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ArcturusXT™ Laser Capture Microdissection System User Guide

1. 93 Additional options 94 Installation Instructions 97 Instructions for lifting and carrying the instrument 97 Preparing for installation 2 2522484 de Phased ble dete RER ead dees 97 General unpacking and installation instructions 98 Installation qualification 2 99 Operational qualification 99 Installing software upgrades 99 Arcturus Laser Capture Microdissection System User Guide Contents APPENDIX C Safety ns en nee en ae E siemens danas 101 Instrumentation Safety 101 Symbols on instruments 101 Safety labels on instruments 103 General instrument safety 103 Physical hazard safety 5e bei eb ae nt rte dut en lee a 104 Electrical Safety s nicieni nn nn ce eee ame ees dee fess Wee en Pen een 104 LASER safety atte te SA ui beh Melt ce a th SE i htt ble dee Beaded Yet 105 Workstation Safety 1 422444 dus ged wees ann a gS eee 106 Safety and electromagnetic compatibility EMC standards
2. 22 Using formalin fixed paraffin embedded tissue samples 23 Using other types of samples 23 Arcturus Laser Capture Microdissection System User Guide 3 Contents CHAPTER 3 CHAPTER 4 Starting the System and Loading Samples 25 Summary of chapter topics 25 Introduction to the Nikon Eclipse Ti E microscope controls 25 Using the front operation panel 25 Using the left operation panel 28 Using the right operation panel 29 Starting the Arcturus operating software 31 Loading materials onto the Arcturus T Instrument 31 Prepare the work surface 31 Optional selecting load options for slides 32 Selecting load options for caps 33 Selecting file path options 33 Selecting static image options 34 Implementing your selections 34 Saving images automatically
3. 61 Setting the IR Capture Spot size 61 Setting up the Test Fire 2202022 ee 61 Checking the spot diameter 62 Working with overlays 63 Savingan overla 2 Ras ne ee SR RE RE dE na 63 Using a saved overlay 63 Working with Capture Groups 22 64 Viewing a capt re groupe nant eee eo ee ee a eee 64 Setting formatting properties for a Capture Group 64 Working with stored positions 65 CHAPTER 6 Microdissecting Cells and Tissue 67 Summary of chapter topics 67 Capturing cells by microdissection 68 Capturing cells in one step 68 Capturing cells using the capture and cutting tools separately 69 Repeating microdissection 70 Inspecting microdissected material 71 Unloading Materials csse er dane ed ee ead ber eet a a ee 72 l ocating the lasers cis ve 4 dt causes igo er Ee Ro aes W
4. 118 Damages claims andreturns 119 Watmages settee atin ee st hohe te CE ae Et re nm edt cel dk Rees a 119 CLAMS 5h Pe ed fae a heed fare mon dariete aa no Seda genes latte min see 119 REtUFNS sise ets pub tte get s oh eda wc tenes dulled Et ae sence 119 Documentation and Support 121 Related documentation 121 HOW P oNN UPPI E trena E parie ana gah A og Anne eae 122 index erin nan iso ie ini ei Mendes as EES 123 Arcturus Laser Capture Microdissection System User Guide 7 Contents 8 Arcturus Laser Capture Microdissection System User Guide About This Guide Purpose of this guide This user guide is intended for use with Arcturus T Laser Capture Microdissection LCM Systems built on the Nikon Eclipse Ti E microscope base If you have a Nikon TE2000 based Arcturus T Instrument please see the specific user guide for that instrument PN 0112 0139 You can view and download both user guides from www appliedbiosystems com Prerequisites This guide is intended for those who perform microdissection using the Arcturus T Instrument Applied Biosystems is not liable for damage or injury that results from use of this manual by unauthorized or untrained parties Instructions in this guide use conventions and terminology that assume a working knowledge of the Microsoft
5. 0 35 Inspecting Slides 2 14 etin esse 37 Summary of chapter topics 37 Using the Inspect tools 37 Viewing the slides 38 Adjusting the brightness 38 Focusing theimage s cice0s dat nr gates odessa sds Bae aes dou 39 Magnifying the image 39 Working with the Bright Field lamp 40 Adjusting the Bright Field lamp 40 Adjusting the video camera properties 41 Changing the autobrightness settings 42 Performing phase contrast and DIC imaging 43 Using phase contrast imaging 43 Using differential interference contrast DIC imaging 45 Working with fluorescence 47 Before youlbeginins svc she mean Pai nce nd cae de Ars er nel T 47 Getting set up for fluorescence 47 Working with fluorescently labeled samples 48 Working with Fluorescence timed exposure
6. 106 Product specific warningS 107 Biological Hazard Safety 108 APPENDIX D Laser Safety Scenarios ss siennes Severe tees 109 APPENDIXE Arcturus Reagent Kits 113 HistoGene LCM Frozen Section Staining Kit 113 HistoGene LCM Immunofluorescence Staining Kit 113 PicoP re RNA SolationiKit lt 4 sce Re ee ee ae RE ee awe 113 PicoPure DNA Extraction Kits users en Sag gene chat ances Mek eee ae EIE N 114 Paradise PLUS FERE KIIS 8 stew teh autre woe Se NS eee fe tovemetens 114 Paradise PLUS FFPE WT RT Kit 2 0 0 00 0 cee ee ect eee 114 RiboAmp PLUS RNA Amplification Kits 115 Turbo Labeling Kits sa manne Giana ae wives np nent a aa E da eh dees 115 APPENDIX F Instrument Warranty Information 117 Computer contig ratio Merss ei eh ha ee en ed Se 117 Limited productwarranty ee dou mi creed eee eed ea ee oe Ae 117 Limited warranty 4 2 2 117 Warranty period effective date 118 Warranty claims sendede reme ti Gods annee eat te aient heres ein 118 Warranty exceptions 25 ss 4 bee E es anne nent Ds 118 Warranty limitations
7. options Description Catalog Number Arcturus LED Illumination Tower 0310 5537 Arcturus Phase Contrast Nikon 0310 5535 Illumination Tower manual Arcturus T Phase Contrast Nikon 0310 5761 Illumination Tower motorized ArcturusX Laser Capture Microdissection System User Guide 93 Appendix A System Specifications Additional options 94 Table 18 Additional upgrades and options Description Catalog Number UV Cutting Arcturus UV Cutting Option 0310 5538 Arcturus Enhanced UV Cutting Option 0310 5950 Fluorescence Arcturus Fluorescence Option Includes fluorescence cubes for excitation with emission at Red Blue and Green 0310 5504 manual 0310 5749 motorized Differential Interference Contrast option for Nikon Illumination Tower Note Life Technologies recommends that you purchase Arcturus Binoculars along with the Phase Contrast Illumination Tower DIC Base 14423 00 Includes 10X and 40X objective sliders Note Optional objective sliders can be purchased in conjunction with the selection of the respective optional objectives e Differential Interference Contrast 20X Slider 6550 0118 e Differential Interference Contrast 60X Slider 14468 00 DIC Analyzer Cube 9000 1055 Note Requires purchase of motorized fluorescence filter turret PN 0310 5749 Optional objective upgrades 4X 14676 00 20X 14658 00 60X 14659 00 100X Dry
8. Laser Capture Microdissection System User Guide 117 Appendix F Instrument Warranty Information Warranty period effective date Warranty claims Warranty exceptions Warranty limitations 118 Applied Biosystems warrants that for a period of ninety 90 days from the date the warranty period begins the tapes diskettes or other media bearing the operating software of the product if any will be free of defects in materials and workmanship under normal use If there is a defect in the media covered by the above warranty and the media is returned to Applied Biosystems within the ninety 90 day warranty period Applied Biosystems will replace the defective media Applied Biosystems does not warrant that the operation of the instrument or its operating software will be uninterrupted or error free Any applicable warranty period under these sections begins on the earlier of the date of installation or ninety 90 days from the date of shipment for hardware and software installed by Applied Biosystems personnel For all hardware and software installed by the buyer or anyone other than Applied Biosystems and for all other products the applicable warranty period begins the date the product is delivered to the buyer Warranty claims must be made within the applicable warranty period or for chemicals or other consumable products within thirty 30 days after receipt by the buyer The above warranties do not apply to defects result
