner Control window Typhoon User s Guide p3 11 Part one Introduction Scanner Control will not allow you to save the modified settings directly back to the original template If you want to save a modified template using the same name you must delete the old template section 3 5 5 N ext select the new settings and save the template using the name of the template you deleted 3 5 4 Selecting a template to use as a default If you want to select a different template to use as the default choose Set D efault Load from the Templates menu and select the template name from the list The Template name and selections appear in the Scanner Control window Each time you start the Scanner Control software
76. as methanol chloroform or acetone Make sure thin layer chromatography TLC plates are completely dry Always remove corrosive liquids before loading the sample Do not use scintillants or enhancers on your sample These compounds interfere with the proper function of the storage phosphor screens Molecular Dynamics and Amersham Pharmacia Biotech are not responsible or liable for any damages caused by or as a consequence of the use of any hazardous material Typhoon User s Guide p2 9 Chapter 3 Getting started This chapter describes the basic procedures for using the Typhoon system The topics in this chapter are e Turning on and warming up the Typhoon instrument section 3 1 e Turning on the computer section 3 2 Opening the Scanner Control software section 3 3 Workflows for scanning section 3 4 Using templates section 3 5 Closing the Scanner Control software section 3 6 Turning off the Typhoon instrument section 3 7 e Turning off the computer section 3 8 3 1 Turning on and warming up the Typhoon instrument To turn on the Typhoon instrument press the on off switch on the lower right side of the instrument figure 3 1 The power indicator light on the front of the instrument turns on and remains red during the self test sequence which takes about 45 seconds The light then turns green After you turn on the Typhoon instrument wait approximately 30 minutes for the instrument to warm up bef
77. ass plate edged with Kapton tape is on the bottom 3 Place one edge of the sandwich gel on the glass platen of the instrument figure 8 3 4 Squirt distilled water on the glass platen The distilled water increases the sensitivity of the scan by filling the space created by the Kapton tape with water 5 Lower the sandwich gel onto the glass platen M ake sure you do not trap air bubbles as you lower the sandwich gel onto the glass platen Do not touch the part of the sandwich gel that will be read by the Typhoon instrument Do not touch the glass platen Oil from fingerprints and powder from gloves even thoroughly washed gloves may leave a print that can be detected 6 When you can no longer lower the sandwich gel using your fingers insert the Wonder Wedge tool between the glass platen and the 3 mm electrophoresis glass plate 7 Lower the sandwich gel onto the Wonder Wedge and then slowly remove the Wonder Wedge M ake sure you do not trap air bubbles between the glass platen and the 3 mm electrophoresis glass plate M ake sure the sandwich gel is aligned on the glass platen 8 Makea note of the grid square coordinates that are covered by the area of the sample you want to scan figure 8 2 You will use this information later in the Scanner Control window section 9 1 3 Typhoon User s Guide p8 9 Part three Scanning in the fluorescence mode SS 9 Close the sample lid and press it down until the latch clicks The sam
78. b page make sure you close the Scanner Control software Leaving Scanner Control running while you use the Web page can cause unpredictable problems in Scanner Control You use the Remove an Optical Filter from the Filter Changer Web page to remove an optical filter If you do not see this page return to section 13 3 1 1 M ake sure the filter changer is in position 2 Slide the filter holder all the way to the right and remove the filter M ake sure you do not touch the glass filter 3 Store the filter in a lint free box 4 Optional To remove additional filters click Eject Filter type the bar code for the next filter you want to remove and then repeat steps 2 and 3 5 Inthe Remove an Optical Filter from the Filter Changer Web page click Done The instrument scans all the bar codes and displays a list of the installed filters 6 Click OK to close the window and return to the Typhoon Direct Instrument Access Web page Use the procedure in section 13 3 5 to replace the front cover 13 3 5 Closing the doors and replacing the front cover You must leave the Typhoon instrument turned on when you change the optical filter selection After you remove the front cover of the Typhoon instrument and open the access and filter doors make sure you follow the procedures in this section to avoid injury Use the following procedure and figure 13 7 to close the access doors and replace the front cover Typhoon User s Guide p13 13
79. briefly in 0 1 Tween or SDS Rinse the gel in distilled water and then scan again If the fingerprints persist you may need to prepare a new gel and handle the new gel more carefully e Dust specks appear on the scan Rinse wet gels in filtered distilled water to remove surface dust prior to scanning Filter liquid reagents used in gels and buffers M ake sure you dissolve agarose completely before pouring the gel Clean the glass platen of the Typhoon instrument with a damp lint free cloth e The tracking dye is fluorescing Place the tracking dye in only one well or dilute the tracking dye with sample buffer e Thesample may have stained unevenly M ake sure you mix staining solutions thoroughly use a large excess of staining solution and rock or shake the gels during staining if possible The image created from a chemiluminescent sample contains streaks or other artifacts e Makesurethe sample is flat on the glass platen of the Typhoon instrument and no air bubbles are trapped under the sample Refer to the manufacturer s documentation for any advice about sample preparation The image created from a storage phosphor screen contains a double image e Thescreen may have been moved after the initial placement in the exposure cassette Always position the screen correctly on the first try and do not readjust the placement If readjustment is absolutely necessary make sure you erase the screen completely before placing it
80. can Teen Armee EO Sewe Mla age L oo Figure 9 7 The ImageQuant Preview window showing one tab containing two linked channels 9 3 3 Viewing the preview image for saturated data While theinstrument is scanning an image appears in the ImageQ uant Preview window that shows the part of the sample that has been scanned Saturated data appear in red in the image If the image appears saturated you may not be able to perform quantitative analysis on the image Because saturation causes a total loss of the ability to distinguish the signal intensity fluctuations a saturated image can be used for viewing only the shapes and positions of the spots on the sample To avoid saturation of the data lower the PM T voltage you selected in the Fluorescence Setup window section 9 2 4 N ote Do not select a PM T voltage below 500V Before repeating the scan verify in ImageQuant that the image is saturated 100 000 rfu and you cannot perform accurate quantitation p9 18 Typhoon User s Guide Chapter 9 Scanning a fluorescent sample 9 3 4 Completing the scan Scanner Control saves the image or images using the file name you typed in the Save As window The mageQ uant Preview window displays a Complete message and then closes The Scan indicator light on the Typhoon instrument turns off At the end of the scan an image of the sample appears automatically in the software you selected from the Image Analysis list If you selected N one
81. changing selection 13 4 cleaning 13 11 filter and holder 13 11 inserting in instrument 13 11 registering in Scanner Control 13 14 removing 13 13 optical system overview 1 3 organic solvents 5 1 orientation of sample 5 8 6 8 8 6 9 4 11 3 12 4 overwriting files 6 13 9 16 12 11 Index P Park Head Optics for Shipment Web page 13 19 phosphor screen see storage phosphor screen photobleaching 8 1 photomultiplier tube see PMT pixel size 6 11 9 6 12 6 PMT overview 1 3 Voltage box 9 9 12 9 polyacrylamide 8 3 powder 8 1 power indicator light description 3 1 troubleshooting A 1 power switch Image Eraser 14 2 Typhoon instrument 3 1 Press Sample option 9 6 12 6 publications related xi Q quick reference for menus and windows B 1 R radiation and storage phosphor screen 4 1 radioactive contamination see contamination radioactive standards 5 2 red excited fluorescence description 1 4 label guidelines 8 2 red excited laser 9 10 references C 1 Remove Optical Filter from Filter Changer Web page 13 7 replacing files 6 13 9 16 12 11 S safety electrical 2 4 fuse 2 5 general precautions 2 1 Typhoon User s Guide Index 5 Index hazardous materials 2 9 high voltage 2 6 interlocks 2 7 laser 2 8 radiation exposure 2 9 sample accurate quantitation 9 9 12 9 enhancers 2 9 5 2 exposing thick 5 6 exposure cassette placement guidelines 5 6 handling 8 8 11 5 intense 9 9 12 9 matrix 8 2 orientation 5 8 6 8
82. conforms to the following Standards EMC LVD Year of CE Marking Supplementary Information Manufacturer s Contact 89 336 EEC 73 23 EEC M olecular Dynamics Inc 928 E Arques Avenue Sunnyvale CA 94086 4536 Typhoon Optical Scanner Typhoon EN 61326 97 A1 98 EN 61010 1 1993 AM 2 1995 EN 60825 1 1994 AM 11 1996 1999 The product complies with the requirements of the EMC Directive 89 336 EEC and Low Voltage Directive 73 23 EEC Product Quality and Regulatory Compliance M anager M olecular Dynamics 928 E Arques Avenue Sunnyvale CA 94086 4536 Telephone 408 773 1222 Fax 408 773 8343 Typhoon User s Guide xiii Preface xiv Typhoon User s Guide Site requirements This section lists the site requirements for the Typhoon system For more information on site requirements see section 13 6 Electrical Power supply 15 amp 100 120 volts 10 amp 220 240 volts Line Frequency 50 to 60 Hz Environmental Temperature 15 C to 35 C H umidity N oncondensing 10 to 80 relative humidity Instrument Weight 160 kg 350 Ib H eight 21 54 cm 19 in Width 118 11 cm 46 5 in Depth 77 47 cm 30 5 in Assistance When calling for assistance be prepared to supply the serial number of your instrument The serial number is located in the Scanner Information area of the Scanner Control window and on the lower right side of the instrument near the plug figure 2 2 Pl
83. ctivity fluorescence or chemiluminescence present in the sample Typhoon User s Guide pl 3 Part one Introduction S 1 3 Typhoon scan acquisition modes The Typhoon instrument scans samples using the following scan acquisition modes Storage phosphor mode C reates images from samples labeled with radioisotopes using storage phosphor technology Part two of the Typhoon User s G uide describes how to use the Typhoon system to scan in the storage phosphor mode Fluorescence mode Creates images from samples labeled with fluorescent dyes The available excitation modes are Green excited mode Creates an image from a sample labeled or stained with a fluorescent dye that is excited at 532 nm Red excited mode Creates an image from a sample labeled or stained with a fluorescent dye that is excited at 633 nm Part three of the Typhoon User s G uide describes how to use the Typhoon system to scan in the green excited or red excited fluorescence mode Chemiluminescence mode Creates images from chemiluminescent samples Part four of the Typhoon User s Guide describes how to use the Typhoon system to scan in the chemiluminescence mode 1 4 Before you begin Before using the Typhoon system become familiar with pl1 4 Typhoon User s Guide Chapter 2 Important safety information Chapter 3 Basic operational instructions for using the Typhoon instrument and the Scanner Control software Part five M
84. d Avoid direct exposure to beam Gefahr Laserstrahlung wenn offen und Verriegelung ausgeschaltet Direkte bestrahlung vermeiden Danger L ouverture du couvercle entra ne une exposition au rayonnement laser si le dispositif de verrouillage a t mis hors service Eviter toute exposition directe au faisceau If a label becomes illegible or is missing for any reason please contact M D Scanner Technical Support for a free replacement label See Assistance in the preface for contact information While waiting for the replacement label copy the labels shown in figure 2 6 and attach the copy of the label to the instrument 2 3 2 Safety precautions The Typhoon instrument has been designed to protect you from the laser beams during normal operation N evertheless laser power from 10 mW to 20 mW can be accessed from within the interior of the instrument The Typhoon instrument contains a red laser and a green laser The output of the red visible laser is specified at a maximum 10 mW at 633 nmin a diverging beam The output of the green visible laser is specified at a maximum 20 mW at 532 nm in a diverging beam p2 8 Typhoon User s Guide Warning A Cautions Chapter 2 Safety Caution Use of controls or adjustments or performance of procedures other than those specified herein may result in hazardous radiation exposure Please observe the following precautions e Do not remove the front cover figure 2 2 of
85. ddition you should place the instrument away from direct sunlight or other very bright light Bright light may cause excessive heat or compromise the light tight operation of the instrument 13 6 2 Preparing to move the Typhoon instrument To prepare the Typhoon instrument for moving you use the Typhoon Direct Instrument Access Web page to park the scan head If you do not park the scan head you can damage the optical system of the Typhoon instrument To park the scan head 1 Use the procedure in section 13 1 to access the Typhoon Direct Instrument Access Web page 2 Inthe Typhoon Direct Instrument Access Web page click Park H ead Optics for Shipment The Park H ead O ptics for Shipment Web page appears figure 13 13 p13 18 Typhoon User s Guide Chapter 13 Maintaining the Typhoon instrument Microsoft Internet Explorer ioj x File Edit View Go Favorites Help St we amp A S amp S Be Fullscreen ck Foward Stop Refresh Home Search Favorites History Channels Address htp 192 168 1 1 v Links Park Head Optics for Shipment Park Head Optics Now Moves the scan head objective and filter to a safe place for shipment ie Done e Internet zone YW Figure 13 13 The Park Head Optics for Shipment Web page 3 Click Park Head Optics N ow The scan head moves to a secure position in the Typhoon instrument 4 Usethe procedures in sections 3 7 and 3 8 to turn o
86. de Chapter 9 Scanning a fluorescent sample Sa 9 1 3 Selecting the scan area The grid in the Scanner Control window allows you to select the area to scan The letter and number markings correspond to the markings on the glass platen of the Typhoon instrument The white rectangle on the grid designates the area you want to scan You can either use the current scan area or select a new one To select a new scan area 1 Place the pointer in the grid square corresponding to the lowest lettered and numbered grid square of the area you want to scan figure 9 2 For example grid square A 1 is the lowest lettered and numbered grid square on the glass platen 2 Drag the pointer to the grid square corresponding to the highest lettered and numbered grid square of the area you want to scan For example R22 is the highest lettered and numbered grid square on the glass platen 3 Releasethe mouse button Thescan area you selected appears in white The Scanning Information area displays the image file size the approximate scan time and the number of lines per channel Ui mam een biyi pi agim IFEN qp hima TIT Tapke a himli oe r m ee a l pce iivr D rs e ul L Dmr aa i Fost ut Fong Parga 1 a fo amp F Lee Cne E b a i Al corner ah majina v See eee i 12d RSP PP DNR RETNA ETER Ti aang ae De Mie ee AT gie Fam frees Ti Cama pre HPS bembe bm OY 2 ra LPa theres KG Ere erie Soe Or
87. e I n 4 4 Coordinates of this grid square 2 2 M10 18 T j Exposure Cassette 18 Bi w am Plastic surface 22 2 z 0 A R Figure 5 2 Placing the sample in the exposure cassette To minimize the size of the image file scan only the grid squares that are covered by the sample If you do not want to scan the entire sample scan the squares that contain the part of the sample you are interested in analyzing To use one screen to expose multiple samples of the same thickness place the samples close together Align the lanes and rows of the sample with the edges of the grid so that the scanned image will be straight as shown in figure 5 2 Typhoon User s Guide p5 7 Parttwo Scanning in the storage phosphor mode Caution Caution 5 5 Placing the sample in the exposure cassette The exposure cassette comes in two styles one for mounted screens and one for unmounted screens Each style of exposure cassette comes in two sizes one for the small screens and one for the large screens Select the exposure cassette size and style that matches the storage phosphor screen you want to use with the sample All exposure cassettes come with grid markings inside to facilitate sample positioning Do not put uncovered wet gels in the exposure cassette For the GP and LE screens make sure wet gels are properly covered Because the TR screen has no protective coating never expose it to wet gels To place the
88. e make sure you are using the red Ethernet cable supplied with the instrument The Ethernet cable may not be properly plugged into the computer or the instrument Check the connections to make sure they are plugged in and are securely fastened figure A 1 If the problem still occurs contact M D Scanner Technical Support See Assistance in the preface for contact information Typhoon User s Guide pA 1 Partsix Appendixes Sample Windows NT configuration Computer Typhoon instrument Shp M am IE Red Ethernet cable To site network Sample Mac OS configuration Typhoon instrument Computer Red Ethernet cable amp ite network Figure A 1 Ethernet connections on the Typhoon instrument and computer pA 2 Typhoon User s Guide Appendix A Troubleshooting A 2 Scanning The instrument will not scan and a warning message tells you that the sample lid is up The sample lid may not be completely closed M ake sure you press the lid all the way down until the latch clicks You start a scan the ImageQuant Preview window remains unchanged for at least three minutes and the pointer no longer responds when you move the mouse The computer has stopped functioning Restart the computer using the manufacturer s instructions Then open t
89. e exposure which eliminates the need for multiple exposures The storage phosphor screens are reusable Caution Chapter 4 About storage phosphor screen autoradiography e The storage phosphor screens are placed on the samples in ambient light and exposure takes place at room temperature e No chemicals darkroom or special treatment is required e Results arein a digital format and can be analyzed qualitatively and quantitatively using ImageQ uant 4 3 Types of storage phosphor screens The storage phosphor screen detects the beta and gamma ionizing radiation from most isotopes You can use any of the three types of storage phosphor screens e General purpose storage phosphor screen section 4 3 1 e Low energy storage phosphor screen section 4 3 2 e Tritium storage phosphor screen section 4 3 3 Unless noted in the descriptions below all three screen types are available mounted on an aluminum backing or unmounted In general you expose the screen to the sample in an exposure cassette See chapter 5 for guidelines on exposing different sample types The exposure cassettes come in four styles Small cassette for small mounted screens Small cassette for small unmounted screens e Large cassette for large and half size mounted screens e Large cassette for large unmounted or macroarray screens 4 3 1 General purpose storage phosphor screen If you use the Typhoon instrument for a wide variety of applications y