9. menus e With the stylus place the stylus on the image press the lower button on the stylus and then select from the menu e With the mouse place the cursor on the image right click and then select from the menu Making menu selections from the main Move stage ima ge v Draw freehand Draw circle To make a menu selection from the main image Draw line of IR Capture spots place the cursor or stylus somewhere in the IR Capture Spot image and either press the lower stylus button or right click the mouse A menu similar to the Select drawing item s one shown in Figure 4 is displayed When you Eraser Ruler click on your selection a checkmark appears to the left In the example Draw freehand has Figure 4 Main image menu been selected 14 Arcturus T Laser Capture Microdissection System User Guide Chapter 1 System Overview Making selections from within a drawing item in the main image To view a menu of commands that allow you to manipulate a drawing item object place the stylus or cursor inside the A Delete drawing item and then press Delete Spots In Object the lower button on the stylus LCM Only OF right click the MOUSE You Move Object s To Capture Group gt will see a menu similar to the IR Capture Objects one shown in Figure 5 In this menu there are no checkmarks The chosen item is highlighted In the example the user has selected the first Figure 5 Drawing item menu from main image opt
10. tt Laser is firing Cap overridden and not detected in beam path 14 xX x x Stand by Stand by P Yellow Stop Clean obj status tt Laser ready to fire Cap overridden but detected in beam path 110 Arcturus Laser Capture Microdissection System User Guide Appendix D Laser Safety Scenarios Laser Laser CapSur L 5 p State Bypass Bypass Cap in aser 9 et Color op Cut Other Tool Tip Messages Button Key in Place State State Laser up Capture Pressed Place 15 xX x x ON Power P tt Laser is firing Cap p Green overridden but detected in beam path Arcturus Laser Capture Microdissection System User Guide 111 Appendix D Laser Safety Scenarios 112 Arcturus T Laser Capture Microdissection System User Guide APPENDIX E Arcturus Reagent Kits Note Only the most frequently used kits are listed here Additional kit configurations are available depending on individual research needs For more information go to www appliedbiosystems com HistoGene LCM Frozen Section Staining Kit The HistoGene LCM Frozen Section Staining Kit is used to process tissue sections for LCM in order to maximize the quality and yield of RNA from the LCM cells The kit comes with all dehydration and staining reagents disposable staining jars specially treated slides and a detailed protocol and troubleshootin
11. Defaut autobghtness ES e EZE Figure 26 The illumination tab depending on your preference and application needs 5 Work with the Intensity and Camera Gain controls here and the Brightness controls in the Inspect tools pane to optimize the image These tips may be useful e Use the Intensity slider to change the bright field lamp intensity Slide the control to the right to increase intensity or to the left to decrease the intensity e Use the Camera Gain slider to adjust the camera gain The camera gain amplifies the signal from the video camera Slide the control to the right to increase the gain or to the left to decrease the gain Eoo 6 To adjust the white balance within the camera settings see Adjusting the video camera properties on page 41 7 Tap or click OK to close the dialog box and save your changes 40 Arcturus T Laser Capture Microdissection System User Guide Adjusting the video camera properties Chapter 4 Inspecting Slides You probably won t need to adjust the video camera often but in case you do this section describes how to work with the video camera properties and set the white balance You adjust the white balance to achieve the proper color representation in your image and for optimal visualization for microdissection To set the white balance 1 To open the Camera DFx 31AF03 Properties x Properties dialog box X clic Device Properties Update I Update continuous Reset t
12. Windows operating system the Internet and Internet based browsers Safety information For general safety information see this section When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety appendix at the end of the manual for the complete alert on the chemical or instrument Safety alert words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices a WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury AN DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for IMPORTANTS each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard symbols that are affixed to Applied Biosystems instruments Arct
13. when you loaded your slides you can change it here at any time except when a L cap is on the slide SlideName The name of this slide You can view properties of a slide its name and any notes in the information area to the right of the slide overview Figure 40 Slide information area image see Figure 40 You can edit the SlideName and or SlideNotes here Editing here is the same as entering the information in the Load Options dialog box 50 Arcturus T Laser Capture Microdissection System User Guide Chapter 4 Inspecting Slides Working with images and videos This section dicusses capturing saving and viewing tiled and static images and videos Capturing and You can capture and save images for later viewing There are two kinds of images saving images e Tiled Images e Static Images Capturing tiled images A tiled image is a snapshot of a selected region of the slide The software captures as many images as needed to span the selected region and stitches them together into one image A tiled image can show a larger region than a static image To capture a tiled image 1 In the main image window a Locate the area for the tiled image b Select the objective for the desired magnification c Adjust the image using the tools in the Inspect tools pane 2 Click the Tiled Image button in the Inspect tools pane 3 In the slide overview image tap or click and then drag the cursor to outline the area
14. 1 Turn on the computer 2 Turn on the Eclipse Ti E microscope base using the switch located on the back of the microscope 3 Turn on the Arcturus Instrument As you face the microscope the power button is located on the left side of the instrument 4 Start the software by clicking the Arcturus icon on the Windows desktop or click Start point to Programs and click the Arcturus option The software opens to fill the screen Loading materials onto the Arcturus Instrument To begin loading materials onto the Arcturus Instrument Prepa re the work 1 In the Setup tool pane click Present surface Stage see Figure 15 The work surface moves forward and to the right 2 If needed remove any slides and caps Figure 15 Present stage from Setup panel left on the work surface 3 Load your slides and caps onto the work surface see Figure 16 4 Push the tension button in and place each slide in a slot 5 Release the tension button 6 Place the caps in the CapSure cassette into the slot on the left side of the work surface Figure 16 Modular stage insert For every cap in the cassette make sure the corresponding cap offload position on the right side of the work surface is empty ArcturusX Laser Capture Microdissection System User Guide 31 Chapter 3 Starting the System and Loading Samples Optional selecting load options for slides 32 Open the Load Options dialo
15. Depending upon the type of slide you have loaded the details of the cutting vary For glass slides the instrument will fire only the IR laser unless you have unchecked LCM Only for this drawing item For membrane glass slides and membrane frame slides the instrument cuts around the region of interest leaving tabs if designated in the Cut and Capture Settings see Setting properties for cut and capture on page 76 2 Inspect the cap and slide See Inspecting microdissected material on page 71 This explains how to capture cells using the IR Capture and UV Cutting tools separately 1 Optional Review the drawing items in each capture group a In the Select tools pane click the letter A B C or D in the Capture Groups list for the capture group of interest b Click each number in the Drawing Items list The stage moves as needed to display the drawing item at the center of the main image 2 Optional If you want to add more IR capture spots to any item a In the Select tools pane click the IR Spot Size control to select a different spot size b Click the IR Spot tool c Click inside any of the items in the main image A spot is drawn where you clicked d Click the Move Stage tool to deactivate the IR Spot tool ArcturusX Laser Capture Microdissection System User Guide 69 o Chapter 6 Microdissecting Cells and Tissue Preparing for Microdissection 1 Click the Place Cap tool to pl
16. KIT0610 Cy5 12 samples Arcturus Laser Capture Microdissection System User Guide 115 AppendixE Arcturus Reagent Kits 116 Arcturus T Laser Capture Microdissection System User Guide APPENDIX F Instrument Warranty Information Computer configuration Applied Biosystems supplies or recommends certain configurations of computer hardware software and peripherals for use with its instrumentation Applied Biosystems reserves the right to decline support for or impose extra charges for supporting nonstandard computer configurations or components that have not been supplied or recommended by Applied Biosystems Applied Biosystems also reserves the right to require that computer hardware and software be restored to the standard configuration prior to providing service or technical support For systems that have built in computers or processing units installing unauthorized hardware or software may void the Warranty or Service Plan Limited product warranty Limited warranty Applied Biosystems warrants that all standard components of its Arcturus Laser Capture Microdissection LCM System will be free of defects in materials and workmanship for a period of one 1 year from the date the warranty period begins Applied Biosystems will repair or replace at its discretion all defective components during this warranty period After this warranty period repairs and replacement components may be purchased from Applied Biosystems
17. Plug the instrument in and turn it on 29200 Ore COR gt Restart the Arcturus software When selecting alternate fluorescence filter cubes be sure that the dichroic and emission filters have at least 65 transmission at 810 nm otherwise the automatic laser location feature will fail when the filter is in the optical path If you want to use a filter with lower transmission you can manually locate the laser Filters should be compatible with the Nikon Eclipse Ti E microscope To replace a fluorescence cube 1 Remove the cover and select the desired filter position from the carousel below the objective turret of the microscope 2 Remove the existing filter cube if present by pulling it straight out 3 Insert the new filter cube by pushing it straight in 4 Replace the cover Arcturus T Laser Capture Microdissection System User Guide Chapter 8 Maintenance and Troubleshooting 8 Replacing the For this procedure you will need the instructions provided with the lamp the new fluorescence lamp fluorescence lamp a 3 mm hex key provided with the instrument and a slotted screwdriver Follow the instructions provided with the lamp Replacing the fuse For this procedure you will need the new fuse 2A time delay 5 x 20 mm and a slotted screwdriver To replace the fuse 1 If needed exit the Arcturus T software 2 Turn the instrument off and unplug it When you are facing the instrument the switch is located on
18. Shin Overviews 1 Click the Image Settings tab Slides Caps File Paths Image Settings E 2 Inthe first panel choose the AutoDocFrefx Lea AutoDocument Filename Settings pen ane a that you want to use a w E Image nnotationColor Black 3 In the Annotatelmage field if you Coo CE want the SlideName and SlideNotes p Sareirell enter No from the Slides tab and the objective in use to be saved in the upper left corner of all image files enter Yes Otherwise enter No a If you entered Yes in the Annotatelmage field enter the color for the annotation in the ImageAnnotationColor field b If you entered Yes in the Figure 20 Image settings AnnotateImage field enter the font for the saved annotation in the ImageAnnotationFont field Click the plus sign to view the formatting options 4 In the ImageFile Extension field choose the file format for saved images You can choose either JPEG jpg or TIFF tif 5 In the SavelmageOverlay field if you want to save drawing items when you save images enter Yes Otherwise enter No Implementing your When you have finished making your choices click the OK button The instrument selections then performs these actions e Moves the work surface to the left and places the 2X objective under the first slide e Displays the selected slide in the main image window e Ifyou selected Load with Overviews automatically acquires and displays the slide overview
19. WARNING PHYSICAL INJURY HAZARD Use this product only as specified in this document Using this instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument Moving and lifting the instrument AN CAUTION PHYSICAL INJURY HAZARD The instrument is to be moved and positioned only by the personnel or vendor specified in the applicable site preparation guide If you decide to lift or move the instrument after it has been installed do not attempt to lift or move the instrument without the assistance of others the use of appropriate moving equipment and proper lifting techniques Improper lifting can cause painful and permanent back injury Depending on the weight moving or lifting an instrument may require two or more persons Moving and lifting stand alone computers and monitors AN WARNING Do not attempt to lift or move the computer or the monitor without the assistance of others Depending on the weight of the computer and or the monitor moving them may require two or more people Things to consider before lifting the computer and or the monitor Make sure that you have a secure comfortable grip on the computer or the monitor when lifting Make sure that the path from where the object is to where it is being moved is clear of obstructions Do not lift an object and twist your torso at the same time Keep your spine in a good neutral position while lifting with your legs Parti