90. e of the exposure cassette opposite the clamp or clamps Place the edge of the screen into the groove The white side of the screen should face down toward the sample The top of the label on the screen should be toward the top of the cassette figure 5 3 4 Gently lower the screen into place in the corner guides making sure the screen fits into the guides correctly Do not reposition the screen after it has touched the sample Because the screen is extremely sensitive adjusting the position of the screen can result in a double image Exposure cassette Phosphor screen Corner guide Labels Swing the clamps out of the way into the position shown Corner guide Phosphor screen white side down label toward the top of the sample Figure 5 3 Loading the large format mounted screen into the exposure cassette Typhoon User s Guide p5 9 Part two Scanning in the storage phosphor mode Close the exposure cassette by rotating each clamp counterclockwise until it stops at the pin figure 5 4 and then flipping the lever over to lock the screen into place Exposure cassette Exposure cassette Screen a E SSS SSS a LD 1 Rotate the lever 90 degrees 2 Flip the lever down over the screen to
91. e to the storage phosphor screen to be equal to an overnight exposure to basic x ray film Because the storage phosphor screen has a wide linear dynamic range it is unlikely that you will overexpose the sample to the screen This allows you to capture both the strong and weak sample signals with only one exposure to the sample Typhoon User s Guide p5 11 Chapter 6 Scanning a storage phosphor screen This chapter provides step by step instructions for scanning a storage phosphor screen The topics in this chapter are e Verifying the Typhoon instrument is clean section 6 1 e Removing the exposed screen from the exposure cassette section 6 2 e Loading the storage phosphor screen into the instrument section 6 3 e Selecting the scan parameters in the Scanner Control window section 6 4 e Starting a scan and checking the progress section 6 5 e Cleaning up after the scan section 6 6 Storing the storage phosphor screen section 6 7 e Analyzing or preprocessing the scanned image section 6 8 6 1 Verifying the Typhoon instrument is clean You should check the Typhoon instrument for all types of contamination before placing the storage phosphor screen in the instrument 6 1 1 Checking for radioactive contamination Periodically you should check the instrument for contamination from radioactive samples To check the instrument for radioactive contamination use the following procedure 1 Select a storage phosphor sc
92. ean the glass first with 75 ethanol and then with distilled water Caution Because window cleaners use ingredients that can fluoresce do not use window cleaners to clean the glass platen In addition the use of acetone or the excessive use of ethanol can shorten the lifetime of the instrument Protect the glass from scratches Scratches interfere with accurate imaging and quantitation p11 2 Typhoon User s Guide Chapter 11 Setting up for chemiluminescence scanning 11 2 2 Cleaning the sample lid The surface of the inner lid should be clean and free of chemiluminescent contamination If necessary clean the surface with a damp not saturated cloth moistened with a small amount of distilled water If visible spots remain clean the surface first with 75 ethanol and then with distilled water To prevent liquid from getting inside the lid or rolling down onto the glass do not spray liquid on the lid 11 3 Loading the sample Before you load the sample determine how to position the sample on the glass platen of the instrument 11 3 1 Determining the sample orientation The Typhoon instrument collects data from underneath the sample Use the following guidelines when determining the sample orientation e Membranes Place the sample face down on the glass platen e Microplate Place the microplate on the glass platen so that the edges of the plate are aligned with the grid markings on the glass platen Note M ake a note of
93. ease use one of the telephone numbers below United States and Canada M olecular Dynamics 928 East Arques Avenue Sunnyvale California 94086 Telephone 1 800 743 7782 or 1 408 773 1222 Fax 1 408 773 0152 Ask for M olecular Dynamics M D Scanner Technical Support United States and Canada 800 Centennial Avenue P O Box 1327 Piscataway N ew Jersey 08855 Telephone 1 800 526 3593 Fax 1 732 457 0557 Assistance United Kingdom Amersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England H P7 9NA Telephone 44 1494 544000 Fax 44 1494 542266 Scandanavia Amersham Pharmacia Biotech AB SE 751 84 Uppsala Sweden Telephone 46 18 612 0000 Fax 46 18 612 1920 Europe Amersham Pharmacia Biotech Europe GmbH M unzinger Strasse 9 Postface 5480 D 790 21 Freiburg Germany Telephone 49 761 490 30 Fax 49 761 490 3159 Eastern Europe and CIS Amersham Pharmacia Biotech Export GmbH Wurzbachgasse 18 A 1152 Vienna Austria Telephone 43 1 982 3826 Fax 43 1 985 8327 M iddle East and Africa Amersham Pharmacia Biotech GmbH P O Box 70051 GR 166 10 Glyfada Greece Telephone 30 1 9600687 Fax 30 1 9600687 Asia Pacific Amersham Pharmacia Biotech Asia Pacific 15 F North Cornwall H ouse Taikoo Place 979 King s Road Quarry Bay H ong Kong Telephone 852 2811 8693 Fax 852 2811 5251 Typhoon User s Guide xv Preface IMM Japan
94. ecting the scan parameters in the Scanner Control window You use the Scanner Control window figure 12 1 to select the parameters you want to use when you scan the chemiluminescent sample After the scan you can view the scan parameters in ImageQ uant The scan parameters include pixel size sensitivity beamsplitter PM T voltage file size image type scan date and time and the instrument make and model For more information see the ImageQ uant User s Guide Typhoon User s Guide pl2 1 Part four Scanning in the chemiluminescence mode Tee et en d Fie Jepa ete err hieraa Hei Pri diie TEDI Saree barir IEE ame diad Saran tmh m z angaia kiyi a ee E Egite x Diir OC l Pau tapk r Fasia Parais k oam mr Liep i mrri i c F da A ii iaa Fe atinin ia rd TT j boney branhii Fie bom Germievip PHI ipite rg See fee bip Th ae I ILF Emi Mea EY Lisi Fa Chae H iena ia cet D 2000 Figure 12 1 The Scanner Control window set for a chemiluminescence scan 12 1 1 Selecting a template If a template exists with the settings you want to use you can select the template review the settings add user comments section 12 1 9 and then start the scan section 12 3 For more information on creating and using templates see section 3 5 To select a template choose Load from the Templates menu and then select the template name from the list If a template does not exist select the settin
95. ectrophoresis glass plate or place a membrane in a low fluorescence plastic bag You place the sample on the glass platen so that the sample or sandwich gel creates a smooth even surface on the glass platen Avoid trapping air bubbles between the sample and the glass platen Air bubbles can appear on the scanned image Before placing a wet sample on the glass platen of the instrument squirt a little buffer or distilled water on the glass platen The buffer or water lowers the chance of trapping air bubbles between the sample and the glass platen Make sure you wipe off the excess buffer or water Using too much buffer or water can cause the sample to move on the glass platen during the scan which affects the quality of the collected data Placing a gel or membrane To place a gel or membrane on the glass platen of the Typhoon instrument 1 If the sample lid is not already open push up on the lid release under the center front of the lid until the lid opens figure 8 1 2 Optional For wet samples squirt a little buffer or distilled water on the glass platen 3 Hold the sample by the edges or use a clean plastic spatula for gels or forceps for membranes to handle the sample Gently lower the sample onto the glass platen starting at the A1 corner Do not trap air bubbles under the sample or scratch the glass Cover a membrane with a glass plate to keep it flat Do not touch the part of the sample that will be read by the Typ
96. een Collects and temporarily stores an image from a radioactive sample Several types and sizes of storage phosphor screens are available section 4 3 e Exposure cassette H olds the storage phosphor screen in a light tight environment Several types and sizes of exposure cassettes are available section 4 3 Additional accessories include an Ethernet cable power cords and tools You connect the Typhoon instrument to a computer running either the M icrosoft Windows NT or Apple M ac OS operating system Typhoon User s Guide pl 1 Part one Introduction a Typhoon instrument b Accessories Image Eraser Exposure cassette for mounted screens Mounted storage phosphor screen Exposure cassette for unmounted screens Unmounted storage phosphor screen Image Eraser Figure 1 1 The Typhoon instrument a and accessories b Note that the exposure cassettes and storage phosphor screens are optional accessories pl 2 Typhoon User s Guide Chapter 1 Introduction to the Typhoon system 1 2 How the Typhoon system works The Typhoon instrument scans and processes samples in the following sequence 1 You place an exposed storage phosphor screen a fluorescent sample or a chemiluminescent sample onto the glass platen of the instr
97. een to the radioactively labeled sample using an exposure cassette An imprint of the sample is created on the screen from the radiation energy that is trapped in the crystal lattice of the screen N ext you use the Typhoon instrument to scan the exposed storage phosphor screen The instrument captures the stored information on the screen in the form of a digital image 4 1 1 Storing the image The storage phosphor screens are composed of fine crystals of BaFBr Eu in an organic binder W hen the screen is exposed to a radioactive sample the radiation excites Eu electrons to a state in which they move easily within the phosphor During the exposure process Eut is oxidized to Eu and BaFBr is reduced to BaFBr These ions remain oxidized and reduced after the screen is removed from the sample In this way the phosphor stores the energy from ionizing radiation M ost of the energy from ionizing radiation stored on the screen remains for as long as several weeks 4 1 2 Releasing the stored information Storage phosphors release stored energy when stimulated by light of appropriate wavelengths The Typhoon instrument scans the screen using a red light at 633 nm The charged BaFBr complexes in the screen absorb light in this range figure 4 1 freeing electrons and reducing the Eu to Eut an Eu ion with an electron in an excited state As the excited electron falls to the ground state it releases energy in the form of blue light
98. eleased by the screen You can use the LE screen to record 3p 355 and 14C emissions Typical samples that can be exposed to an LE screen include 7 S sequencing gels 35S protein gels and 33P macroarrays You should handle the LE screen carefully Scratching the surface of the screen will remove the thin protective overlay Always follow the guidelines and instructions provided in chapter 5 when handling the LE screen You can use any of the following four screen sizes with the Typhoon instrument e Thesmall screen which is 20 cm x 25 cm e The half size screen which is 17 5 cm x 43 cm e Thelarge screen which is 35 cm x 43 cm e The macroarray screen which is 24 cm x 30 cm available in unmounted style only 4 3 3 Tritium storage phosphor screen You use the tritium TR storage phosphor screen to record 3H emissions Because the screen is not coated with the protective cellulose acetate overlay the TR screen can detect the weak energy emitted by 3H Typical samples that can be exposed to a TR screen include tissue sections on glass and whole body autoradiography The TR screen is available in one size 19 cm x 24 cm p4 4 Typhoon User s Guide Caution Chapter 4 About storage phosphor screen autoradiography You should handle the TR screen carefully Because you cannot clean a contaminated TR screen make sure the sample is as dry as possible before exposing it to the TR screen You can erase an exposed TR screen that is
99. els or thick samples in a light tight drawer or other light tight place Check that your exposure cassette closes properly pA 4 Typhoon User s Guide Appendix A Troubleshooting The image created from a storage phosphor screen shows a loss of resolution The sample may not be placed directly against the screen Use the exposure cassette whenever possible Do not expose wet gels or thick samples in exposure cassettes Cover a wet gel with plastic wrap or polyester film to protect the general purpose GP and low energy LE screens The image created from either a storage phosphor screen or fluorescent sample contains streaks or other artifacts The instrument may not have been warmed up before the sample was scanned If the Typhoon instrument has been turned off allow 30 minutes warmup time Diagonal streaks may indicate a light leak during scanning Check for damaged panels on the instrument Contact M D Scanner Technical Support See Assistance in the preface for contact information The glass platen may be scratched If possible scan the sample on another portion of the glass Contact M D Scanner Technical Support to order replacement glass and arrange for a service call See Assistance in the preface for contact information The storage phosphor screen may be contaminated See section 5 2 3 for details The image created from a storage phosphor screen contains streaks or other artifacts Static electricity may have accu
100. endix C for a list of references Typhoon User s Guide p 1 Part three Scanning in the fluorescence mode E a Cy3 b Cy5 650 532330 570 633 667 q A Ly ALSAN A AEAN Ca EE i x i l ON S LAN wt i weet x VS A 300 400 500 600 700 800 300 400 500 600 700 800 Wavelength Wavelength Figure 7 1 Absorption and emission spectra of the fluorescent product of Cy3 a and of Cy5 b The wavelength with maximum absorption or emission is shown above the curves The vertical line shows the excitation wavelength These curves are approximations based on data collected at Molecular Dynamics 7 2 Advantages of direct fluorescence Scanning samples labeled with dyes excitable by the 532 nm green or the 633 nm red laser provides results in a digital image format T hese results can be analyzed qualitatively and quantitatively using ImageQ uant Scanning direct fluorescence on the Typhoon system offers several advantages p7 2 Typhoon User s Guide Nucleic acid and protein gels can be analyzed shortly after electrophoresis Soak the gels in a dye solution and rinse away the excess dye as you would for the well known ethidium bromide and Coomassie protocols Some of these same gels can be used for follow on blot experiments You can analyze fluorescently labeled samples that can be detected directly and do not require staining You can analyze nucleic acid and protein
101. ent turned on when you change the optical A filter selection After you remove the front cover of the Typhoon instrument and open the access and filter doors make sure you follow the procedures in this section to avoid injury When you received the Typhoon instrument the instrument contained a standard set of six optical emission filters that you use for fluorescence scanning section 7 3 The filter changer in the Typhoon instrument can contain a maximum of 17 filters To purchase additional filters contact M D Scanner Technical Support See Assistance in the preface for contact information The Typhoon instrument uses a bar code to identify each optical filter installed in the filter changer Bar codes 1 through 20 are reserved for optical filters defined by M olecular Dynamics Bar codes 21 through 62 can be used for optical filters that you define If you purchase custom optical filters from another source and you do not have a sheet of bar code labels contact M D Scanner Technical Support You use the Typhoon Direct Instrument Access Web page to insert or remove optical filters To access the Web page see section 13 1 13 3 1 Moving the filter changer to the access door The filter changer which is attached to the slow stage of the Typhoon instrument is stored at the left side of the Typhoon instrument The access door for changing the filters is at the right side of the instrument Before you can install or remove an optical fi
102. er should be serviced only by an authorized M D Scanner Technical Support representative If you have problems with the instrument or eraser contact M D Scanner Technical Support immediately See Assistance in the preface for contact information When you call MD Scanner Technical Support you will be asked for the serial number of your instrument The serial number of the Typhoon instrument is shown in the Scanner Information area of the Scanner Control window and on Typhoon User s Guide p2 1 Part one Introduction SSS the serial number certification label figure 2 1 The serial number certification label also displays the model number and CDRH Center for Devices and Radiological H ealth compliance information The label is located on the lower right side of the Typhoon instrument near the plug Figure 2 2 shows the exact location of the labels on the Typhoon instrument The serial number of the Image Eraser is located on the right side of the eraser near the plug Figure 2 3 shows the exact location of the labels on the Image Eraser a Typhoon instrument 928 E Arques Ave wo Sunnyvale CA 94086 MODEL TYPHOON 8600 100 240V 5A 50 60Hz SERIAL E AAAA ra This product conforms to the applicable requirements of 21CFR subchapter J at the date of manufacture a Image Eraser 928 E Arques Ave molecular dynamics Sunnyvale CA 94086 part of Amersham Pharmacia Biotec MODEL
103. es screen contamination with minimum attenuation of the signal e You can expose a wrapped wet gel in the exposure cassette but do not lock the clamps on the exposure cassette to secure the screen Doing so can cause liquid to leak into the exposure cassette If possible dry a thin wet gel on blotting paper such as Grade 3MM Chr by Whatman Incorporated before placing the gel in the exposure cassette e Some wet gels are too thick for use in the exposure cassette Expose these wet gels in a light tight drawer or other light tight enclosure e Because you cannot clean a tritium TR screen do not expose a wet gel to aTR screen TheTR screen does not have the cellulose acetate layer that protects the GP and LE screens 5 1 3 Using radioactive standards By exposing your sample in conjunction with a set of radioactive standards you can convert Typhoon system counts which are arbitrary units to the units of your choice such as disintegrations per minute Keep the following in mind when using standards e Therange of activities of the standards should bracket expected levels in your sample p5 2 Typhoon User s Guide Important Chapter 5 Preparing for storage phosphor screen autoradiography e Increasing the number of steps in the standard series will increase accuracy in the conversion process e For accurate quantitation load the standards as part of the sample e If you cannot load the standards as part of the sample
104. ess of the Typhoon instrument Press the ENTER key Windows NT or the RETURN key M ac OS The Typhoon Direct Instrument Access Web page appears in the browser window figure 13 1 The Typhoon Direct Instrument Access Web page lists the maintenance procedures If you want to e Select a Laser Shut Off Time go to section 13 2 Insert or Eject O ptical Filters go to section 13 3 e Park Head Optics for Shipment go to section 13 6 Eile Edit View Go Favorites Help El 2 4 9 BD A e f 3 218 Beck Komad Stop Refresh Home Search Favorites History Channels Fullscreen Address hitp 192 168 1 1 7 Links Typhoon Direct Instrument Access NOTE Please turn off the Scanner Control Software before using these functions Insert or Eject Optical Filters Park Head Optics for Shipment Select a Laser Shut Off Time g Done e Internet zone YW Figure 13 1 The Typhoon Direct Instrument Access Web page p13 2 Typhoon User s Guide Chapter 13 Maintaining the Typhoon instrument Se 13 2 Selecting a laser shut off time Shutting down the lasers after the instrument has been idle helps to prolong the life of the lasers You can use the Typhoon Direct Instrument Access Web page to select the amount of time you want the instrument to remain idle before the lasers shut off When you turn on the instrument or start a scan the lasers automatically turn on Caution Before using the T