20. alee Ne 12V100W r an Figure 13 Nikon Eclipse Ti E left operation panel Selecting the resolution for vertical movement with the Focus Selection switch Use the Focus Selection switch to select the resolution for the vertical movement of the nosepiece when using the focus knobs on the instrument Press the switches up and down to toggle between coarse fine and extra fine resolutions The indicator will light up next to the selected resolution Note The Coarse Fine Extra Fine Focus Selection switch is only relevant when you are manually adjusting focus using the knobs on the instrument The Arcturus operating software uses its own z step settings When you use the focus buttons in the Arcturus operating software only those z step settings are applied regardless of the focus resolution chosen on the operation panel Moving the nosepiece using the Focus knob Use the Focus knob to move the nosepiece up and down for focus adjustment If you use the Focus knob while using the Arcturus the software will track the position The numbers displayed in the Arcturus software interface will continue to match the numbers displayed in the Status Display window in the front operation panel Changing objectives without the Objective switch Whenever you use the Arcturus Instrument the Objective switch is disabled To change objectives you use the Arcturus operating software If you are operating the microscope in manual mode i e
21. as or to the extent set forth above In such countries and jurisdictions the limitation or exclusion of warranties liability remedies or damages set forth above shall apply to the fullest extent permitted by law and shall not apply to the extent prohibited by law Damages claims and returns Damages If shipping damage to the product is discovered contact the shipping carrier and request inspection by a local agent Secure a written report of the findings to support any claim Do not return damaged goods to Applied Biosystems without first securing an inspection report and contacting Applied Biosystems Technical Support for a Return Authorization RA number Claims After a damage inspection report is received by Applied Biosystems Applied Biosystems will process the claim unless other instructions are provided Returns Do not return any material without prior notification and authorization If for any reason it becomes necessary to return material to Applied Biosystems contact Applied Biosystems Technical Support or your nearest Applied Biosystems subsidiary or distributor for a return authorization RA number and forwarding address Place the RA number in a prominent location on the outside of the shipping container and return the material to the address designated by the Applied Biosystems representative Arcturus Laser Capture Microdissection System User Guide 119 Appendix F Instrument Warranty Information 120 Arctur
22. e The Microscope Lamp Intensity in the Fluorescent tab can contribute to the photobleaching of the sample even at minimal settings Ensure that the Microscope Lamp is OFF unchecked when fluorescence is not in use e Use the Gain setting next to Timed Exposure see Figure 36 on page 48 only with the Timed Exposure feature Adjusting this setting outside of Timed Exposure will not result in a change Working with When working with the IFuorescence lamp samples can become photo bleached if Fluorescence they are exposed for too long to the light source You can limit a sample s exposure to timed exposure the fluorescence lamp by using timed exposures in the Arcturus T software To set up for timed exposure 1 To work with a static image rather than the main image click Timed Exposure in the lower left corner of the Inspect Options screen Note The instrument opens the shutter only briefly to illuminate the slide when a snapshot is taken to prevent photo bleaching of the sample The snapshot is then used to indicate areas for capture 2 Click OK to close the dialog box and save your changes The Arcturus Instrument will use the lamp and camera settings you have set for the fluorescence mode 3 Click the Camera button to capture a static image for tissue selection 4 Draw on the snapshot to indicate areas for capture 5 When you have identified another area for a timed exposure tap or click the Camera button again to acquire
23. without the Arcturus operating software you click the toggle button on the Arcturus track ball to change objectives Arcturus Laser Capture Microdissection System User Guide Chapter 3 Starting the System and Loading Samples Turning the lamp On Off through software controls The Dia Illumination button is disabled when you are using the Arcturus Instrument You control the illumination lamp through theArcturus Instrument control box To turn on the illumination lamp you must first turn on the Eclipse Ti E microscope base and then turn on the ArcturusXT Instrument Adjusting the lamp brightness through software controls When you use the Arcturus operating software the Lamp Brightness Control knob is disabled You adjust the lamp brightness using the ArcturusXT software controls However if you are in manual mode i e not using the Arcturus operating software you can use the Brightness Control knob to increase or decrease microscope illumination intensity Using the right The right operation panel contains a large Focus knob a Focus Selection switch operation panel Escape and Refocus buttons and several controls that are not used with the Arcturus Instrument These features are described below and shown in Figure 14 Focus selection switch j manual Epi shutter button mode only disabled Fluorescence block switch Focus knob disabled Refocus button Escape button Figure 14
24. 14662 00 100X Oil 14661 00 Binoculars Microscope Binoculars 0200 6228 Note Life Technologies recommends that you purchase Arcturus Binoculars along with the Phase Contrast Illumination Tower Second Camera High Resolution 5 MegaPixel Camera with MetaVue 14379 00 Imaging System Note Life Technologies recommends that you purchase a second monitor when you purchase a high resolution camera This way you can view the camera output on one monitor and use the other to run the operating system software Arcturus T Laser Capture Microdissection System User Guide Appendix A System Specifications Description Catalog Number LCD Flat screen monitor for dual monitor operation 10904 00 Excitation 340 390 nm emission gt 410 nm 9000 1034 Excitation 385 400 475 493 545 565 6530 0056 Emission 450 465 503 533 582 622 AutoScan Image Analysis Software Module 9050 0005 Large Format Slide Stage Insert 0310 5401 Petri Dish Stage Insert 0310 5631 ol ArcturusX Laser Capture Microdissection System User Guide 9 96 Appendix A System Specifications Arcturus T Laser Capture Microdissection System User Guide APPENDIX B Installation Instructions Instructions for lifting and carrying the instrument The Arcturus Instrument is shipped from the factory in two or more boxes depending upon the configuration you have ordered Use proper lifting techniques when unpacking and installing the instrume