105. fe Pew r Deaan Wanni Saray ca Figure 9 4 The Fluorescence Setup window with Scan 1 active 9 2 1 Displaying the Fluorescence Setup window To display the Fluorescence Setup window make sure Fluorescence is the selected acquisition mode and then click the Setup button in the Acquisition M ode area in the Scanner Control window figure 9 1 9 2 2 Selecting the number of scans The Fluorescence Setup window displays the parameters for all four scans As a default Scanner Control activates the parameters for Scan 1 only as shown in figure 9 4 To activate the parameters for Scan 2 click the Use check box for Scan 2 The parameters for Scan 2 activate Continue to click the Use check box for each scan you want to perform 9 2 3 Selecting the emission filter You select an emission filter from the Emission Filter list for each scan you want to perform Each list displays the filters that are installed on the Typhoon instrument If you do not want to use an emission filter select None from the list For a description of the emission filters see section 7 3 When you select a filter Scanner Control automatically suggests the laser you should use with the emission filter you selected p9 8 Typhoon User s Guide Important Chapter 9 Scanning a fluorescent sample 9 2 4 Setting the PMT voltage For fluorescent samples you can alter the PM T voltage to improve the signal collection capabilities of the Typhoon instrumen
106. ff the instrument and computer Warning Do not disconnect the cables with the power on A 5 Disconnect the power cords and the Ethernet connection s 6 Wait for authorized Amersham Pharmacia Biotech or M olecular Dynamics personnel to move the instrument to the new location Typhoon User s Guide p13 19 Part five Maintaining the Typhoon system hardware od 13 6 3 Completing the installation after the move N ote If you connect the Typhoon instrument to a different computer than the one used previously you must make sure that the computer has a dedicated Ethernet card to communicate with the Typhoon instrument If you use an Ethernet connection for your local area network LAN you will need two Ethernet cards one for communicating with the Typhoon instrument and one for communicating with your building network See the M olecular Dynamics Web page www mdyn com for the Approved Computer list For the Ethernet configuration procedure see the M olecular Dynamics Web page and the instructions provided with the Ethernet cards After the instrument and computer have been moved to the new location you should 1 Reconnect the instrument and computer 2 Makesure you plug the instrument and computer into a surge protector which is plugged into a properly grounded outlet 3 Use the procedures in sections 3 1 and 3 2 to turn on the instrument and computer 4 Use the procedure in section 3 3 to start the Scanner Control softw
107. filter 670 DF 30 H as a transmission peak centered at 670 nm and transmits light between 655 nm and 685 nm Use this filter with fluorochromes such as Cy5 526 nm short pass filter Transmits light below 526 nm Use this filter with fluorochromes such as fluorescein 560 nm long pass filter Transmits light above 560 nm Use this filter with fluorochromes such as TRITC For more information on emission filters see the M olecular Dynamics Technical N ote 58 Using D yes and Filters in the Fluorl mager System See appendix C for a list of references You use the Fluorescence Setup window in the Scanner Control software to select the appropriate filter section 9 2 Section 13 3 describes how to install and remove emission optical filters Typhoon User s Guide p 3 Part three Scanning in the fluorescence mode 7 4 Beamsplitters The Typhoon instrument contains two beamsplitters You can use the beamsplitters with multilabel fluorescent samples to create multichannel images The two beamsplitters are e 560 nm dichroic Reflects light of wavelengths shorter than 560 nm and passes light longer than 560 nm 580 nm dichroic R eflects light of wavelengths shorter than 580 nm and passes light longer than 580 nm The instrument uses one of these beamsplitters when you link two scans together in the Fluorescence Setup window in the Scanner Control software section 9 2 7 You link scans that contain si
108. ge If you decrease the voltage below 400 you may lose linearity Increasing the PMT voltage increases the limit of detection For the best sensitivity use the High sensitivity setting with a medium PMT voltage 600 volts to 800 volts If you change your sample matrix you may need to test several PM T settings between 400 volts and 1000 volts to determine which setting gives an acceptable background As a suggested starting point use 600 volts Typhoon User s Guide p9 9 Part three Scanning in the fluorescence mode E Procedure for changing the PMT voltage To set the voltage in the PM T Voltage box e Click the box to select it and then type the value you want maximum setting is 1 000 volts e Usethe up or down arrows to change the value in 5 volt increments To approximately double the signal intensity increase the PM T voltage by 50 volts If you are performing multiple scans select a PM T voltage for each scan 9 2 5 Selecting the laser Based on the emission filter you select Scanner Control selects the laser you should use for the scan You can change the laser displayed in the Laser list to the other nonoptimal choice The choices are the Green 532 or Red 633 laser 9 2 6 Setting the limit of detection You use the sensitivity setting controls to define the limit of detection of the Typhoon instrument The sensitivity setting controls how long the Typhoon instrument collects data from each pixel The Sensi
109. ge phosphor 4 1 to 4 2 emission filter see also optical filter description 7 3 overview 1 3 selecting for fluorescence scanning 9 8 enhancers 2 9 5 2 erase time 5 5 Ethernet 1 1 13 20 A 1 excitation description 7 1 fluorescence labeling 8 2 exposure determining time 4 2 guidelines for storage phosphor screen 5 6 temperature 5 8 time for storage phosphor screen 5 11 exposure cassette cleaning and protecting 14 6 closing 5 10 coordinates 5 7 guidelines for placing sample 5 6 large and small sizes 5 8 opening 6 4 placing mounted screen 5 8 to 5 9 placing sample 5 8 placing unmounted screen 5 10 to 5 11 types 4 3 using with wet gel 5 2 F fatal scan errors 6 14 9 17 12 13 File menu B 1 file type data file gel 6 12 9 15 12 10 dataset file ds 9 15 12 10 filter changer accessing 13 9 moving to access door 13 4 Filter Changer Maintenance Web page 13 5 filter changer see also optical filter filters see emission filter and optical filter fingerprint oil 5 4 6 2 8 4 11 1 fluorescence see also sample advantages of 7 2 beamsplitters 7 4 contamination 8 4 8 5 description 7 1 green excited description 1 4 labeling 8 2 overview 7 1 red excited description 1 4 Index saturated data 9 18 scan acquisition mode 1 4 9 2 selecting a template 9 2 workflow 3 5 Fluorescence Setup window 9 8 Auto Link button 9 11 closing 9 14 disable link warning 9 13 emission filter list 9 8 laser list 9 10 Link Wit
110. grid markings on the exposure cassette and how you aligned the A1 corner of the screen with the grid markings on the glass platen Figure 6 6 describes each sample orientation option Important Make sure you accurately match the orientation of the sample on the screen and the orientation of the screen on the glass platen before you select an orientation button Depending on how you positioned the sample on the screen and how you positioned the screen on the glass platen the top of the sample may be rotated several times p6 8 Typhoon User s Guide Chapter 6 Scanning a storage phosphor screen SEE Orientation buttons Ore Deserta Ft RA H j FAS ES Sample placement in the Screen placement on the For face up sample For face down sample exposure cassette glass platen choose choose S Al corner pe i a ea ea dg Al corner Al comer E ee ieii R Al Top H bags tc al Re EYI Al corner Al corner Meee ys i Pa tl a IT Al corner Figure 6 6a Determining which orientation button to use Typhoon User s Guide p6 9 Part two Scanning in the storage phosphor mode Orientation buttons
111. gs using the instructions in sections 12 1 2 through 12 2 4 12 1 2 Selecting the chemiluminescence scan acquisition mode Storage phosphor is the default scan acquisition mode when you first start the Scanner Control software To change to the chemiluminescence mode select C hemiluminescence from the Acquisition M ode list The selections for the chemiluminescence scan acquisition mode appear in the Scanner Control window figure 12 1 and in the additional Chemiluminescence Setup window figure 12 4 p12 2 Typhoon User s Guide Chapter 12 Scanning a chemiluminescent sample E 12 1 3 Selecting the scan area The grid in the Scanner Control window allows you to select the area to scan The letter and number markings correspond to the markings on the glass platen of the Typhoon instrument The white rectangle on the grid designates the area you want to scan You can either use the current scan area or select a new one To select a new scan area 1 Place the pointer in the grid square corresponding to the lowest lettered and numbered grid square of the area you want to scan figure 12 2 For example grid square A 1 is the lowest lettered and numbered grid square on the glass platen 2 Drag the pointer to the grid square corresponding to the highest lettered and numbered grid square of the area you want to scan For example R22 is the highest lettered and numbered grid square on the glass platen 3 Releasethe mouse button
112. h area 9 11 linking scans 9 11 to 9 12 PMT Voltage box 9 9 quick reference B 4 selecting beamsplitter 9 11 selecting number of scans 9 8 Sensitivity setting 9 10 Unlink All button 9 11 fluorescent indicator dyes 8 1 fluorochrome 7 5 FluorSep 7 5 focal plane Platen setting 9 6 12 7 3 mm setting 9 6 12 7 fuse Image Eraser changing 14 4 to 14 5 location 14 2 type 2 5 repeated replacement 2 5 Safety 2 5 Typhoon instrument changing 13 16 to 13 17 type 2 5 gel see also sample handling 8 8 placing on glass platen 8 8 using 8 2 Typhoon User s Guide Index 3 Index glass platen cleaning 6 2 8 4 11 1 contamination 6 1 placing multiple samples 8 7 11 4 placing sample on 8 6 11 3 solvents 6 2 8 5 11 2 gloves 8 1 GP screen see also storage phosphor screen cleaning 5 4 description 4 3 using plastic wrap 5 2 green excited fluorescence description 1 4 label guidelines 8 2 green excited laser 9 10 grid see scan area H hardware components 1 1 hazardous materials safety 2 9 Help menu B 2 high voltage hazard 2 6 to 2 7 image double 8 6 sample orientation 6 8 9 4 12 4 saturation 9 9 12 9 troubleshooting A 4 Image Analysis Software box 6 11 9 7 12 7 Image Eraser caution 2 7 changing bulb 14 2 to 14 4 changing fuse 14 4 to 14 5 cleaning 14 1 fuse location 14 2 fuse type 2 5 high voltage hazard 2 6 power switch 14 2 serial number and label 2 2 Index 4 Typhoon User s Guide ImageQuant Preview
113. hanol and then with distilled water To prevent liquid from seeping inside the lid or rolling down onto the glass platen do not spray liquid on the lid Typhoon User s Guide p6 3 Part two Scanning in the storage phosphor mode Important 6 2 Removing the exposed screen from the exposure cassette The image on an exposed storage phosphor screen is light sensitive When transferring the screen from the exposure cassette to the instrument keep the screen face down After placing the screen on the glass platen close the lid immediately Exposure to direct light will erase some of the signal on the screen To remove the storage phosphor screen from the exposure cassette 1 Open the latch on the cassette For mounted screens Flip the lever s on the cassette figure 6 2 and then rotate the clamp s clockwise so that they no longer cover the screen For unmounted screens O pen the latch es and raise the lid of the cassette Exposure cassette Exposure cassette Screen a meee 1 Flip the lever up away from the screen 2 Rotate the lever 90 degrees Figure 6 2 Opening the exposure cassette for mounted screens 2 Remove the screen H andle the screen by the edges only Do not touch the white surface of the screen If the sample sticks to the screen gently peel off the sample N ote To keep unmounted screens from moving and causing a double image press down on one edge of the screen
114. hat the scanned image will be straight Top view Back of the Typhoon instrument Coordinates of this grid square L10 Coordinates of this grid square Al Front of the Typhoon instrument Figure 11 2 Sample placement on the glass platen of the Typhoon instrument 11 3 3 Placing a sample on the glass platen You can place the sample directly on the glass platen If you are scanning a membrane place the membrane inside transparency material sheet protectors or a plastic bag and then place this on the glass platen You place the sample on the glass platen so that the sample creates a smooth even surface on the glass platen Avoid trapping air bubbles between the sample and the glass platen Air bubbles can appear on the scanned image p11 4 Typhoon User s Guide Chapter 11 Setting up for chemiluminescence scanning Before placing a wet sample on the glass platen of the instrument squirt a little buffer or distilled water on the glass platen The water lowers the chance of trapping air bubbles between the sample and the glass platen Important Make sure you wipe off the excess water Using too much water can cause the sample to move on the glass platen during the scan which affects the quality of the collected data To place a sample on the glass platen of the Typhoon instrument 1 If the sample lid is not already open push up on the lid release under the center front of the lid until the lid open
115. he Scanner Control software reselect the scanner settings and start the scan again You start a scan and the software aborts the scan and displays the following message Scan aborted hardware error please try to scan again The first time this happens start the scan again If the scan aborts again turn off the power switch on the right side of the Typhoon instrument wait a few seconds and then turn the instrument back on If it happens repeatedly contact M D Scanner Technical Support See Assistance in the preface for contact information You start a scan the software aborts the scan and either e The software displays a message that does not give instructions for resolving the problem note the number of the message e Thered Scan indicator light blinks Restart the scan as follows 1 Close the Scanner Control window Leave the computer on 2 Turn off the power switch on the right side of the Typhoon instrument Wait a few seconds and turn it on again 3 Open the Scanner Control software reselect the settings and rescan If the problem occurs repeatedly contact MD Scanner Technical Support See Assistance in the preface for contact information Typhoon User s Guide pA 3 Partsix Appendixes SS A 3 Image The image has a high background or inaccurate readings The instrument may not have been warmed up before the sample was scanned If the Typhoon instrument has been turned off allow 30 minutes w
116. he fluorescence mode ee Table 7 1 Fluorochrome absorption and emission maxima Fluorochrome Absorption maximum nm Emission maximum nm Acridine Orange dsDNA 502 526 BODIPY 493 503 493 503 BODIPY FL 503 512 Cy3 550 570 Cy5 649 670 DDAO Phosphate 646 660 ECF Substrate 440 560 Ethidium Bromide EtBr 526 610 FAM 495 535 Fluorescein FITC 490 520 HEX 529 560 NanoOrange 485 590 Phycoerythrin R 480 578 PicoGreen dsDNA 480 520 Rhodamine 6 G R6G 546 560 RiboGreen 500 520 ROX 590 605 SYBR Green I dsDNA 497 520 SYBR Green II 497 513 SYBR Gold 495 537 SYPRO Orange 472 570 SYPRO Red 530 625 TAMRA 555 580 TET 519 545 Texas Red 595 615 TOTO 1 dsDNA 513 532 TRITC 547 572 Vistra Green 490 520 Values listed are from the manufacturers catalogs or from data collected at Molecular Dynamics SS p7 6 Typhoon User s Guide Chapter 8 Setting up for fluorescence scanning This chapter describes how to prepare the sample for fluorescence scanning and how to place the sample in the Typhoon instrument The topics in this chapter are e Preparing the sample section 8 1 e Cleaning the glass platen and sample lid section 8 2 e Loading the sample section 8 3 8 1 Preparing the sample The following sections provide guidelines to use when preparing samples for fluorescence scanning 8 1 1 General guidelines Dust f
117. he fluorescence mode Important e Transparency support To avoid contaminating the glass platen and sample lid you can place a sample such as a membrane in a plastic bag between two pieces of transparency material or between sheet protectors section 8 3 3 You should check the background generated by the material to make sure it is low enough for your purposes e Solvents Spectroscopic grade solvents have the lowest autofluorescence 8 2 Cleaning the glass platen and sample lid You should clean the glass platen and sample lid before placing your sample on the plate In addition you should protect the glass from scratches because these interfere with data collection and quantitation If you use the Typhoon instrument for storage phosphor scanning you should periodically check the instrument for contamination from radioactive samples section 6 1 1 8 2 1 Cleaning the glass platen The glass platen of the Typhoon instrument should be kept free of sources of fluorescent background such as dust dried buffer and skin oils It is good practice to clean the glass platen before and after you scan each sample To clean the glass 1 Push up on the lid release under the center front of the sample lid until the lid opens figure 8 1 p8 4 Typhoon User s Guide Chapter 8 Setting up for fluorescence scanning Push up on the lid release eae Sample lid open Lid release HATTON LNT MTOM
118. he optical filter e Usethe procedure in section 13 3 5 to close the filter and access doors and replace the front cover 13 3 2 Accessing the filter changer You must leave the Typhoon instrument turned on when you change the optical filter selection After you remove the front cover of the Typhoon instrument and open the access and filter doors make sure you follow the procedures in this section to avoid injury To access the filter changer you remove the front cover of the Typhoon instrument and open the access and filter doors 1 Makesure the slow stage has stopped moving and the Scan indicator light is no longer blinking 2 Usea 9 64 inch hex wrench to loosen the two socket head screws on the bottom of the front panel figure 13 7 Turn the screws several times but do not remove them Pull off the front cover 3 Usea 9 64 inch hex wrench to loosen the screw on the access door and open the access door 4 Loosen the fingernut on the filter door and open the filter door If the slow stage has not moved the filter changer into position call MD Scanner Technical Support See Assistance in the preface for contact information Typhoon User s Guide p13 9 Part five Maintaining the Typhoon system hardware Typhoon instrument Front cover b Opening the access door Screw ie c Opening the filter door Slide the filter holder Fingernut all the way to the left
119. heck the Scanning Information area in the Scanner Control window for the time required to complete the scan e High Collects data from each pixel eight times and averages the results Using the High setting to detect weak signals improves the sensitivity by reducing the background noise N ote Selecting H igh increases the scanning time but does not increase the size of the image file Check the Scanning Information area in the Scanner Control window for the time required to complete the scan p12 8 Typhoon User s Guide Important Chapter 12 Scanning a chemiluminescent sample 12 2 3 Selecting a beamsplitter for a multichannel scan The Typhoon instrument contains two beamsplitters that you can use with chemiluminescent detection The two beamsplitters are e 560 nm dichroic R eflects light of wavelengths shorter than 560 nm and passes light longer than 560 nm 580 nm dichroic Reflects light of wavelengths shorter than 580 nm and passes light longer than 580 nm If you want to scan the sample and create a multichannel image you must select a beamsplitter from the Beamsplitter list The default selection is N one which is the correct setting for a single channel chemiluminescent scan 12 2 4 Setting the PMT voltage For chemiluminescent samples you can alter the PM T voltage to improve the signal collection capabilities of the Typhoon instrument Guidelines for changing the PMT voltage For accurate quantitation of
120. hoon instrument Do not touch the glass platen Oil from fingerprints and powder from gloves even thoroughly washed gloves may leave a print that can be detected 4 Makea note of the grid square coordinates of the area you want to scan figure 8 2 You will use this information later in the Scanner Control window section 9 1 3 5 Close the sample lid and press it down until the latch clicks The sample is ready to scan p8 8 Typhoon User s Guide SEER Important Important Chapter 8 Setting up for fluorescence scanning Placing a sandwich gel Make sure the low fluorescence electrophoresis glass plate that you place on the glass platen is 3 mm thick The 3 mm focal plane setting is optimized for 3 mm thick glass You can use Kapton tape to help prevent an interaction between the glass platen and the 3 mm electrophoresis glass plate The Kapton tape prevents the two pieces of glass from coming in contact with each other Before assembling the sandwich gel place two strips of Kapton tape oneon each long side of the 3 mm electrophoresis glass plate M ake sure each piece of Kapton tape covers at least 75 of the long edge of the plate To place a sandwich gel on the glass platen of the Typhoon instrument 1 If the sample lid is not already open push up on the lid release under the center front of the lid until the lid opens figure 8 1 2 Hold the sandwich gel by the edges and make sure the 3 mm electrophoresis gl
121. in clean the surface first with 75 ethanol and then with distilled water Do not use powdered detergents Any undissolved particles can scratch the surface of the eraser 3 Verify that the eraser surface is free of contamination from radioactive samples 4 Reconnect the power cord and turn on the Image Eraser Typhoon User s Guide p14 1 Part five Maintaining the Typhoon system hardware Important Front view Side view Image Eraser Fuse window Power switch Figure 14 1 The Image Eraser 14 2 Changing the bulbs in the Image Eraser The Image Eraser contains four warm white compact fluorescent bulbs W hen one of the bulbs fails replace all four bulbs to provide uniform light intensity across the screen To order replacement bulbs contact M D Scanner Technical Support or your local service office see the manufacturer part number on the bulb See Assistance in the preface for contact information If you are replacing the bulbs shipped with the Image Eraser you must order replacement bulbs and fixtures from MD Scanner Technical Support The bulbs shipped with the Image Eraser are epoxied to the fixture to avoid damage during shipment The replacement fixture and bulbs are not epoxied p14 2 Typhoon User s Guide Chapter 14 Maintaining the Image Eraser and exposure cassettes To replace the bulbs 1 Turn off the Image Eraser and disconnect the power cord figure 14 1
122. in the Setup window figure 3 3 For more information on selecting options see section 9 2 fluorescence or section 12 2 chemiluminescence Typhoon User s Guide p3 9 Part one Introduction a Fluorescence Setup window 1 Select the number 2 For each scan select 3 Link scans of scans Emission filter PMT voltage Laser Sensitivity 4 Click OK b Chemiluminescence Setup window 1 Select sensitivity 2 Select beamsplitter 3 Select PMT voltage Doe 2 Ofer Chelona scence Sahip for byphoon lo 4 Click OK Figure 3 3 Selecting options in the a Fluorescence Setup window or b Chemiluminescence Setup window p3 10 Typhoon User s Guide Important Chapter 3 Getting started 3 Choose Save As Template from the Templates menu The Save As Template window appears figure 3 4 Save As Template x Save the current scanning protocol as a template named I Set as Default Template Cancel Figure 3 4 The Save As Template window 4 Typea new name for the template in the box N ote Because the Template list displays all the available templates you may want to label the templates by scan acquisition mode For example a template for storage phosphor can be named TemplateN ame _Phos A template name can be a maximum of 32 characters and you should type an underscore instead of a space 5 Optional If you want the template to become the new default click the Set
123. ing and protecting the exposure cassettes 14 6 Typhoon User s Guide ix Table of Contents x Typhoon User s Guide Partsix Appendixes Appendix A Troubleshooting A 1 Power and communication 0 cece eee eee A 22 SCANNING seek Mie doe an ind Oe atime ea are Ses Mats AZ MaE A n e ce eA a aa a aE a tena ee Appendix B Quick reference for menus and windows Bak MENUS S iaa aan EAE AI E R Ot basen wean a Yin mew SE B 2 7 WNdOWS e eiw anaa a a aaa we a T E y Appendix C Literature references Glossary Warning A Preface About this user s guide The Typhoon User s G uide provides information on how to use the Typhoon system to scan fluorescent samples chemiluminescent samples and storage phosphor screens that have been exposed to radioactive samples You connect the Typhoon instrument to a computer running the M icrosoft WindowsNT or the Apple Mac OS operating system e Part one Introduction D escribes the Typhoon instrument the safety issues and basic instructions for using the instrument and the Scanner Control software Part two Scanning in the storage phosphor mode Describes how to use the Typhoon system to scan a storage phosphor screen Part three Scanning in the fluorescence mode D escribes how to use the Typhoon system to scan a fluorescent sample Part four Scanning in the chemiluminescence mode D escribes how to use the Typhoon
124. ing the glass platen and sample lid o an naaa 12 14 12 6 Analyzing or preprocessing the scanned image 12 14 Part five Maintaining the Typhoon system hardware Chapter 13 Maintaining the Typhoon instrument 13 1 Starting the Typhoon Direct Instrument Access Web page 13 1 13 2 Selecting a laser shut off time nauan 13 3 13 3 Changing the optical filter selection n naaa 13 4 13 3 1 Moving the filter changer to the access door 13 4 13 3 2 Accessing the filter changer s a a naana 13 9 13 3 3 Inserting a new optical filter 0 00 13 11 13 3 4 Removing an optical filter 0 0000 13 13 13 3 5 Closing the doors and replacing the front cover 13 13 13 3 6 Registering a new optical filter in Scanner Control 13 14 13 4 Changing the definition of an optical filter 0c eae 13 15 13 5 Changing the fuses 0 ee 13 16 13 6 Moving the Typhoon instrument 00 000 e eee eee 13 18 13 6 1 Determining a new location for the instrument 13 18 13 6 2 Preparing to move the Typhoon instrument 13 18 13 6 3 Completing the installation after the move 13 20 Chapter 14 Maintaining the Image Eraser and exposure cassettes 14 1 Cleaning the Image Eraser nananana ee 14 1 14 2 Changing the bulbs in the Image Eraser 00005 14 2 14 3 Changing the fuses in the Image Eraser cee eee eee 14 4 14 4 Clean
125. ing windows and window options are used in the Scanner Control software Scanner Control window Option Description Scanner Information area Displays the model number IP address and serial number of the Typhoon instrument Template name Displays the template name for the settings displayed in the Scanner Control window and Setup window fluorescence and chemiluminescence only If no template is selected the default settings appear in the windows Scan area grid Displays the selected scan area in white and allows you to change the selected area by redrawing the white rectangle Setup area Displays the current user name User Name Setup area Allows you to select the scan acquisition mode The Acquisition Mode list choices are Storage Phosphor default Fluorescence and Chemiluminescence Setup area Setup button Depending on the selected acquisition mode displays the Setup window for fluorescence or chemiluminescence Setup area Options area Orientation button Click to display the eight orientation options Allows you to select the option that matches how you placed the sample or storage phosphor screen on the glass platen of the instrument pB 2 Typhoon User s Guide Appendix B Quick reference for menus and windows Option Description Setup area Options area Press Sample check box Available in the fluorescence and chemiluminescence modes A
126. int of the laser beam to the area just above the glass platen of the Typhoon instrument e 3 mm Use this setting if you are scanning a microplate or you want to place the membrane on a piece of 3 mm electrophoresis glass plate before setting the membrane on the glass platen of the Typhoon instrument Scanner Control adjusts the focal point of the laser beam 3 millimeters higher than the glass platen to accommodate the height of the electrophoresis glass plate used to support the membrane 12 1 8 Selecting the image analysis software If you want to analyze the image after you finish scanning you can select the image analysis software you want to use from the Image Analysis list in the Setup area of the Scanner Control window figure 12 1 After the instrument finishes scanning the selected application opens and displays the image If you select N one from the Image Analysis list the Scanner Control window remains active so that you can perform another scan using the current scan settings 12 1 9 Entering user comments optional The user comments are not saved as part of a template If you want to include comments you must type the comments each time you scan In the User Comment box type any comment you want to save with the image approximately 4 000 characters including spaces The comments are for reference only and do not affect the scan After the scan you can view but not change the comments in mageQ uant 12 2 Using
127. ion of important labels on the Image Eraser 2 2 Electrical safety You should follow the electrical safety information provided in this section to make sure you are operating the Typhoon instrument and Image Eraser safely 2 2 1 Electrical connections Before you turn on the Typhoon instrument Image Eraser or computer check that the instrument eraser computer and monitor are plugged into surge protectors and the surge protectors are plugged in and turned on Plug the Typhoon instrument into a grounded circuit capable of delivering at least e 15 amps for a 100 volt to 120 volt power source 10 amps for a 200 volt to 240 volt power source Do not use circuits shared by equipment containing compressors such as refrigerators and centrifuges M ake sure power cords are in good condition and are not frayed p2 4 Typhoon User s Guide Caution Warning A Chapter 2 Safety 2 2 2 Fuses When you turn on the Typhoon instrument the instrument automatically switches to the correct operating voltage The Image Eraser does not automatically switch to the correct operating voltage You must manually select the correct voltage section 14 3 Before turning on the Image Eraser make sure that the correct operating voltage appears in the fuse window Using the wrong voltage can severely damage the eraser Both the Typhoon instrument and the Image Eraser have fuses in a fuse holder On the Typhoon instrument T he fu
128. k one of the link buttons that has a colored dot Figure 9 5 b shows the Fluorescence Setup window after the scans have been linked N ote that both scans now display a 1 as the scan number Figure 9 5 also shows that you could link scan 2 with either scan 1 or scan 3 Because there is no colored dot for scan 2 linking scan 2 would not be the best link To link scan 2 with scan 1 click the scan 1 link button in the Link With area for scan 2 After you click the link button brackets appear connecting the Use check boxes for the original scan 1 and 2 In addition the Scan numbers change to indicate which scans you combined into one scan After you link nonoptimal scans and click OK Scanner Control warns you that your selection is not optimal saves the settings and closes the Fluorescence Setup window If you do not want Scanner Control to warn you that you selected nonoptimal links click the Disable the link warning check box Otherwise after you click OK to save the settings and close the Fluorescence Setup window Scanner Control will display a message warning you of the nonoptimal links Typhoon User s Guide p9 13 Part three Scanning in the fluorescence mode Important Warning A Cautions Unlinking scans If you want to unlink all the linked scans click the Unlink All button Scanner Control removes all the links You no longer see the brackets connecting the Use check boxes and the Scan numbers return to their origin
129. le of the company within the Amersham Pharmacia Biotech group that supplies them A copy of these terms and conditions is available on request Amersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Pharmacia Biotech AB SE 751 84 Uppsala Sweden Amersham Pharmacia Biotech Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Molecular Dynamics Inc 928 East Arques Avenue Sunnyvale CA 94086 USA Molecular Dynamics Inc 1999 All rights reserved November 1999 256 031 Table of Contents Preface About this user s guide 2 2 eect tee xi Related publications 0 2 0 cece eee xi Sally f 26 eset etad nn taetine A ed Brae S aie we anes beara nae xi Trained operator cea eee eed ee Seka ood endo xi Special safety text 1 tees xii ASSUMPUONS ia ace erties wieder ee teow Ieee Waitara ties xii CE declaration secs 4c sad Ae ae A ee ea A ee ee xiii Site requirements cree Sata Canin win ea Ais eka ne tata xiv EICCIrIGA we rirdi ueo wig Be eas Hens sea Gea yeaa xiv Environmental ssi Pacha owe ed a ew ae E A E xiv ASHU MENT ia idin aer pe aaoi eaa aih eiei uA ELEA xiv ASSISTANCE extn i i gee ee e n a tate S S ra wey nace xiv Partone Introduction Chapter 1 Introduction to the Typhoon system 1 1 Typhoon system hardware components 0 0es eee 1 1 1 2 How the
130. les choose the 200 microns setting which provides the fastest scan time and the smallest image file size The 200 micron pixel size produces 50 data points per centimeter and 100 data lines per grid square For samples requiring high resolution such as DN A sequences choose the 100 microns setting The 100 micron pixel size produces 100 data points per centimeter and 200 data lines per grid square For samples requiring very high resolution such as whole body autoradiography choose the 50 microns setting The 50 micron pixel size produces 200 data points per centimeter and 400 data lines per grid square After you change the pixel size Scanner Control updates the image size the scan time and the number of data lines per channel in the Scanning Information area figure 6 4 6 4 6 Selecting the image analysis software If you want to analyze the scanned image after you finish scanning you can select the analysis software you want to use from the Image A nalysis list in the Setup area of the Scanner Control window figure 6 4 After the instrument finishes scanning the selected application opens and displays the image If you select N one from the Image Analysis list the Scanner Control window remains active so that you can perform another scan using the current scan settings Typhoon User s Guide p6 11 Parttwo Scanning in the storage phosphor mode Important Warning A Cautions 6 4 7 Entering user commen
131. llows you to select that you want the sample pressed during scanning Pressing is automatic in the storage phosphor mode Setup area Options area Pixel Size list Displays a list of pixel size options The choices are 200 microns for normal samples 100 microns when a higher resolution is required and 50 microns when very high resolution is required Focal Plane area Available in the fluorescence and chemiluminescence modes Allows you to change the focal plane of the laser to match the sample type You can choose either Platen sample is on glass platen or 3 mm sample is 3 mm higher than glass platen Setup area User Comment box Allows you to save comments maximum 4 000 characters with the image file Setup area Image Analysis list Allows you to select the image analysis software to open after scanning Choose None to leave Scanner Control active Setup area Scan button Click to start the scan If you have not provided aname for the scanned image you are prompted to provide one before the scan begins Scanning Information area Displays the settings selected for the scan and the approximate scan time and image file size required for the scan Typhoon User s Guide pB 3 Part six Appendixes Fluorescence Setup window Option Description Use check box Allows you to select the number of scans to perform Also displays scans that are linked Scan box
132. lly select the settings make sure Storage Phosphor is the selected scan acquisition mode Then select the grid area pixel size sample orientation and image analysis software Type comments you want saved with the image 4 Start the scan Click Scan Type a file name and click 6 5 Save Windows NT or Open Mac OS The ImageQuant Preview window appears While the Typhoon instrument scans the screen an image of the screen appears in the window 5 Evaluate the results During scanning check the image in the 6 5 3 ImageQuant Preview window for saturation Saturated pixels appear in red If the image appears saturated you may need to expose a clean screen to the sample for a shorter amount of time If the image appears faint you may need to expose a clean screen for a longer amount of time If the image appears usable continue with the next scan If you are scanning another screen make sure you clean the glass platen and sample lid before you place the next screen in the instrument p3 4 Typhoon User s Guide Chapter 3 Getting started Table 3 1 Storage phosphor scanning workflow continued Task Description Section 6 Clean up after the Remove the sample from the exposure cassette 6 6 scan and dispose of the sample using the appropriate 6 7 laboratory procedures Decontaminate and clean the Typhoon instrument exposure cassette and Image Eraser Decontaminate clean and erase a GP or LE screen Erase a
133. locates another filter that uses the same bar code as the one you are installing O ptional To install additional filters click Step 1 Access Filter Rod and then repeat steps 1 through 7 Click Done The Typhoon instrument scans the bar codes of all the installed filters and records the changes After a minute you see a list of all the filters installed in the Typhoon instrument Click OK to close the window and return to the Typhoon Direct Instrument Access Web page Use the procedurein section 13 3 5 to replace the front cover and then use the procedure in section 13 3 6 to define the filter in Scanner Control p13 12 Typhoon User s Guide Warning A Caution Warning A Chapter 13 Maintaining the Typhoon instrument 13 3 4 Removing an optical filter You must leave the Typhoon instrument turned on when you change the optical filter selection After you remove the front cover of the Typhoon instrument and open the access and filter doors make sure you follow the procedures in this section to avoid injury You can remove an optical filter you no longer want to use M ake sure that this filter is not used in any active templates created using the Scanner Control software section 3 5 If the filter is used in a template you should modify or delete the template If you do not Scanner Control removes the template from the template list and you cannot use it Before using the Typhoon Direct Instrument Access We
134. lter you must move the filter changer to the access door p13 4 Typhoon User s Guide Chapter 13 Maintaining the Typhoon instrument Sa Moving the filter changer into position to install an optical filter The following procedure moves the slow stage and positions the filter changer at the access door so that you can add an optical filter Caution Before using the Typhoon Direct Instrument Access Web page make sure you close the Scanner Control software Leaving Scanner Control running while you use the Web page can cause unpredictable problems in Scanner Control 1 Inthe Typhoon Direct Instrument Access Web page figure 13 1 click the Insert or Eject O ptical Filters option The Filter Changer M aintenance Web page appears figure 13 3 Eile Edit View Go Favorites Help El a S g fay Q Ke Q E Back oryverd Stop Refresh Home Search Favorites History Channels Fullscreen Address htip 192 168 1 1 Links Filter Changer Maintenance Page Please refer to the users manual for detailed instructions Install a New Optical Filter Remove an Optical Filter e Done a Internet zone Figure 13 3 The Filter Changer Maintenance Web page Typhoon User s Guide p13 5 Part five Maintaining the Typhoon system hardware E 2 Make sure the sample lid is closed and then click Install a N ew O ptical Filter The Insert a N ew Optical Filter into the Filter Changer Web page appea