25. 4 Click OK to close the dialog box and save your changes Figure 25 Autofocus Magnifying the The microscope has an option that allows you to increase the magnification of the image current 1 0X objective to 1 5X The dial for this setting is located on the front of the instrument For the instrument and the software to properly align you must indicate in the Arcturus software that you are using this feature To use the 1 5X magnification feature 1 On the front panel of the microscope turn the magnification knob to the 1 5X position 2 Click on the 1 5X box in the Inspect tools pane see Figure 21 on page 37 to indicate that this feature has been selected Note You can also indicate that you have enabled the 1 5X magnifier in the Inspect Options dialog box on the Magnification tab ArcturusX Laser Capture Microdissection System User Guide 39 Chapter 4 Inspecting Slides Working with the Bright Field lamp This section explains how to adjust the settings on the bright field lamp how to adjust the properties of the video camera and how to work with the autobrightness settings Adjusting the To adjust the bright field lamp Bright Field lamp 1 Click the Information i button in the upper right SA corner of the Inspect tools pane see Figure 21 on noe page 37 to open the Inspect Options dialog iv box 2 Click the Illumination tab gt Diffuser setting oz Save autbrighiness
26. Cut and capture Move cap to QC Figure 50 The Microdissect tools pane IR cap UY Cutting Speed 526 pmis i UV Cutting Speed I 52 1000 UY Laser Current 10 UV cut UV Laser Current ao gt lio 1 100 0 O ArcturusX Laser Capture Microdissection System User Guide 67 6 Chapter 6 Microdissecting Cells and Tissue Capturing cells by microdissection This section explains how to capture cells in one step how to capture cells in two steps and how to repeat a microdissection Capturing cells in 1 Optional Review the drawing items in each capture group one step a In the Select tools pane click the letter A B C or D in the Capture Groups list for the capture group of interest b Click each number in the Drawing Items list The stage moves as needed to display the drawing item at the center of the main image 2 Optional If you want to add more IR capture spots to any item a In the Select tools pane click the IR Spot Size control to select a different spot size b Click the IR Spot tool c Click inside any of the items in the main image A spot is drawn where you clicked d Click the Move Stage tool to deactivate the IR Spot tool Preparing for Microdissection 1 Locate the laser s as appropriate a See Locating the UV cutting laser on page 73 b See Locating the IR capture laser on page 74 2 Move the stage so the cap is on an area a
27. Nikon Eclipse Ti E right operation panel Selecting the resolution for vertical movement with the Focus Selection switch You use the Focus Selection switch to select the resolution for the vertical movement of the nosepiece when using the focus knobs on the instrument Press the switches up and down to toggle between Coarse Fine and Extra Fine resolutions The indicator will light up next to the selected resolution Note The Coarse Fine Extra Fine Focus Selection switch is only relevant when you are manually adjusting focus using the knobs on the instrument The Arcturus operating software uses its own z step settings When you use the focus buttons in the Arcturus operating software only those z step settings are applied regardless of the focus resolution chosen on the operation panel ArcturusX Laser Capture Microdissection System User Guide 29 Chapter 3 Starting the System and Loading Samples 30 Controlling the lamp through the software or through the control box The Epi Shutter button is disabled on the Arcturus Instrument The Arcturus Instrument uses an external fluorescence lamp which you control through either the Arcturus operating software or the fluorescence control box For more information about using Epi fluorescence with the Arcturus Instrument see Working with fluorescently labeled samples on page 48 Rotating the fluorescence filter turret by hand The Fluorescence Block switch is disabled on th
28. Pop up Cut Capture Other Tool Tip Messages 5 x On Stand by Orange Stop Clean obj status tt Laser disabled because cap is out of beam path and override is off popup Lasers have been disabled because cap is out of beam path and override is off Change cap placement or enable override to continue 6 x x Stand by Stand by U Yellow Stop Clean obj status tt Laser ready to fire Cap placed in beam path Power Green tt Laser is firing Cap placed in beam path 8 x Stand by Stand by U Red Stop Clean obj status tt Laser disabled because cap is out of beam path and override is off popup Lasers cannot be enabled because the hardware bypass key is absent Stand by Red Stop Clean obj status tt Laser disabled because cap is out of beam path and override is off popup Lasers cannot be enabled because the hardware bypass key is absent tt Laser ready to fire Cap placed in beam path Power p Green tt Laser is firing Cap placed in beam path popup Laser interlock cannot be bypassed because the hardware key is not detected 12 x Xx Stand by Stand by PF Yellow Stop Clean Obj Status tt Laser ready to fire Cap overridden and not placed in beam path Power p PF Green
29. The following list of steps provides a broad outline of the microdissection process The chapters in this guide are keyed to this list 1 Prepare samples See Chapter 2 Preparing Samples on page 21 2 Load slides and caps See Chapter 3 Starting the System and Loading Samples on page 25 3 Locate the cells of interest See Chapter 4 Inspecting Slides on page 37 4 Mark the cells and tissue for capture See Chapter 5 Selecting Cells for Microdissection on page 55 5 Capture the tissue See Chapter 6 Microdissecting Cells and Tissue on page 67 6 Unload the samples and extract the tissue See Chapter 7 Extracting Cells and Tissue on page 79 Arcturus T Laser Capture Microdissection System User Guide Chapter 1 System Overview Using the Arcturus operating software To start the software you click the Arcturus icon on the Windows desktop see Figure 1 The system will display Al the primary screen which is shown in Figure 2 This Z application facilitates the microdissection workflow ArcturusXT Figure 1 Arcturus icon The primary screen The most prominent feature in the primary screen is the main image window that shows the live microscope image To the side of the screen is the tool panel which contains tool panes arranged from top to bottom in the order of the steps for laser microdissection 1 Setup 2 Inspect 3 Select 4 Microdissect By default the main image
30. User Guide Delete All 7 Drawing Items list Capture Groups list Delete All drawing items AutoScanXT image analysis software module 55 Chapter 5 Selecting Cells for Microdissection Marking cells for microdissection 56 You use the Select tools pane see Figure 42 on page 55 to mark the cells for microdissection Note If your instrument is equipped with a UV cutting laser you should mark your cells and perform microdissection using the same objective This ensures that the UV cutting laser will most accurately follow the dissection marks To mark cells for microdissection 1 Click the desired size small medium or large in the IR Spot Sizes control in the Select tools pane see Figure 42 on page 55 to choose the relative size of the IR capture spots The IR capture spot size is one of the factors that determines how the IR capture spots are placed in a drawing item to identify where the IR capture laser will be fired 2 Tap or click any drawing tool to activate it The tool remains active until you tap or click another tool For information on each of these tools see Table 9 Note When you are using the Freehand and Defined Circle Area drawing tools the system will place IR capture spots depending on the slide type selected during set up If you are using a glass slide drawing items using these tools will be filled in with IR capture spots and the UV laser will not be used If you are using glas