135. luoresces and scatters light which causes artifacts on images and can interfere with quantitation To avoid this Wear powder free gloves Because most powder used in laboratory gloves fluoresces you should wear powder free gloves Always rinse the gloves with distilled or tap water before handling the sample and before preparing the reagent Filter solutions R emove dust particles by filtering all the stock solutions used to prepare the sample sample matrix and buffers Use clean rinsed containers Some reagents such as fluorescent labels are supplied dust free and require no further filtration W hen diluting clean reagents to working concentration use distilled water collected in a rinsed container so that you do not have to filter the solutions again e Avoid fluorescent indicator dyes M any of the commonly used electrophoresis tracking dyes fluoresce such as bromophenol blue or xylene cyanol Whenever possible put the tracking dye in a separate lane Alternatively use a dye in the sample loading buffer that is either nonfluorescent or does not enter the gel e Avoid excessive exposure to light Fluorochromes differ greatly in their sensitivity to light induced degradation photobleaching When working with sensitive fluorochromes and fluorescently labeled samples use low light conditions Typhoon User s Guide p8 1 Part three Scanning in the fluorescence mode E 8 1 2 Label guidelines Use the followi
136. milar settings so that you can acquire signal from two different fluorochromes during one scan This optimizes scanning of samples containing multiple fluorochromes by reducing the scan time Similar to a long pass emission filter the beamsplitter separates the emissions from the linked scans so that one PM T collects the longer wavelength emissions while the other PM T collects the shorter wavelength emissions figure 7 2 Emission filter Mirror A cate Short wavelength Emitted light Long wavelength Beamsplitter p Emission filter Figure 7 2 How the beamsplitter separates short and long wavelength emissions Scanner Control determines that two scans can be linked if they Use the same laser Use the same sensitivity setting Use two different filters or at least one uses no emission filter p7 4 Typhoon User s Guide Chapter 7 About fluorescence scanning e Usea beamsplitter that has a wavelength between the wavelengths of the two emission filters and the wavelength of the beamsplitter is at least 5 nm from each filter Based on the selected emission filters and laser Scanner Control selects the appropriate beamsplitter to use with the linked scans The possible links appear in the Fluorescence Setup window figure 9 5 You select whether or not to activate the link 7 5 Multilabel multichannel experiments You can label the sample with multiple fluorescent dyes and then create a
137. miluminescent experiment section 10 3 Beamsplitters section 10 4 10 1 How chemiluminescence works Chemiluminescence is the release of light energy from a chemical reaction Chemiluminescent reactions used in biological assays typically emit visible light from the enzymatic conversion of a synthetic substrate M ost common chemiluminescent substrates react with enzymes such as peroxidase alkaline phosphatase or galactosidase Each enzyme can initiate multiple chemiluminescent reactions Depending on the system you use the reaction kinetics and the wavelength of the emitted light will vary To assure saturation kinetics you use an excessive amount of the substrate In addition environmental influences such as temperature affect the efficiencies of some chemiluminescent reactions Refer to the manufacturer s documentation for information about how the environment affects the chemistry you are using You can detect the light emission from a chemiluminescent reaction using film autoradiography or using a light sensitive instrument such as the Typhoon system 10 2 Advantages of chemiluminescence Chemiluminescence is a popular and convenient alternative to radioactive fluorescent and colorimetric detection methods Unlike colorimetric or fluorescent measurements chemiluminescent samples contribute very little native background because an exogenous light source illumination or excitation is not required for detection The l
138. mulated on the screen Clean the GP and LE screens with an intensifying screen cleaner Do not attempt to clean the tritium TR screen The screen may be contaminated with radioactive material The source of this contamination could be the surfaces of the eraser or the exposure cassette Clean the GP or LE screen exposure cassette and eraser surfaces section 5 2 The screen may have been exposed to too much light between the time you removed it from the exposure cassette and the time you put it in the instrument for scanning Keep the screen face down in subdued light until you place it in the instrument The screen may not have been erased immediately before exposure Cosmic radiation will generate a background signal on screens left unused for long periods of time Typhoon User s Guide pA 5 Partsix Appendixes e Thescreen may not have been completely erased Select a higher setting and erase again section 5 2 4 e Thescreen may be scratched If possible expose the sample on another portion of the screen For large samples you will need to use a different screen e Fingerprints may be on the screen Clean the GP and LE screens with an intensifying screen cleaner Do not attempt to clean the TR screen Instead use a new TR screen The image created from a fluorescent sample contains streaks or other artifacts e Fingerprints appear in the scan Clean the glass platen If the fingerprints are on the gel rinse the gel
139. n 9 1 8 Selecting the image analysis software If you want to analyze the image after you finish scanning you can select the image analysis software you want to use from the Image Analysis list in the Setup area of the Scanner Control window figure 9 1 After the instrument finishes scanning the selected application opens and displays the image If you select N one from the Image Analysis list the Scanner Control window remains active so that you can perform another scan using the current scan settings 9 1 9 Entering user comments optional The user comments are not saved as part of a template If you want to include comments you must type the comments each time you scan In the User Comment box type any comment you want to save with the image approximately 4 000 characters including spaces The comments are for reference only and do not affect the scan After the scan you can view but not change the comments in mageQ uant 9 2 Using the Setup window to select the fluorescence para meters You use the Fluorescence Setup window to select the fluorescence parameters to use with the sample You can select from one through four sets of parameters to perform one through four scans The following sections describe how to use the Fluorescence Setup window figure 9 4 Typhoon User s Guide p9 7 Part three Scanning in the fluorescence mode Laim e n x 0m eens feo x iF fame Pere fore r i pee Pw S
140. n the membrane place the membrane between two pieces of transparency material or between sheet protectors Alternatively you can place the membrane in a plastic bag to keep the sample wet and surrounded by substrate 11 2 Cleaning the glass platen and sample lid You should clean the glass platen and sample lid before placing the sample on the plate In addition you should protect the glass from scratches because these interfere with accurate imaging and quantitation If you use the Typhoon instrument for storage phosphor scanning you should periodically check the instrument for contamination from radioactive samples section 6 1 1 11 2 1 Cleaning the glass platen The glass platen of the Typhoon instrument should be kept free of sources of contamination such as substrate It is good practice to clean the glass platen before and after you scan each sample Typhoon User s Guide p11 1 Part four Scanning in the chemiluminescence mode es To clean the glass 1 Push up on the lid release under the center front of the sample lid until the lid opens figure 11 1 Push up on the lid release i 4 Sample lid open Lid release Sample lid TT TTTT__wTw OOOO KENNT www Figure 11 1 Releasing and opening the sample lid 2 Optional Check the glass periodically for chemicals 3 Clean the glass with distilled water and a clean lint free cloth or paper If visible spots remain cl
141. ner section of the lid to hold the screen flat 5 Close the sample lid and press it down until the latch clicks The screen is ready to scan 6 4 Selecting the scan parameters in the Scanner Control window You use the Scanner Control window figure 6 4 to select the scan parameters you want to use when you scan the storage phosphor screen The scan parameters include pixel size PM T voltage file size image type scan date and time and the instrument make and model After the scan you can view the scan parameters in ImageQ uant For more information see the mageQ uant User s Guide If the Scanner Control window is not open see section 3 3 fee Prphoen Soenran Ceetred C08 i iemp cca like gal De Tersplaien Hobs ogres shear behest phiana i Abimi HIB Tmi a ji empiu hel ih eT A Linai Fiii T shop sive hai a Ene Frenin Dra fi H L E 4 i H f E a E B a Ay iPad Fb aaa H Sata ary i Oe fie Low imiy Pll Seite Toi canes Tina es S Se t M birsi Pa Chaa 1H rage berre Dkp TEIA Figure 6 4 The Scanner Control window set for a storage phosphor scan p6 6 Typhoon User s Guide Chapter 6 Scanning a storage phosphor screen In the storage phosphor scan acquisition mode two features cannot be changed The Press Sample option is selected so that the screen is pressed tightly against the glass platen for scanning Platen is selected as the Focal Plane so that the Typhoon instrument
142. ng guidelines when selecting the label for the fluorescence scanning Green excited fluorescence D etects samples labeled with fluorochromes that are excited at 532 nm Red excited fluorescence D etects samples labeled with fluorochromes that are excited at 633 nm M ultilabel fluorescence D etects samples labeled with two three or four fluorochromes and creates a multichannel image To minimize the spectral overlap from the fluorochromes the emission peaks should be greater than 20 nm apart Use both lasers and up to four different filters to create the multichannel image The multichannel image contains one image for each fluorochrome Using ImageQ uant you can view these images separately or overlaid N ote T he instrument contains a built in filter for the laser lines If you do not select an emission filter the built in filter blocks the laser light at 532 nm 8 nm and 635 nm 10 nm 8 1 3 Low fluorescence sample support guidelines For fluorescent samples requiring high sensitivity or highly accurate quantitation the following material sources have been tested and found to have low background p8 2 Typhoon User s Guide Ges Background fluorescence contributed by the gel matrix increases with gel thickness Always use the thinnest gel practical for your experiment especially for agarose gels Agarose FM C SeaKem Gold and LE have low background If another type of agarose has properties that are useful for
143. not contaminated and reuse it Always follow the guidelines and instructions provided in chapter 5 when handling the TR screen Typhoon User s Guide p4 5 Chapter 5 Preparing for storage phosphor screen autoradiography This chapter provides guidelines for sample preparation and instructions on exposing the sample to both mounted and unmounted storage phosphor screens The topics in this chapter are Guidelines for preparing the samples section 5 1 Preparing the storage phosphor screen for exposure section 5 2 Guidelines for exposing storage phosphor screens section 5 3 Guidelines for placing the sample in the exposure cassette section 5 4 Placing the sample in the exposure cassette section 5 5 Placing the screen in the exposure cassette section 5 6 Exposing the storage phosphor screen section 5 7 5 1 Guidelines for preparing the samples The following sections provide guidelines to use when preparing samples for use with the storage phosphor screen 5 1 1 General Use the following guidelines when preparing the samples Follow all the laboratory procedures for the type of sample you are preparing M ake sure that thin layer chromatography TLC plates are completely dry before placing them with the screen To keep flecks off the screen cover the dry TLC plate with plastic wrap or strong thin polyester film such as 5 mm M ylar If you also use the Typhoon instrument for fluorescence scanning make
144. ore you start the first scan Scanning before the instrument is warmed up can affect the accuracy of the collected data Power indicator light m Figure 3 1 Right side view of the Typhoon instrument Typhoon User s Guide p3 1 Part one Introduction 3 2 Turning on the computer You can turn on and use the computer before you turn on the Typhoon instrument H owever before the computer can communicate with the Typhoon instrument the instrument must be turned on and the power indicator light must be green Use the computer manufacturer s instructions for turning on the computer 3 3 Opening the Scanner Control software After the computer is turned on use the following instructions to display the Scanner Control window e Windows NT Log on to Windows NT Locate and double click the Scanner Control shortcut icon on the desktop Alternatively you can select Scanner Control using the Start menu e Mac OS Locate and double click the Scanner Control icon in the hard drive window The Scanner Control window appears 3 4 Workflows for scanning You can scan three types of samples on the Typhoon instrument e A storage phosphor screen that has been exposed to a radioactive sample section 3 4 1 e A fluorescent sample section 3 4 2 e A chemiluminescent sample section 3 4 3 This section provides an overview of the workflow for each sample type and indicates where in this user s guide you should look
145. orientation buttons appear M ove the pointer to the button that represents how you aligned the sample on the glass platen and release the mouse button During the scan the software maps the pixels to display the image of your sample face up and top end up p12 4 Typhoon User s Guide Chapter 12 Scanning a chemiluminescent sample Orientation buttons Op Drinistor F Ris FAA Bis Sample placement on the For face up sample For face down sample glass platen choose choose Al corner Figure 12 3 Determining which orientation button to use Typhoon User s Guide p12 5 Part four Scanning in the chemiluminescence mode Important Caution 12 1 5 Selecting the Press Sample option The Press Sample option is not saved as part of a template If you are using a template you must select this option before you scan If you are scanning a dry membrane that does not lie flat against the glass platen of the instrument you can select the Press Sample check box When you scan the sample the inner lid of the Typhoon instrument lowers to press the sample flat Alternatively lay an electrophoresis glass plate over the sample after you place the sample on the glass platen of the instrument If you do not select Press Sample the inner lid remains retracted during the scan Do not select Press Sample if you are scanning wet membranes Pressing wet membranes that are notin a plastic bag can damage the instrument
146. ote Identifies items of general interest Assumptions The software related instructions in this user s guide assume you have basic computer skills You should be familiar with the Windows NT or MacOS graphical user interface You should also know how to usea mouse If you do not have these skills refer to the documentation or the online H elp for WindowsNT or MacOS The following terms are used in this guide e Click Press and then quickly release the mouse button e Double click Press and release the mouse button twice e Drag Hold down the mouse button while moving the mouse e Pointer An object such as an arrow that moves on the screen as you move the mouse e Point M ove the mouse until the pointer is positioned on the desired object N ote Unless stated otherwise the Windows NT mouse button instructions refer to the left button The Scanner Control examples in the Typhoon User s Guide display the Windows NT version of the Scanner Control software If the Windows NT and M ac OS versions of Scanner Control differ the example displays both versions of the software CE declaration CE declaration oO molecular dynamics part of Amersham Pharmacia Biotech Declaration of Conformity according to ISO IEC Guide 22 and EN 45014 Application of Council Directive Manufacturer s Name Manufacturer s Address declares that the product Product Name Product Description Model Number s
147. ou should use the general purpose GP storage phosphor screen The GP screen is protected from radioactive contamination by a durable cellulose acetate overlay that covers the phosphor layer The phosphor layer of the GP screen can detect energy from most isotopes The isotopes include but are not limited to 32P 33p 1251 35s and 14C Typical samples that can be exposed to a GP screen include 22P Northern blots 32P Southern blots 1251 Western blots and a variety of gels You should handle the GP screen carefully Scratching the surface of the screen will remove the protective overlay Always follow the guidelines and instructions provided in chapter 5 when handling the GP screen Typhoon User s Guide p4 3 Parttwo Scanning in the storage phosphor mode Caution You can use any of the following four screen sizes with the Typhoon instrument e Thesmall screen which is 20 cm x 25 cm The half size screen which is 17 5 cm x 43 cm e Thelarge screen which is 35 cm x 43 cm e The macroarray screen which is 24 cm x 30 cm available in unmounted style only 4 3 2 Low energy storage phosphor screen If you use the Typhoon instrument for applications that require more sensitivity you should use the low energy LE storage phosphor screen The phosphor layer of the LE screen is coated with a thin layer of the durable cellulose acetate overlay and a small amount of iodide which increases the efficiency of the energy stored and r
148. ouble image 8 3 1 Determining the sample orientation Determine whether to place the sample face up or face down on the glass platen The Typhoon system illuminates the sample and collects data from underneath the sample e For a one sided opaque sample such as a membrane or TLC plate place the sample face down e For a sandwich gel make sure you create the sandwich gel using a 3 mm electrophoresis glass plate Place the 3 mm electrophoresis glass plate on the glass platen of the instrument e If the sample is physically uneven on one side such as an agarose gel place the flat side down This allows the sample to lie flat on the glass platen Fora microplate or microplate strip place the plate or strip on the glass platen so that the edges of the plate or strip align with the grid markings on the glass platen e Asareference point for the sample orientation you can cut a corner of the gel or sample 8 3 2 Determining the sample placement Use the following guidelines and figure 8 2 to determine where on the glass platen to place the sample To minimize the scan time Place the sample near the A1 corner of the grid Each lettered or numbered grid segment is marked by two dots Position a rectangular sample so that the shortest edge is along the numbered side of the glass platen as shown in figure 8 2 Because each pass of the scan head moves the entire length of the glass platen from front to back placing
149. pen Figure 14 3 Opening the fuse box 8 Snap the cover of the fuse box back into place Check that the correct voltage rating for your power source appears in the voltage window If the voltage is incorrect for your power source remove the fuse holder rotate the holder 180 so that the correct voltage rating is right side up and reinsert the fuse holder Caution Before you turn on the Image Eraser after you change the fuse make sure that the correct operating voltage appears in the fuse window Selecting the wrong voltage can severely damage the eraser 9 Reconnect the power cord and turn on the Image Eraser figure 14 1 Typhoon User s Guide p14 5 Part five Maintaining the Typhoon system hardware 14 4 Cleaning and protecting the exposure cassettes To protect the exposure cassettes from contamination and damage observe the following precautions and cleaning procedure Do not place uncovered wet gels in the exposure cassette Wet gels can permanently contaminate the cassette For information on using wet gels see section 5 1 2 Do not place sharp or heavy objects inside the cassette A crease or dent in the cassette lining causes uneven pressure on the sample e Keep the foam inside the cassette dry e Immediately before placing the sample in the cassette clean the grid surface of the cassette with a damp cloth moistened with a small amount of distilled water If
150. placed the upper left corner of the sample on the B2 grid square in the exposure cassette and then aligned the A1 corner of the screen with the Al corner of the glass platen place the pointer on the B2 grid square on the grid 2 Drag the pointer to the grid square corresponding to the highest lettered and numbered grid square of the area you want to scan figure 6 5 Typhoon User s Guide p6 7 Parttwo Scanning in the storage phosphor mode SSS 3 Releasethe mouse button Thescan area you selected appears in white The status bar displays the approximate image file size the approximate scan time and the number of lines per channel es ees Ce Tee a Di mmm 1 mam ries tay ae pT afim FET Sead Marshes TIT Vane Diet bir dais f hopes iinr Fetoa E Diran a r Fimi ory Foi Pire Eu zl I Lae Commas E zl anona enr ErOTEF A A1 corner i ee CCETEI Bajpai ivs ktera r pen phe Mit Bue es MTA Each atc tte ECEN j Oe lie aa FT ee mF Bel Biel Tastan Te Soames Fe Lea Pe Dare FE iamar mh pra harei E E Figure 6 5 Selecting the scan area 6 4 4 Selecting the sample orientation You choose the orientation from the O ptions area in the Scanner Control window figure 6 4 The orientation buttons allow you to change how the scanned image will be displayed in ImageQ uant To do this you select an icon that represents how you aligned the upper left corner of the sample with the
151. plate to use as adefault 3 12 3 5 5 Deleting a template 0 cc eee eee 3 12 3 6 Closing the Scanner Control software 0c cece eee 3 13 3 7 Turning off the Typhoon instrument 0 000 eae 3 13 3 8 Turning off the computer 0 cece eee 3 13 Parttwo Scanning in the storage phosphor mode Chapter 4 About storage phosphor screen autoradiography 4 1 How Typhoon generates an image 00 0 e eee eee 4 1 4 1 1 Storing the image 6 0 eee 4 1 4 1 2 Releasing the stored information 0 000 4 1 4 2 Advantages of storage phosphor screen autoradiography 4 2 4 3 Types of storage phosphor screens 0 0c eee eee eee 4 3 4 3 1 General purpose storage phosphor screen 4 3 4 3 2 Low energy storage phosphor screen 00 4 4 4 3 3 Tritium storage phosphor screen 0 cece eee eee 4 4 Table of Contents Chapter5 Preparing for storage phosphor screen autoradiography 5 1 Guidelines for preparing the samples 0 0000 ee eaue 5 1 5k Generali sc aiaa tere ede te Meta here a Pat 5 1 5 1 2 Using wet gels with the storage phosphor screen 5 2 5 1 3 Using radioactive standards 00 c cece ee eee 5 2 5 2 Preparing the storage phosphor screen for exposure 5 3 5 2 1 Protecting the storage phosphor screen 5 3 5 2 2 Verifying that the storage phosphor screen is clean 5 3 5 2 3 Cle
152. plays the Save As window so that you can type a name for the image you are about to create Exit Quit Closes the Scanner Control software Templates menu Command Description Load Displays a list of all the templates for the Typhoon instrument After you select a template from the list the settings for that template appear in the Scanner Control window and if the template is a fluorescence or chemiluminescence template in the Setup window Set Default Load Displays a list of all the templates After you select a template from the list the settings for that template become the default and appear when you open the Scanner Control software Save as Template Displays the Save As Template window so that you can type a name for the template and save the settings Delete Template Displays the Delete Template s window which lists all the templates except the template displayed in the Scanner Control window and Setup window fluorescence and chemiluminescence only Allows you to select and delete a template or templates Typhoon User s Guide pB 1 Part six Appendixes Help menu Command Description Contents and Index Displays a Help window that contains topics about how to use the Scanner Control software About Displays the About Typhoon Scanner Control window which contains the copyright version number of the Scanner Control software and contact information B 2 Windows The follow
153. ple is ready to scan Top view Back of the Typhoon instrument Sandwich gel HEW Wonder Wedge Front view Sandwich gel Wonder Wedge Sandwich gel 3 mm glass plate ZO L Kapton tape Z_ Squirt distilled water here Glass platen Glass platen Figure 8 3 Aligning a sandwich gel on the glass platen of the Typhoon instrument p8 10 Typhoon User s Guide Chapter 9 Scanning a fluorescent sample This chapter provides step by step instructions for scanning a fluorescent sample The topics in this chapter are Selecting the scan parameters in the Scanner Control window section 9 1 e Using the Setup window to select the fluorescence parameters section 9 2 e Starting a scan and checking the progress section 9 3 e Removing the sample from the Typhoon instrument section 9 4 e Cleaning the glass platen and sample lid section 9 5 e Analyzing or preprocessing the scanned image section 9 6 9 1 Selecting the scan parameters in the Scanner Control window You use the Scanner Control window figure 9 1 to select the scan parameters you want to use when you scan the fluorescent sample After the scan you can view the scan parameters in ImageQ uant The scan parameters include pixel size PM T voltage sensitivity emission filters lasers beamsplitter file size image type scan date and time and the instrument make and model For more information see the mage
154. pter 13 Maintaining the Typhoon instrument Sa Moving the filter changer into position to remove an optical filter The following procedure moves the slow stage and positions the filter changer at the access door so that you can remove an optical filter Caution Before using the Typhoon Direct Instrument Access Web page make sure you close the Scanner Control software Leaving Scanner Control running while you use the Web page can cause unpredictable problems in Scanner Control 1 Inthe Typhoon Direct Instrument Access Web page figure 13 1 click the Insert or Eject O ptical Filters option The Filter Changer M aintenance Web page appears figure 13 3 2 Makesurethe sample lid is closed and then click Removean O ptical Filter The Remove an Optical Filter from the Filter Changer Web page appears figure 13 5 Eile Edit View Go Favorites Help Bee ee res a 3 Q jaie Ronverd Stop Refresh Home Search Favorites History Channels Fullscreen Address htip 192 168 1 1 z Links Remove an Optical Filter from the Filter Changer Eject filter Positions a filter at the door so you may remove it DONE Does a complete inventory of the filter changer to record all changes Takes about a minute e Internet zone Figure 13 5 The Remove an Optical Filter from the Filter Changer Web page Typhoon User s Guide p13 7 Part five Maintaining the Typhoon system hardware Sey 3 Click Ejec
155. r channels are created using a combination of fluorescent dyes emission filters and beamsplitters chemiluminescence the emission of light by a dye molecule as a result of a chemical reaction datasets the files and folders that make up a multichannel image default template the template and settings that appear in the Scanner Control window when the software is first opened electrophoresis glass plate a 3 mm thick low fluorescence piece of glass used to create sandwich gels emission the release of light from a dye molecule when an electron falls from an excited to a lower energy state emission spectrum a plot of emitted light wavelength versus relative intensity of emitted light excitation the absorption of light energy by a molecule so that an electron is boosted to a higher level energy state excitation spectrum a plot of incident light wavelength versus relative intensity of emitted light fluorescence the emission of light by a dye molecule as a result of absorption of laser light Typhoon User s Guide Glossary 1 Glossary GP screen general purpose storage phosphor screen glass platen the area of the Typhoon instrument on which you set the sample or storage phosphor screen grid coordinates the markings on the exposure cassette and glass platen of the instrument that allow you to position the sample The coordinates correspond to markings in the Scanner Control window L
156. raser Erase the screen immediately before exposure as well as after scanning the screen in the instrument Cosmic radiation creates background on screens left unused for long periods of time Erase the screen until the recorded signal is fully removed Because you cannot decontaminate a TR screen you should protect the eraser and the screen from possible contamination by placing plastic wrap or polyester film between the TR screen and the eraser surface To erase the screen H old the screen by its edges with the white side facing the eraser Place the screen on the eraser as shown in figure 5 1 Rest the screen on the lip of the eraser To erase two small screens at once suspend an unmounted screen from the clip at the top of the eraser Caution Do not use the clip on the TR screen The clip can damage the surface of the screen p5 4 Typhoon User s Guide Chapter 5 Preparing for storage phosphor screen autoradiography 3 Set the eraser time by pressing the Time button Figure 5 1 shows the button positions for Normal out and Extended in Select Normal for typical samples The N ormal setting takes approximately 10 minutes Extended if the background or residual image is high For example the original image contained readings of 104 counts and higher The Extended setting takes approximately 20 minutes 4 Press the Erase button If the Image Eraser is unavailable you can use a fluorescent light box and
157. reen that is free from radioactive contamination 2 Erase the screen scan the screen to make sure it is not contaminated and then erase the screen again If during scanning an image that looks like a gel or a blot appears in the ImageQ uant Preview window the screen may be contaminated Erase the screen Rotate the screen or place the screen on a different part of the glass platen and scan the screen again If the same image appears at the new position in the mageQ uant Preview window the screen is contaminated Select a different screen and repeat this step 3 Place the white side of the screen on the glass platen of the Typhoon instrument 4 Leave the screen in the instrument overnight Typhoon User s Guide p6 1 Parttwo Scanning in the storage phosphor mode Se 5 Scan the screen If an image appears in the ImageQ uant Preview window that looks like a gel or blot the instrument is contaminated N ote If a white or gray image appears the image was probably created by background contamination and the instrument is not contaminated Use sections 6 1 2 and 6 1 3 to clean the instrument 6 1 2 Cleaning the glass platen The glass platen of the Typhoon instrument should be kept free of contamination from a radioactive sample In addition you should clean the glass platen before and after you scan each sample To clean the glass platen 1 Push up on the lid release under the center front of the sample lid until the
158. rred in the scan you may want to repeat the exposure and scan again If the scan was almost finished you might be able to use the data To cancel the scan click Cancel Scan in the mageQ uant Preview window Scanner Control saves the data collected before you canceled the scan p6 14 Typhoon User s Guide Important Chapter 6 Scanning a storage phosphor screen EE E Red 7 ae F Ay Linea Scored Total Soin Teen leer fans Soames Margen SS Figure 6 8 The ImageQuant Preview window for a storage phosphor scan 6 5 3 Viewing the preview image for saturated data While the instrument is scanning an image appears in the mageQ uant Preview window The image shows the part of the sample that has been scanned Saturated data appear in red in the image If the image appears saturated you will not be able to perform quantitative analysis on the portions of the image that are saturated Because saturation causes a total loss of the ability to distinguish the signal intensity fluctuations a saturated image can be used only for viewing the shapes and positions of the spots on the sample Because the scanning process destroys the signal on the storage phosphor screen you may want to finish the scan If the image appears unusable expose a clean erased storage phosphor screen to the original sample and expose the screen for a shorter time Alternatively you can prepare a new sample and expose a clean erased storage phosphor
159. rs figure 13 4 3 Click Step 1 Access Filter Rod The slow stage begins to move from the left to the right side of the instrument and the Scan indicator light blinks red After the slow stage stops moving and the Scan indicator light stops blinking e Usethe procedure in section 13 3 2 to remove the front cover and open the access and filter doors e Usethe procedure in section 13 3 3 to insert a new optical filter e Use the procedure in section 13 3 5 to close the filter and access doors and replace the front cover e Use the procedure in section 13 3 6 to define the filter in Scanner Control s Microsoft Internet Explorer mi x Eile Edit View Go Favorites Help 2 ie ow O A a i Beck Eonverd Stop Refresh Home Search Favorites History Channels Fullscreen Address htip 192 168 1 1 v Links Insert a New Optical Filter into the Filter Changer Step 1 Access Filter Rod This moves the filter rod out to the door so you will be able to install a new filter Step 2 Insert Filter After you place the new filter at the back of the first rod press this to pull the filter into instrument and sean the barcode DONE Does a complete inventory of the filter changer to record all changes Takes about a minute E Done e Internet zone WY Figure 13 4 The Insert a New Optical Filter into the Filter Changer Web page p13 6 Typhoon User s Guide Cha
160. s figure 11 1 2 Optional For a wet sample squirt a little buffer or distilled water on the glass platen 3 Hold the sample by the edges or use forceps to handle the sample Gently lower the sample onto the glass platen starting at one edge Do not trap air bubbles under the sample or scratch the glass Important Do not touch the part of the sample that will be read by the Typhoon instrument Do not touch the glass platen Oil from fingerprints and powder from gloves even thoroughly washed gloves may leave a mark that affects the quality of the image 4 Makea note of the grid square coordinates of the area you want to scan figure 11 2 You will use this information later in the Scanner Control window section 12 1 3 5 Close the sample lid and press it down until the latch clicks The sample is ready to scan Typhoon User s Guide p11 5 Chapter 12 Scanning a chemiluminescent sample This chapter provides step by step instructions for scanning a chemiluminescent sample The topics in this chapter are e Selecting the scan parameters in the Scanner Control window section 12 1 e Using the Setup window to select the chemiluminescence parameters section 12 2 Starting a scan and checking the progress section 12 3 e Removing the sample from the Typhoon instrument section 12 4 e Cleaning the glass platen and sample lid section 12 5 e Analyzing or preprocessing the scanned image section 12 6 12 1 Sel
161. s Guide p9 11 Part three Scanning in the fluorescence mode a Before linking Link llth J Nese a fan fern a eed nop fi TS Tae Reed CIS CE aj al a NS OS fom o FE Beo e E Fos D Dii Link iang TE Airdri pag a b After linking Fhargpreonte Seber he yphern thh Link with Bracket indicates F i fol am ie a icra al z linked scans sonnel a mel vora E a wife ey Cy a oS poeme 4 fee 2 fF D Derai Link warara iiri i are i Canal Figure 9 5 The Fluorescence Setup window Window a shows the scans that can be linked Scans 1 and 3 display dots indicating optimal links Window b shows that the first and third scans have been linked and both scans are labeled Scan 1 Note that the last scan is now labeled Scan 3 and has not been selected Automatically linking scans The Fluorescence Setup window allows you to link scans automatically After you select the scans you want to perform and select the settings for each scan Scanner Control determines which scans can be linked activates the Link With area and displays colored dots above the scan numbers to show the optimal links For optimal linking Scanner Control requires more than 5 nm between the peak of a bandpass filter and the edge of the beamsplitter or more than p9 12 Typhoon User s Guide Chapter 9 Scanning a fluorescent sample 5 nm between the edge of a short pass or long pass filter and the edge of the
162. s to the correct voltage automatically The fuse box is located to the right of the power switch figure 13 11 The Typhoon instrument takes two 5A 250V T5A 250V slow acting fuses The fuse holder is designed to accept both 0 5 inch x 1 5 inch English fuses designated 3AG fuses and 5 mm x 20 mm metric fuses Front Back Fuse box eea iD Figure 13 11 Right side view of the Typhoon instrument p13 16 Typhoon User s Guide Chapter 13 Maintaining the Typhoon instrument SSS To change a fuse 1 Turn off the Typhoon instrument section 3 7 and disconnect the power cord 2 Place the end of a small flathead screwdriver into the groove to the right of the fuse box and pry open the fuse box figure 13 12 3 Place the screwdriver in the groove on the side of the fuse holder and pry out the fuse holder W hen the holder is loose pull it out of the fuse box 4 Oneor both fuses may be blown Replace the blown fuse s with new fuse s of the same type and rating Fuse specifications are listed at the beginning of this section and on the serial number and fuse labels located on either side of the fuse box 5 Insert the fuse holder into the fuse box 6 Snap the cover of the fuse box back into place 7 Reconnect the power cord and turn on the Typhoon instrument section 3 1 Fuse cover a Groove A Le J Pry here to open the fuse cover CGE Pry here to remove CEEA IDOOIOOIICUONOGOU
163. sample 12 1 Selecting the scan parameters in the Scanner Control window 12 1 12 1 1 Selecting a template 0 eee eee 12 2 12 1 2 Selecting the chemiluminescence scan acquisition mode 12 2 12 1 3 Selecting the scan area 1 2 eee 12 3 12 1 4 Selecting the sample orientation 00 12 4 12 1 5 Selecting the Press Sample option 12 6 12 1 6 Selecting the pixel size 2 2 eee 12 6 12 1 7 Selecting the focal plane 2 0 0 cee eee eee 12 7 12 1 8 Selecting the image analysis software 12 7 12 1 9 Entering user comments optional 00 eee 12 7 12 2 Using the Setup window to select the chemiluminescence param terS srs ete ack ete ee ae Pe Rak Re a waa ease Wied 12 7 12 2 1 Displaying the Chemiluminescence Setup window 12 8 12 2 2 Setting the limit of detection 0 00 00 c eee 12 8 12 2 3 Selecting a beamsplitter for a multichannel scan 12 9 12 2 4 Setting the PMT voltage 0 eee eee 12 9 12 2 5 Closing the Chemiluminescence Setup window 12 10 Table of Contents 12 3 Starting a scan and checking the progress 000 12 10 12 3 1 Starting a Scan cece 12 11 12 3 2 Monitoring the scan progress saana 12 12 12 3 3 Viewing the preview image for saturated data 12 14 12 3 4 Completing the scan saanunna 12 14 12 4 Removing the sample from the Typhoon instrument 12 14 12 5 Clean
164. sample in the exposure cassette 1 Useadamp cloth to clean the grid surface inside the exposure cassette and remove any contamination section 14 4 2 Place the sample on the grid using the guidelines in section 5 4 3 Makea note of the grid coordinates of the area you want to scan as shown in figure 5 2 The grid coordinates in the exposure cassette also appear in the Typhoon instrument section 6 3 and in the Scanner Control window section 6 4 3 5 6 Placing the screen in the exposure cassette You should perform storage phosphor screen autoradiography at room temperature Exposing the screen at subzero temperatures provides no advantage Condensation can destroy the screen If you are exposing a screen to a frozen sample place the screen ina sealed dry environment such as a sealed bag After exposure allow the screen to come to room temperature before removing it from the bag and scanning Make sure you place the screen in the correct position when you first set it down Because the screen is extremely sensitive adjusting the position of the screen can result in a double image 5 6 1 Placing a mounted screen To place a mounted screen in the exposure cassette 1 Makesure the clamp or clamps on the cassette are rotated out of the way 2 Remove the screen from the eraser p5 8 Typhoon User s Guide Caution Chapter 5 Preparing for storage phosphor screen autoradiography 3 Note the groove along the sid