31. another snapshot of the new area Arcturus Laser Capture Microdissection System User Guide 49 Chapter 4 Inspecting Slides Note If you want to control the shutter manually do not check Timed Exposure The shutter will open when you tap the Fluorescence button Each time you want to open or close the shutter you will need to open the Inspect Options dialog box and tap or click Open Shutter in the Fluorescence tab Otherwise the shutter will only close when you revert back to bright field illumination Working with slides This section provides information about selecting and viewing slides Displaying a To display a different slide different slide 1 To the left of the slide overview image in the cap and slide handling area at the bottom of the screen tap the Slide button see Figure 39 for the slide of interest Load Caps Slides Os A MEM 0 Macro ia FRM The stage will move the slide over the objective The O e ES slide overview and the main image update to show O this slide MEM O c FRN 2 If there is not already a slide overview image right click in the slide overview and select Reacquire Overview Image Figure 39 Cap and slide handling area Viewing slide PPPOE EEEE You can view the slide type in the cap and properties EEF a slide handling area to the left of the slide Attributes overview image see Figure 39 SlideName Slide C SlideNotes If you did not select the correct slide type
32. be damaged Try another cap Cannot locate IR Laser No cap on slide Place a cap on the slide Bias or brightness settings are too low Open the IR locate tab in the Microdissect dialog box The Bias setting should be at 60 You may also need to adjust the Brightness setting in the Inspect tool pane to darken the field of view and visualize the IR laser guide light IR laser is out of the field of view 1 Fire IR laser using the test fire button located in the Microdissect tool pane 2 Move around under the cap to locate a wetted spot 3 Right click in the center of the spot and select the IR spot located from the pop up window If this doesn t work the IR laser location may need adjustment Contact technical support to arrange for service Tissue is thick or dark Move to a thinner tissue area or a clear area on the slide IR Capture laser fires off target IR laser location is set incorrectly Relocate the IR laser Arcturus T Laser Capture Microdissection System User Guide Solving problems With UV Laser cutting Solving problems with image quality Chapter 8 Maintenance and Troubleshooting 8 Table 11 Possible problems with UV Laser cutting SYMPTOM POSSIBLE CAUSE REMEDY UV Laser does not cut Neutral density filters may be in place Verify that the ND filters are in the out position levers tilted to the right The ND filters are locate
33. configuring your workstation to promote neutral or relaxed working positions CAUTION MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by potential risk factors that include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors To minimize musculoskeletal and repetitive motion risks e Use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard monitor and mouse e Position the keyboard mouse and monitor to promote relaxed body and head postures This section provides information on e U S and Canadian safety standards e Canadian EMC standard e European Safety and EMC standards e Australian EMC standards U S and Canadian safety standards The instrument has been tested to and complies with standard e UL61010 1 CSA C22 2 No 61010 1 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements e FDA Radiation Control for Health and Safety Act of 1968 Performance Standard 21 CFR 1040 10 and 1040 11 as applicable The instrument has been tested to and complies with the Radiation Control for Health and Safety Act of 1968 Performance Standard 21 CFR 1040 10 and 1040 11 as applicable Canadian EMC standard This instrument has been tested to a
34. dialog box Phase annulus Ensure that the proper phase annulus is chosen for the objective in use PhL 4x Ph1 10x and 20x Ph2 40x and 60x Annular diaphragm Ensure that the annular diaphragm is centered The DIC image does not appear optimal The diffuser Ensure that the diffuser is in the OUT position in the Select Options dialog box Condenser turret position Ensure that the condenser is turned to the DIC N1 position Polarizer and Ensure that the polarizer and analyzer are analyzer both fully pushed into the IN positions Condenser Ensure that the condenser is properly alignment centered and focused DIC Slider Ensure that the DIC Slider has been inserted beneath the objective in use Note For more information on Phase Contrast and DIC as well as complete instructions for use please refer to the Nikon user guides provided with the Arcturus Instrument or visit www microscopyu com ArcturusX Laser Capture Microdissection System User Guide 89 8 Chapter 8 Maintenance and Troubleshooting Solving general problems 90 Table 15 Possible problems with the instrument in general SYMPTOM POSSIBLE CAUSE REMEDY Microdissection UV or IR is not aligned to markings of drawing item s Magnification of image does not match the software operation Check the position of the Intermediate magnification dial and compare against the magnification
35. for the tiled image The selected area is outlined with SSSR i e If this area is not correct select Cancel and repeat steps 2 and 3 If this area is correct select Take Tiled Image of Selected Area A tiled image of the selected region is acquired and opens in a new window Note If the software cannot capture a tiled image due to limited memory select a smaller region on the slide overview and or change to a lower magnification objective and try again 4 Work with the tiled image as you would with the main image to identify and mark tissue for microdissection 5 To close the tiled image tap the close box in the upper right corner of the static image window 6 The stitched image is saved in the folder specified in the File Paths tab of the Load Options dialog box It is saved in the format specified in the Image Setting tab of the Load Options dialog box ArcturusX Laser Capture Microdissection System User Guide 51 Chapter 4 Inspecting Slides Capturing static images 52 A static image is a snapshot of the area visible in the main image window You select the image file format and other options for static images in the Image Settings tab of the Load Options dialog box You set the location for the file in the File Paths tab of the Load Options dialog box To capture a static image 1 2 3 Move the stage so the main image window displays the area that you want to save as an image The static i
36. tab located in the microscope dialog box Both of these items should match at 1X or 1 5X UV or IR laser is not located properly Use the IR or UV locate options found in the microdissection dialog box Brightfield light is flashing either slow or fast Stage bump 1 Re align the stage so that all front edges of the stage are parallel 2 Close down the software and turn off the instrument 3 Restart the instrument and re initiate the software Limit for the cap robot arm has been exceeded 1 Lower the cap fork by turning the lead screw counter clockwise 3 mm slotted screwdriver until the motor takes hold 2 Close down the software and turn off the instrument 3 Restart the instrument and re initiate the operating software Microdissection process does not initiate within AutoScanXT by using the Harvest button Live image was moved away from where static image was taken Move the cap to the area containing the items for microdissection and click the Harvest button again Arcturus T Laser Capture Microdissection System User Guide APPENDIX A System Specifications This appendix provides the specifications for the Arcturus Laser Capture Microdissection LCM System with a Nikon Eclipse Ti E microscope base Depending on the configuration of your system not all options listed below may be included with your instrument Instrument specifications Table 16 Descri