165. scan area 6 7 9 3 12 3 selecting template 3 11 starting scan 6 12 9 14 12 10 User Comments area 6 12 9 7 12 7 using templates 3 8 scanning aborting scan 6 14 9 17 12 13 accurate quantitation 9 9 12 9 canceling 6 14 9 17 12 13 chemiluminescence workflow 3 6 cleaning after 6 16 9 19 12 14 end of scan 6 16 9 19 12 14 fatal errors 6 14 9 17 12 13 fluorescence workflow 3 5 ImageQuant Preview window 6 14 9 17 12 12 intense sample 9 9 12 9 minimizing image file size 8 6 8 7 11 3 minimizing time required 8 6 11 3 modes 1 4 naming 6 12 9 15 12 10 nonfatal errors 6 14 9 17 12 13 overview 1 3 sample orientation 6 8 8 6 9 4 11 3 12 4 saturated data 6 15 9 18 12 14 starting 6 12 9 14 12 10 storage phosphor workflow 3 3 troubleshooting A 3 weak sample 9 9 scintillants 2 9 5 2 screen cleaner 5 4 screen see storage phosphor screen sensitivity of storage phosphor screen 4 2 sensitivity setting 9 10 12 8 serial number Image Eraser 2 2 Typhoon instrument 2 1 service Calling for xiv short pass emission filter 7 3 shutoff time laser 13 3 site requirements xiv solutions filtering 8 1 solvent organic 2 9 5 1 spectroscopic grade 8 4 special safety text definitions xii standards radioactive 5 2 Stokes shift 7 1 storage phosphor description 4 1 scan acquisition mode 1 4 6 7 selecting a template 6 7 workflow 3 3 storage phosphor screen advantages 4 2 background 5 5 5 8 checking for contamination 5 3
166. scans and renumbers the scans Link With area UnLink All button Allows you to remove the selected links Scanner Control removes the brackets connecting the Use check boxes of the scans and renumbers the scans Disable Link Warning check box Allows you to turn off the warning message that appears if you select nonoptimal links Cancel button Closes the Fluorescence Setup window without saving any changes OK button Saves the changes and closes the Fluorescence Setup window pB 4 Typhoon User s Guide Appendix B Quick reference for menus and windows Chemiluminescence Setup window Option Description Sensitivity list Allows you to select the sensitivity limit of detection to use for each scan Normal collects one data point from each pixel Medium collects four data points from each pixel and averages the results High collects eight data points from each pixel and averages the results Beamsplitters list Allows you to select a beamsplitter if you want to perform a multichannel scan You can choose either 560 or 580 If you are performing a single scan leave the setting at None PMT Voltage list Allows you to select the PMT voltage to use Values range from 0 to 1 000 and are in 5 volt increments If you selected None in the Beamsplitter list you see one PMT Voltage list box If you selected 560 or 580 you see two PMT Voltage list boxes Cancel button Closes the
167. scence mode Warning A Cautions Procedure for changing the PMT voltage To set the voltage in the PM T Voltage box e Click the box to select it and then type the value you want maximum setting is 1 000 volts e Usethe up or down arrows to change the value in 5 volt increments To approximately double the signal intensity increase the PM T voltage by 50 volts If you are using a beamsplitter to perform a multichannel scan you can select a PMT voltage for each scan 12 2 5 Closing the Chemiluminescence Setup window After you finish selecting the chemiluminescence settings click OK to save the settings and close the Chemiluminescence Setup window If you do not want to save the settings click Cancel to return to the Scanner Control window The Scanning Information area in the Scanner Control window displays the settings you selected in the Chemiluminescence Setup window 12 3 Starting a scan and checking the progress Do not attempt to defeat the safety interlocks on the sample lid These interlocks are designed to protect you from laser light exposure Never turn off the Typhoon instrument or disconnect the Ethernet cable while scanning You can severely damage the internal mechanism of the instrument Keep the sample lid closed during the scan Opening the lid causes Scanner Control to abort the scan and save the data already collected During the scanning process the chemiluminescent sample emits light which is
168. screen to the new sample Before repeating the exposure and the scan verify in ImageQuant that the image is saturated and that you cannot perform accurate quantitation Typhoon User s Guide p6 15 Parttwo Scanning in the storage phosphor mode 6 5 4 Completing the scan Scanner Control saves the image using the file name you typed in the Save As window The ImageQuant Preview window displays a Complete message and then closes The Scan indicator light on the Typhoon instrument turns off At the end of the scan an image of the sample appears automatically in the software you selected from the Image Analysis list If you selected N one the Scanner Control window remains active for more scanning 6 6 Cleaning up after the scan After the Typhoon instrument completes the scan open the sample lid and remove the storage phosphor screen Avoid touching the white side of the screen To lift a large mounted screen pull up on the round tabs attached to the metal plate and then slide a finger under the edge of the metal backing If your screen does not have tabs or the tabs are damaged you should replace the tabs Contact M D Scanner Technical Support See Assistance in the preface for contact information After each scan you should e Remove the sample from the exposure cassette and dispose of the sample using the established procedure in your laboratory e Check the storage phosphor screen for contamination section 5 2 2
169. se holder is on the lower right side of the Typhoon instrument and contains two 5A 250V T5A 250V slow acting fuses The fuse label contains the fuse specifications figure 2 4 The fuse holder is designed to accept both 0 5 inch x 1 5 inch English fuses designated 3AG fuses and 5 mm x 20 mm metric fuses On the Image Eraser T he fuse holder is on the right side of the Image Eraser and contains two 1 5A 250V T1 5A 250V slow acting fuses The fuse holder is designed to accept both 0 5 inch x 1 5 inch English fuses designated 3AG fuses and 5 mm x 20 mm metric fuses T5A 250V Figure 2 4 The fuse label on the Typhoon instrument To change the fuses on the Typhoon instrument follow the instructions in section 13 5 To change the fuses on the Image Eraser follow the instructions in section 14 3 If you need to change the fuses in the instrument or eraser turn off the instrument or eraser and disconnect the power cord If a fuse requires repeated replacement the instrument or eraser could have an electrical problem Do not use the instrument or eraser You could expose yourself to electrical shock Contact MD Scanner Technical Support See Assistance in the preface for contact information Typhoon User s Guide p2 5 Part one Introduction Warning Warning A Caution Attention Warnung A 2 2 3 High voltage hazard and precautions Inside the Typhoon instrument Image Eraser computer
170. ssion spectrum longer wavelength lower energy storage phosphor screen autoradiography the emission of light by a radioactive molecule which is collected on a phosphor coated screen instead of on film template scan settings that are saved in Scanner Control using a unique name Templates can be saved for storage phosphor fluorescence and chemiluminescence TR screen tritium storage phosphor screen Glossary 2 Typhoon User s Guide Index A abort scan 6 14 9 17 12 13 absorption fluorescence 7 1 to 7 2 storage phosphor 4 1 to 4 2 acetic acid 5 1 acquisition mode see scan acquisition mode agarose 8 2 alkaline gels 5 1 artifacts image 8 1 assistance xiv assumptions xii Auto Link button in Fluorescence Setup window 9 11 background chemiluminescence 11 1 12 9 cosmic radiation 5 4 fluorescence 8 2 8 4 8 6 9 9 storage phosphor screen 5 5 5 8 6 2 troubleshooting A 4 bandpass emission filter 7 3 bar code optical filter 13 4 Beamsplitter list Chemiluminescence Setup window 12 9 Fluorescence Setup window 9 11 beamsplitters chemiluminescence 10 3 fluorescence 7 4 browser 13 1 bulb changing 14 2 to 14 4 C cancel scan 6 14 9 17 12 13 caution definition xii hazardous materials 2 9 CE declaration xiii Typhoon User s Guide Index 1 Index chemiluminescence see also sample advantages 10 1 beamsplitters 10 3 contamination 11 1 11 3 optimizing experiment 10 2 overview 10 1 prep
171. sure you wear powder free gloves when handling the samples Always rinse the gloves thoroughly with distilled or tap water before handling the samples Dust and powder fluoresce and scatter light which can affect the quality of images created from fluorescent samples N eutralize alkaline denaturing gels and make sure the sample is free from acetic acid vapors and organic solvents T hese materials are harmful to the Typhoon User s Guide p5 1 Parttwo Scanning in the storage phosphor mode Caution storage phosphor screens and can penetrate the plastic wrapping or polyester film on wet samples Do not use scintillants or enhancers such as PPO or Amersham Pharmacia Biotech s Amplify These products interfere with the proper function of the screen 5 1 2 Using wet gels with the storage phosphor screen In addition to the general guidelines in section 5 1 1 use the following guidelines when exposing the screen to a wet gel Do not directly expose the storage phosphor screen to wet chemicals of any kind especially organic solvents Because organic solvents penetrate plastic plastic wrap will not protect the storage phosphor screens from organic solutions e Protect the general purpose GP and low energy LE screens from contamination by separating a wet gel from the screen with a piece of plastic wrap or polyester film Wrap the plastic wrap completely around the gel so that liquid cannot leak out This precaution reduc
172. system to scan a chemiluminescent sample e Part five M aintaining the Typhoon system hardware D escribes how to maintain the Typhoon instrument Image Eraser and exposure cassettes Part six Appendixes Includes troubleshooting and reference information Related publications In addition to the Typhoon U ser s Guide and Typhoon Installation Instructions you should be familiar with the image analysis software documentation Safety Trained operator The operator of the Typhoon instrument is assumed to be trained in the correct operation of the instrument and the safety issues Throughout the Typhoon User s Guide the word you refers to this trained operator Typhoon User s Guide xi Preface xii Warning AN Caution Important Typhoon User s Guide Chapter 2 in this guide provides important safety information that should be used when operating the Typhoon instrument Before using the Typhoon instrument read and understand the safety information thoroughly Special safety text M ake sure you follow the precautionary statements presented in this guide Indicates a possibility of physical injury or death to the user or other persons if the precautions or instructions are not observed Indicates that damage to the instrument loss of data or invalid data could occur if the user fails to comply with the advice given Highlights information that is critical for optimal performance of the system N
173. t Guidelines for changing the PMT voltage For accurate quantitation of intense samples pixel intensities in your scanned image should be within a specific range To determine the correct PM T voltage scan a test sample and display the image in ImageQ uant Use the Gray Color Adjust feature to display the intensity values of the image See the mageQ uant User s Guide or online H elp for more information e If the values are more than 1 count and less than 100 000 counts all spots in your image are within the dynamic range of the instrument If some of the values are equal to or greater than 100 000 counts part of your image is at or near saturation Lower the PMT voltage rescan and confirm that the image is not saturated If your experiment includes only very weak samples or only very intense samples you can adjust the voltage to extend the limit of detection e For weak samples increase the PMT voltage If you increase the PM T voltage you may lose the accuracy of the quantitation at the upper end of the signal range If the sample background is very low above 900 volts you may lose the sensitivity because of increased PM T noise N ote that you cannot exceed 1 000 volts e For intense samples that saturate the system decrease the PMT voltage to bring high intensity signals into the linear range of the instrument Otherwise you may lose the sensitivity and the accuracy of the quantitation at the lower end of the signal ran
174. t Filter The Select Filter ID to Eject Web page appears and displays a list of the bar codes for the installed filters figure 13 6 Q felect Fake bd in ajani Mieicnali Intranet E mplases Ee Edt Vee Ge Fyre Halo foo A 2 8 eo ee Buc Eim E 916 T 1 Aarcha ape OD he lal TEN ec esta CT ba piia Ud Select Filter d be pytt katali Filbert 16 117 73 14 03 10 13 Diela Filter Daigre echo Pc re AC Fater ee Fy Local inane gory EJ Figure 13 6 The Select Filter ID to Eject Web page 4 Inthe Filter box type the bar code for the filter you want to remove If you do not know the bar code click Display Filter Descriptions to view a list of the installed filters Click Back to return to the Select Filter ID to Eject Web page and type the bar code 5 Inthe Select Filter ID to Eject Web page click Done to return to the Remove an Optical Filter from the Filter Changer Web page p13 8 Typhoon User s Guide Warning A Chapter 13 Maintaining the Typhoon instrument The slow stage begins to move from the left to the right side of the instrument and the Scan indicator light blinks red You can lift the sample lid and watch the slow stage move into position After the slow stage stops moving and the Scan indicator light stops blinking e Usethe procedure in section 13 3 2 to remove the front cover and open the access and filter doors e Use the procedure in section 13 3 4 to remove t
175. ta If you cannot use the image for quantitation you must scan again To cancel the scan click Cancel Scan in the mageQ uant Preview window Scanner Control saves the data collected before you canceled the scan If you are scanning a sample using a beamsplitter the ImageQ uant Preview window displays the two channels in one tab Typhoon User s Guide p12 13 Part four Scanning in the chemiluminescence mode tlmportant 12 3 3 Viewing the preview image for saturated data While theinstrument is scanning an image appears in the mageQ uant Preview window that shows the part of the sample that has been scanned Saturated data appear in red in the image If the image appears saturated you may not be able to perform quantitative analysis on the image Because saturation causes a total loss of the ability to distinguish the signal intensity fluctuations a saturated image can be used for viewing only the shapes and positions of the spots on the sample To avoid saturation of the data lower the PM T voltage you selected in the Chemiluminescence Setup window section 12 2 4 Before repeating the scan verify in ImageQuant that the image is saturated 100 000 rfu and you cannot perform accurate quantitation 12 3 4 Completing the scan Scanner Control saves the image or images using the file name you typed in the Save As window The mageQ uant Preview window displays a Complete message and then closes The Scan indicator light on
176. ted If the error is a fatal error the instrument aborts the scan and Scanner Control saves the data collected before the fatal error occurred You should check the image in ImageQ uant Depending on where the fatal error occurred in thescan you might be able to use the data If you cannot use the image for quantitation you must scan again To cancel the scan click Cancel Scan in the mageQ uant Preview window Scanner Control saves the data collected before you canceled the scan If you are performing a multichannel scan the ImageQ uant Preview window displays a tab for each scan For example if you selected settings for four unlinked scans in the Fluorescence Setup window the instrument scans the sample four times and you see four tabs in the ImageQ uant Preview window one for each scan or channel If you used the link feature to combine two scans you see one tab that contains two channels You see one channel image that was created using one emission filter and beamsplitter and you see one channel image that was created using a second emission filter and beamsplitter figure 9 7 If you create a multichannel image from both linked and unlinked scans you see two channel images in the tab for the linked scan and you see one channel image and a blank space in the tab for each of the unlinked scans Typhoon User s Guide p9 17 Part three Scanning in the fluorescence mode Important eyciluent Prya Linen iem ZF Tot S
177. ted If you type a name and select the gel extension and that name has already been used with the ds file extension a message appears and asks you to change the file name 4 Optional Change to a different folder O therwise the software saves the image in the Data folder Typhoon User s Guide p12 11 Part four Scanning in the chemiluminescence mode Caution Using a network to save the scanned data to a folder located on a remote computer can cause a loss of data For the best results choose a folder on the local hard drive After you scan the sample move the image file s from the folder on the local hard drive to a folder on the remote computer For dataset image files make sure you move both the ds file and the dir directory into the new folder 5 To start the scan Windows NT Click Save e MacOS Click Open If you selected Press Sample in section 12 1 5 the inner lid of the Typhoon instrument lowers to hold the sample flat against the glass platen of the instrument Before the Typhoon instrument begins the scan an initialization process occurs Depending on the state of the instrument and the scan parameters you chose in Scanner Control the initialization can take as long as 3 minutes The initialization procedures include e Moving the scan head into position e Changing the filters to match the scan parameters e Stabilizing the laser if the laser has been idle shut down or recently turned on
178. ter and holder with the bar code label attached to the top of the holder and the metal notch to the left Typhoon User s Guide p13 11 Part five Maintaining the Typhoon system hardware Write down the bar code and the wavelength of the filter you are installing After you install the filter you will need this information to register the filter in the Scanner Control software section 13 3 6 M ake sure the filter changer is in position H old the optical filter so that the bar code is facing up and the metal notch on the holder is on the left figure 13 8 Align the holes in the filter holder with the prongs on the filter changer figure 13 7 Slide the filter holder all the way to the left M ake sure the bar code is on the top and the metal notch is on the left Important Insert one filter at a time 7 10 In the Insert a N ew O ptical Filter into the Filter Changer Web page figure 13 4 click Step 2 Insert Filter The filter changer moves back inside the Typhoon instrument The instrument scans the bar code and moves the filter holder to a slot farther back in the changer M essages appear for the following e If the instrument cannot insert the filter e If you inserted the filter holder backwards e If you did not push the filter holder all the way to the left e If the filter holder does not have a bar code label e If you clicked Step 2 Insert Filter before you inserted the filter If the instrument