37. the left side The plug is on the back of the instrument 3 Use the slotted screwdriver to open the fuse holder The fuse is located in the plug socket 4 Remove the fuse and replace it with the new one 5 Plug in the instrument and turn it on Arcturus T Laser Capture Microdissection System User Guide 85 8 Chapter 8 Maintenance and Troubleshooting Troubleshooting tips If you encounter a problem that you cannot resolve using this troubleshooting section contact your Life Technologies service representative Solving problems Table 10 Possible Problems with IR Laser Capture LCM with IR Laser Capture LCM 86 Symptom Possible cause Remedy Cells do not adhere to the CapSure Cap Inadequate IR laser power or duration settings Open the IR Settings tab in the Microdissect dialog box and adjust the power and duration settings until proper wetting is achieved Tissue preparation Debris or loose tissue may be present Use a PrepStrip to remove the debris or loose tissue from the slide prior to microdissection Folds may be present Position the Cap such that it is not placed on the fold Alternatively increase IR laser power and duration settings to achieve proper wetting Tissue not dehydrated Ensure that the section is properly dehydrated Proper dehydration is 100 ethanol for 1 minute followed by Xylenes for 5 minutes and then air dry for 5 minutes Cap The CapSure cap may
38. the tissue image is called an Saving an overlay To save an overlay 1 Load your slides and identify the tissue you want to capture with the drawing tools 2 Choose Save and then Markup from the File menu 3 Enter a file name in the dialog box and click Save Note Do not use any of the following file names for the markup file capturegroupattributes bin containers bin cutncapture bin drawingitems bin imagesettings bin and observesettings bin These names are reserved for system files required by the Arcturus software The overlay is saved as a bin file in the C Program Files Life Technologies ArcturusXT folder The overlay file contains the drawing items and their locations Using a saved To use a saved overlay overlay 1 Choose Open and then Markup from the File menu 2 Navigate to the bin file containing the overlay and click Open The drawing items appear in the main image window in the original locations Any drawing items in the main image window remain there You can microdissect at this stage or you can work with the drawing items as needed ArcturusX Laser Capture Microdissection System User Guide 63 Chapter 5 Selecting Cells for Microdissection Working with Capture Groups Tools in the Select tools pane enable you to designate capture groups Viewing a capture To move the stage to view a specific drawing item in a capture group group 1 In the Select tools pane click the letter A B
39. time values increase image pixilation b Set the Brightness exposure time see Figure 36 to 1s or less Higher values result in significant delay in live image updates c If the live image displays with very bright fluorescence intensity lower the CR Figure 56 48 Arcturus Laser Capture Microdissection System User Guide Chapter 4 Inspecting Slides Toggling between fluorescence and Bright Field illumination If you need to toggle between Fluorescence and Bright Field illumination for example when performing IR test fires use the camera gain and microscope intensity settings on the Fluorescence tab not the Illumination tab 1 Set Camera Gain see Figure 36 on page 48 for optimal fluorescence image 2 Adjust the Microscope Lamp Intensity see Figure 36 on page 48 A good starting point for the Microscope Lamp Intensity setting is 30 Adjust from this point to get enough light Interactions between Fluorescence and Illumination tab settings This section explains how the Fluorescence and Illumination tabs work e The Microscope Lamp Intensity and Camera Gain settings in the Fluorescence tab are independent of those in the Illumination tab Changes to these settings apply only while using fluorescence illumination Once you switch off the fluorescence the settings in the Illumination tab are applied e Camera Gain in the fluorescence tab controls the gain of both brightfield and fluorescence illumination
40. tool see Figure 42 on page 55 then click in the main image window Click a single drawing item to select it or drag the stylus to select more than one drawing item Hold down the cursor within the selected object s The cursor changes to a four headed arrow In the main image window drag the stylus to the new location for the drawing item s and release it The drawing item s move to the new location To move a drawing item to a different capture group 1 Click the Select Object s tool see Figure 42 on page 55 then click in the main image window Click a single drawing item to select it or drag the stylus to select more than one drawing item Right click within the selected object s A pop up window will appear Select Move Selected Object s to Group and select the desired capture group The selected drawing items move to the new capture group To delete a single drawing item e Inthe Drawing Items list tap or click the number of the drawing item then right click the stylus button or mouse and select Delete Object or e In the live image tap the item right click and select Delete Object s from the pop up menu To delete multiple drawing items e Select a group of drawing items by tapping and dragging with the Select Object s tool tap within one of the selected objects and right click Then choose Delete Object s from the pop up menu or Hold down the Ctrl button on the keyboard an
41. turn on the EXFO box after initiating the software you will be prompted with a pop up window asking if you would like the Arcturus Instrument to take control of the illumination source Click Yes or No and proceed with the steps below Getting set up for Prior to starting a fluorescence experiment fluorescence 1 Make sure that the fiber optic cable is fully inserted into the EXFO cone attached to the scope and is fully inserted into the back of the EXFO box 2 Make sure that the EXFO cone is properly seated into the insert attachment on the scope 3 Make sure that the see Figure 35 4 Make sure that the 5 Make sure the DIC Shutter Aperature set Neutral density i screws fluorescence prisms are removed filters When the DIC option is Figure 35 Fluorescence components installed the DIC prisms are beneath the 10x 20x 40 and 60x objectives 6 Make sure that the fluorescence aperture is open pulled out and centered Center the aperture using the 2 set screws see Figure 35 7 Make sure that th see Figure 35 Arcturus Laser Capture Microdissection System User Guide 47 Chapter 4 Inspecting Slides Working with 1 Click on the Fluorescence icon on the right side of the Inspect tool pane see fluorescently Figure 21 on page 37 labeled samples Note If you have the motorized fluorescence filter turret in place the filter selection buttons will become active when the fluorescence module is turne