179. tes reappear in the list 3 5 1 Creating a new template To create a template 1 In the Setup area of the Scanner Control window figure 3 2 select the options you want saved with the template N ote that comments in the Thanks TamaUser Comment box and the Press Sample option are not saved with the template For more information on selecting options see section 6 4 storage phosphor section 9 1 fluorescence or section 12 1 chemiluminescence p3 8 Typhoon User s Guide 1 Select the scan area amp T phann Gears Coded l MIA ae qran bin ri De Jepi jiin oo erm kiemai Meie Pari ideme TEID nrgi bair 11i Tia Fee ae aana Sar 3 j ERN E i 3 Select the sample Fill a atv inne Ten RT Un E J Dipti orientation JONES GEG T E T A E A ee Fim aes Foira 4 Select the pixel mri n size E E E E pre 5 Select the focal eee ee eee plane eere eer eree J i s E g1 1 1 I C l 6 Select the image l tt Sia Feah Th 119 te TF 18 a analysis software Dia raig bT daai Toia Scope Fog 0 A a Lisi Fa Charie Bll iea ha Dee OD al Chapter 3 Getting started 2 Select the scan acquisition mode fen Fae Lom Bedii PHT Spt E SG eime Ao EY Ee r hra Geam mi Hea EY Figure 3 2 Selecting options in the Setup area of the Scanner Control window 2 Fluorescence and Chemiluminescence O nly Click the Setup button and select the settings
180. the Scanner Control window remains active for more scanning 9 4 Removing the sample from the Typhoon instrument After you finish scanning you should remove the sample from the Typhoon instrument and dispose of the sample using the established procedures in your laboratory M ake sure the Scan indicator light turns off before you open the sample lid and remove the sample 9 5 Cleaning the glass platen and sample lid To remove fluorescent contamination you should clean the glass platen and sample lid after you finish each scan See section 8 2 for details 9 6 Analyzing or preprocessing the scanned image You should display the image of the sample to determine the quality of the scan If you selected image analysis software in the Scanner Control window the software opens and displays the image Refer to the applicable user s guide or online H elp for the selected software for detailed information If you created a multichannel image the channel numbers displayed in the Scanning Information area of the Scanner Control window are the sameas the channel numbers displayed in ImageQ uant Typhoon User s Guide p9 19 Part four Scanning in the chemiluminescence mode Chapter 10 About chemiluminescence scanning This chapter describes the theory behind chemiluminescence The topics in this chapter are How chemiluminescence works section 10 1 e Advantages of chemiluminescence section 10 2 e How to optimize the che
181. the screen 4 After storage scan the screen and examine the image If no hot spots appear on the image erase the screen again and begin the next exposure Typhoon User s Guide p5 3 Parttwo Scanning in the storage phosphor mode Saas 5 2 3 Cleaning the storage phosphor screen Use the cleaning method appropriate for the type of screen you are cleaning GP and LE screens Use a soft cotton cloth and an intensifying screen cleaner for example Kodak Intensifying Screen Cleaner Follow the directions on the bottle Alternatively use a small amount of alcohol and distilled water Do not use organic solvents such as ethanol Do not use a powdered detergent Any undissolved particles can scratch the surface of the screen This cleaning procedure removes dust fingerprints static electricity and mild contamination from radioactive samples TR screen Use a gentle gas stream or soft brush to remove any particulate matter from the unprotected surface of the screen Caution To avoid damaging the TR screen never expose the screen to liquids of any sort 5 2 4 Erasing the storage phosphor screen After you clean the storage phosphor screen you use the Image Eraser to erase any residual signal from the screen Use the following guidelines when erasing the screen To avoid contaminating the screen or the eraser make sure that both the screen and eraser are free from radioactive contamination before placing the screen on the e
182. the Setup window to select the chemiluminescence parameters You use the Chemiluminescence Setup window to select the chemiluminescence parameters to use with the sample The following sections describe how to use the Chemiluminescence Setup window figure 12 4 Typhoon User s Guide p12 7 Part four Scanning in the chemiluminescence mode 1 ar Chonnal Chamilenindicenes Sep bat Typhiendsll GEA Figure 12 4 The Chemiluminescence Setup window contains parameters for scanning a chemiluminescent sample 12 2 1 Displaying the Chemiluminescence Setup window To display the Chemiluminescence Setup window make sure Chemiluminescence is the selected acquisition mode and then click the Setup button in the Acquisition M ode area in the Scanner Control window figure 12 1 12 2 2 Setting the limit of detection You use the sensitivity settings to define the limit of detection of the Typhoon instrument The sensitivity setting controls how long the Typhoon instrument collects data from each pixel The Sensitivity list contains three choices e Normal lsthe default setting and samples each pixel once In general you should use the N ormal setting e Medium Collects data from each pixel four times and averages the results Using the M edium setting to detect weak signals improves the sensitivity by reducing the background noise N ote Selecting M edium increases the scanning time but does not increase the size of the image file C
183. the Typhoon instrument turns off At the end of the scan an image of the sample appears automatically in the software you selected from the Image Analysis list If you selected N one the Scanner Control window remains active for more scanning 12 4 Removing the sample from the Typhoon instrument After you finish scanning you should remove the sample from the Typhoon instrument and dispose of the sample using the established procedures in your laboratory M ake sure the Scan indicator light turns off before you open the sample lid and remove the sample 12 5 Cleaning the glass platen and sample lid To remove chemiluminescent contamination you should clean the glass platen and sample lid after you finish each scan See section 11 2 for details 12 6 Analyzing or preprocessing the scanned image You should display the image of the sample to determine the quality of the scan If you selected image analysis software in the Scanner Control window the software opens and displays the image Refer to the applicable user s guide or online H elp for the selected software for detailed information p12 14 Typhoon User s Guide Part five Maintaining the Typhoon system hardware Important Caution Chapter 13 Maintaining the Typhoon instrument This chapter provides information about maintaining the Typhoon instrument The topics in this chapter are e Starting the Typhoon Direct Instrument Access Web page section 13 1
184. the grid square coordinates covered by the sample You will use this information in the Scanner Control window section 12 1 4 11 3 2 Determining the sample placement Use the following guidelines and figure 11 2 to determine where on the glass platen to place the sample To minimize the scan time Place the sample near the A1 corner of the grid Each lettered or numbered grid segment is marked by two dots Position a rectangular sample so that the shortest edge is along the numbered side of the glass platen as shown in figure 11 2 Because each pass of the scan head moves the entire length of the glass platen from front to back placing the longest edge of the sample along the scan head path minimizes the scan time e To minimize the size of the image file scan only the number of grid squares covered by the sample If you do not want to scan the entire sample scan the squares that contain the part of the sample you are interested in analyzing Typhoon User s Guide p11 3 Part four Scanning in the chemiluminescence mode Se To record multiple samples during the same scan place the samples close together M ake sure the samples are the same thickness If the samples are not the same thickness the thinner sample will not contact the glass platen uniformly which can result in a bad scan poor image and poor quantitation results e Align the lanes and rows of the sample with the edges of the grid on the glass platen so t
185. the instrument figure 10 1 Substrate 2 Signal Substrate 1 intensity Time hr Figure 10 1 How signal intensity of a substrate and time affect the scan p10 2 Typhoon User s Guide Chapter 10 About chemiluminescence scanning Scanner Control settings T he settings you choose in Scanner Control affect how long the instrument needs to scan the sample The settings that can increase the scan time are sensitivity section 12 2 2 which sets the limit of detection and pixel size section 12 1 6 Sample size and position T he size of the scan area and the position of the sample on the glass platen also affect the amount of time the Typhoon instrument needs to scan the sample section 11 3 When selecting the settings in the Scanner Control window and the Chemiluminescence Setup window note the time change in the Scanning Information area at the bottom of the Scanner Control window If the signal intensity of the sample diminishes significantly before the instrument finishes the scan the accuracy of the collected data may be affected 10 4 Beamsplitters The Typhoon instrument contains two beamsplitters You can use the beamsplitters with chemiluminescent detection to collect light emitted at different wavelengths The two beamsplitters in the Typhoon instrument are e 560 nm dichroic R eflects light of wavelengths shorter than 560 nm and passes light longer than 560 nm e 580 nm dichroic
186. the scan time The beamsplitter separates the emissions from the linked scans so that one PM T collects the longer wavelength emissions while the other PM T collects the shorter wavelength emissions For a description of the beamsplitters in the Typhoon instrument see section 7 4 Determining if scans can be linked Scans can be linked if they meet the following criteria The scans use the same laser e The scans use different emission filters or at least one uses no emission filter e The scans use the same sensitivity setting e The wavelength of one of the beamsplitters is between the wavelengths of the two emission filters and the wavelength of the beamsplitter is at least 5 nm from each filter If Scanner Control determines that scans can be linked the Link With area becomes active Scan number buttons appear in the Link With area for the scans that can be linked figure 9 5 and the Auto Link button becomes active After you link two scans the Unlink All button becomes active so that you can remove the links if you do not want to use them The Link With area displays the scans that can be linked and uses colored dots above the scan numbers to show the optimal link for each scan line Scanner Control selects the optimal link by determining which scans provide the best limit of detection and the best spectral separation The colored dots also represent which scans will be linked if you click the Auto Link button Typhoon User
187. the settings for the template you selected as the default appear in the Scanner Control window If the default template uses the fluorescence or chemiluminescence scan acquisition mode the settings also appear in the Setup window 3 5 5 Deleting a template If you want to delete a template choose D elete from the Templates menu The Delete Template s window appears figure 3 5 Select the template name that you want to delete and click D elete N ote Because you cannot delete the template displayed in the Scanner Control window this template name does not appear in the list To delete the template in use close the Delete Template s window select a different template from the Load list and then choose Delete again You should see the template name in the list To delete all the templates except the template displayed in the Scanner Control window click Select All and then click Delete Delete Template s x Templates Fluor_Large Fluor Small Select All Stor_Lrg Stor_small Cancel Delete Figure 3 5 The Delete Template s window Note The Templates list does not include the template currently displayed in the Scanner Control window p3 12 Typhoon User s Guide Important Chapter 3 Getting started 3 6 Closing the Scanner Control software Although it is not necessary you can close the Scanner Control software after you finish scanning Before installing or removing an emission filter you must
188. tivity list contains the following three choices e Normal s the default setting and samples each pixel once In general you should use the N ormal setting e Medium Collects data from each pixel four times and averages the results Using the M edium setting to detect weak signals improves the sensitivity by reducing the background noise N ote Selecting M edium will increase the scanning time but does not increase the size of the image file Check the Scanning Information area in the Scanner Control window for the time required to complete the scan e High Collects data from each pixel eight times and averages the results Using the High setting to detect weak signals improves the sensitivity by reducing the background noise N ote Selecting H igh will increase the scanning time but does not increase the size of the image file Check the Scanning Information area in the Scanner Control window for the time required to complete the scan If you are performing multiple scans you set sensitivity settings for each scan If you select the High setting and the smallest pixel size for four scans the total scan time can be over 21 hours p9 10 Typhoon User s Guide Chapter 9 Scanning a fluorescent sample 9 2 7 Linking scans You link scans that contain similar settings so that you can acquire signal from two different fluorochromes during one scan This optimizes the scanning of samples containing multiple fluorochromes by reducing
189. ts optional The user comments are not saved as part of a template If you want to include comments you must type the comments each time you scan In the User Comment box type any comment you want to save with the image approximately 4 000 characters including spaces The comments are for reference only and do not affect the scan After the scan you can view but not change the comments in mageQ uant 6 5 Starting a scan and checking the progress Do not attempt to defeat the safety interlocks on the sample lid These interlocks are designed to protect you from laser light exposure Never turn off the Typhoon instrument or disconnect the Ethernet cable while scanning You can severely damage the internal mechanism of the instrument Keep the sample lid closed during the scan Opening the lid causes the instrument to close the laser shutter and block the laser light Then Scanner Control aborts the scan and saves the data already collected During the scanning process light illuminates each position on the sample only while the corresponding pixel is being recorded The position is only minimally exposed during the rest of the scan Scanner Control creates an image file and stores it using the data file gel file type 6 5 1 Starting a scan To start a scan 1 Make sure that the storage phosphor screen is in place the sample lid is shut and all the settings are correct in the Scanner Control window 2 Inthe Scanner
190. ument You use the Scanner Control software to select settings that are appropriate for the type of sample you are scanning and to start the scan The screen or sample emits light in three ways e When the red 633 nm laser illuminates each one pixel section of an exposed storage phosphor screen the phosphor emits blue light e When the green 532 nm or red 633 nm laser illuminates each one pixel section of a fluorescent sample the fluorochrome emits light with a characteristic spectrum e Thechemiluminescent sample emits light with a characteristic spectrum The optical system collects the emitted light If you select one or more emission filters the optical system directs the light through the selected emission filter s Each filter allows only the emitted light within the filter s bandwidth to pass through to the designated photomultiplier tube PM T The PM T converts the light to an electric current which varies with the intensity of the light collected by the PM T The analog signal from the PM T is then converted into digital information and the digital information is stored on the hard disk of the computer After the scan you use the mageQ uant or equivalent software to map the information to the appropriate pixel location on the monitor and produce an accurate image of the original sample ImageQ uant enables you to quantitate variations in the signal The level of signal is proportional to the amount of radioa
191. use type 2 5 high voltage hazard 2 6 laser safety 2 8 moving completing 13 20 determining new location 13 18 preparing for 13 18 securing scan head 13 18 optical filter access door 13 10 accessing filter changer 13 9 changing definition 13 15 changing selection 13 4 changing illustrated 13 10 closing access door 13 13 Index 8 Typhoon User s Guide closing filter door 13 13 inserting 13 11 moving filter changer to access door 13 4 registering in Scanner Control 13 14 removing 13 13 replacing front cover 13 13 overview 1 3 power indicator light 3 1 A 1 power switch 3 1 serial number 2 1 serial number label 2 2 setting laser shutoff time 13 3 turning off 3 13 turning on 3 1 Typhoon system counts 5 2 hardware components 1 1 to 1 2 U uneven samples placing 8 6 unit of measure 5 2 Unlink All button in Fluorescence Setup window 9 11 unlinking fluorescent scans 9 14 unmounted storage phosphor screen 4 3 5 3 5 8 5 10 6 4 6 5 Use check box in Fluorescence Setup window 9 8 User Comments area 6 12 9 7 12 7 V voltage safety 2 4 selecting for Image Eraser 14 5 Ww warning definition xii fuse 2 5 general safety 2 1 label replacement 2 8 laser light 2 7 radiation exposure 2 9 Web pages Filter Changer Maintenance 13 5 Insert New Optical Filter into Filter Changer 13 6 Park Head Optics for Shipment Web page 13 19 Remove Optical Filter from Filter Changer 13 7 Typhoon Direct Instrument Access 13
192. window 6 14 9 17 12 13 important definition xii Insert New Optical Filter into Filter Changer Web page 13 6 L labels high voltage hazard 2 6 laser warning 2 8 location of 2 3 replacing 2 8 lasers safety precautions 2 8 selecting for fluorescence scanning 9 10 setting shutoff time 13 3 troubleshooting A 3 types 2 7 warning label 2 8 LE screen see also storage phosphor screen cleaning 5 4 description 4 4 using plastic wrap 5 2 lid see sample lid light excessive exposure 8 1 limit of detection setting 9 10 12 8 linear dynamic range phosphor screen 4 2 link warning disable in Fluorescence Setup window 9 13 Link With area in Fluorescence Setup window 9 11 linking fluorescent scans 9 11 to 9 14 low fluorescence sample support 8 2 M membrane handling 8 8 11 5 placing on glass platen 8 8 using 8 3 menus File B 1 Help B 2 microplate placing on glass platen 8 6 11 3 using 8 3 microplate strip placing on glass platen 8 6 using 8 3 mounted storage phosphor screen 4 3 5 8 6 4 6 16 moving instrument 13 18 to 13 20 multichannel description 7 5 label guidelines 8 2 multilabel description 7 5 guidelines 8 2 network remote scanning 6 13 9 16 12 12 setting connection for browser 13 1 noise see background nonfatal scan errors 6 14 9 17 12 13 note definition xii 0 opaque samples placing 8 6 optical filter see also emission filter bar code for 13 4 changing 13 10 changing filter definition 13 15
193. yphoon Direct Instrument Access Web page make sure you close the Scanner Control software Leaving Scanner Control running while you use the Web page can cause unpredictable problems in Scanner Control To select a shut off time for the lasers 1 Inthe Typhoon Direct Instrument Access Web page click the Select a Laser Shut O ff Time option The Select a Laser Shut Off Time Web page appears figure 13 2 Microsoft Internet Explorer iojx File Edit View Go Favorites Help ee aE E Q amp amp Bact Ronverd Stop Refresh Home Search Favorites History Channels Fullscreen Address htip 192 168 1 1 Links Select a Laser Shut Off Time Minutes 60 minimum DONE e Done Internet zone 7 Figure 13 2 The Select a Laser Shut Off Time Web page Typhoon User s Guide p13 3 Part five Maintaining the Typhoon system hardware SSS 2 In the Minutes box type the number of minutes you want the instrument to remain idle before the lasers shut off The minimum value is 60 minutes 3 Click Done to set the shut off time and return to the Typhoon Direct Instrument Access Web page Whenever the Typhoon instrument is idle for the time period you specified the lasers shut off To reactivate the lasers you can either start a scan or turn the instrument off and then turn the instrument back on 13 3 Changing the optical filter selection Warning You must leave the Typhoon instrum

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