42. type of caps you are loading OC to jeu kei i vi Figure 18 Load options for caps To enter information about where image files should be saved click the File Paths tab see Figure 19 In the AutomaticFilename field enter Yes if you want the file name e For saved images to be the date and a number e g 2007 01 23_X tif where X is an incrementing number e For tiled images to be a date and a number e g 2007 01 23_002_S tif e For videos to be a date and a number e g 2007 01 23_X avi Enter No if you want to be prompted for a file name when you save an image In the StudyFolder field enter the Figure 19 File path options name of the folder where saved images videos and reports are to be located Click Browse to select the location In the ImageSubfolder enter the name of the folder inside the StudyFolder where image files are to be saved In the ReportSubfolder enter the name of the folder inside the StudyFolder where the cap interaction history files are to be saved A cap interaction history is generated when you off load a cap after microdissection In the VideoSubfolder field enter the name of the folder inside the StudyFolder where video files are to be saved Arcturus Laser Capture Microdissection System User Guide 33 Chapter 3 Starting the System and Loading Samples Selecting static To enter information related to static CG x image options mages
43. 49 Working with slides 2 50 Displaying a different slide 50 Viewing slide properties 50 Working with images and videos 51 Capturing and saving images 51 Capturing saving and viewing videos 53 Arcturus Laser Capture Microdissection System User Guide Contents CHAPTER 5 Selecting Cells for Microdissection 55 Summary of chapter topics 55 Marking cells for microdissection 56 Working with drawing items 2 58 Moving drawing items 58 Moving drawing items to a different Capture Group 58 Deleting drawing items 58 Deleting IR Capture Spots from a drawing item 59 Changing the microdissection properties of drawing items 59 Viewing information about a drawing item 60 Setting drawing tools options 60 Measuring distances and objects
44. 831 6844 then select option 5 ArcturusX Laser Capture Microdissection System User Guide 23 Chapter 2 Preparing Samples 24 Arcturus T Laser Capture Microdissection System User Guide CHAPTER 3 Starting the System and Loading Samples Chapter contents Summary of chapter topics 25 Introduction to the Nikon Eclipse Ti E microscope controls 25 Starting the ArcturusXT operating software 31 Loading materials onto the ArcturusXT Instrument 31 Saving images automatically 0 eee eee ee 35 Summary of chapter topics This chapter explains how to use the three operation panels on the Nikon Eclipse Ti E microscope the front panel the right panel and the left panel It explains which controls will be disabled when the Arcturus operating software application is running during an LCM session This chapter also provides instructions for starting the Arcturus T operating software and for loading samples on the ArcturusXT Instrument Finally it lists the steps for saving images automatically Introduction to the Nikon Eclipse Ti E microscope controls Using the front operation panel The Arcturus Instrument is built around a Nikon Eclipse Ti E research microscope There are three operation panels located on the microscope base each containing switches knobs and buttons which you use to cont
45. AL DAMAGES SUSTAINED BY THE BUYER OR ANY OTHER PERSON OR ENTITY WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES INCLUDING WITHOUT LIMITATION DAMAGES ARISING FROM OR RELATED TO LOSS OF USE LOSS OF DATA FAILURE OR INTERRUPTION IN THE Arcturus Laser Capture Microdissection System User Guide Appendix F Instrument Warranty Information OPERATION OF ANY EQUIPMENT OR SOFTWARE DELAY IN REPAIR OR REPLACEMENT OR FOR LOSS OF REVENUE OR PROFITS LOSS OF GOOD WILL LOSS OF BUSINESS OR OTHER FINANCIAL LOSS OR PERSONAL INJURY OR PROPERTY DAMAGE NO AGENT EMPLOYEE OR REPRESENTATIVE OF APPLIED BIOSYSTEMS HAS ANY AUTHORITY TO MODIFY THE TERMS OF THIS LIMITED WARRANTY STATEMENT OR TO BIND APPLIED BIOSYSTEMS TO ANY AFFIRMATION REPRESENTATION OR WARRANTY CONCERNING THE PRODUCT THAT IS NOT CONTAINED IN THIS LIMITED WARRANTY STATEMENT AND ANY SUCH MODIFICATION AFFIRMATION REPRESENTATION OR WARRANTY MADE BY ANY AGENT EMPLOYEE OR REPRESENTATIVE OF APPLIED BIOSYSTEMS WILL NOT BE BINDING ON APPLIED BIOSYSTEMS UNLESS IN A WRITING SIGNED BY AN EXECUTIVE OFFICER OF APPLIED BIOSYSTEMS THIS WARRANTY IS LIMITED TO THE BUYER OF THE PRODUCT FROM APPLIED BIOSYSTEMS AND IS NOT TRANSFERABLE Some countries or jurisdictions limit the scope of or preclude limitations or exclusion of warranties of liability such as liability for gross negligence or wilful misconduct or of remedies or damages
46. C or D in the Capture Groups list for the capture group you want to view 2 In the Drawing Items list tap or click the number of the item you want to see The stage moves so that the selected item is highlighted and centered in the main image window Setting formatting To set formatting properties of a capture group properties for a 1 Click the Information button in the upper right corner of the Select tools pane to Capture Group open the Select Options dialog box 2 Click the Capture Groups tab see Figure 49 aQ Select Options x Tools Options ll IR Spot Sizes Capture Groups Drawing Items Capture Groups D 3 Click the radio button for the capture group of interest Group A OB OC On 4 Inthe Attributes area set options A for the drawing items within the Name Capture group Annotation Default capture group current capture group IR SpotColor E Rea UV CutColor EM Red aa Ta FiLIR S N Information supplied in this field ac s appears in the cap interaction history Name The name of the capture group If you use the Default capture group then enter these fields a Name The name of the x canei capture group This appears in the cap interaction history Figure 49 Capture Groups tab b Annotation This appears in the cap interaction history c IR SpotColor The color of the IR capture spots in the main image d UV CutColor The color of the UV cut line i
47. Capture Microdissection System User Guide 65 Chapter 5 Selecting Cells for Microdissection 66 Arcturus T Laser Capture Microdissection System User Guide CHAPTER 6 Microdissecting Cells and Tissue Chapter contents Summary of chapter topics 67 Capturing cells by microdissection 68 Inspecting microdissected material 71 Unloading materials ici sce Gia e is as teens 72 Locating the lasers 73 Selecting preferences for cut and capture 75 Working With Caps essuie maar een siens a 77 Using the Laser Bypass feature 78 Summary of chapter topics This chapter explains how to microdissect cells and tissue inspect caps and slides after microdissection and unload the caps and slides It also tells you how to locate both the IR Capture Laser and the UV Cutting Laser and how to set parameters for IR capture and for UV cutting It covers setting the order and selecting properties for cut and capture as well as viewing and updating cap information It explains how and when to use the laser bypass feature You access all of these features from the Microdissect tools pane see Figure 50 Microdissect a Information button ser Place cap Laser Bypass a lid IR laser test
48. If you do not click the OK at the bottom of the dialog box the changes will not be saved You can also locate the IR laser on the top level of the software user interface To locate the IR laser 2 Fire the IR laser using the IR Capture Test Spot icon located in the Microdissect tool pane 3 Place the cursor at the center of the wetted IR spot right click and select IR Laser Capture Laser Here The blue plus sign will relocate itself to the center of the LCM spot Selecting preferences for cut and capture This section explains how to switch the order of the cut and capture tasks and how to set up the properties for the cut and capture Changing the cut By default the Arcturus T Instrument performs an IR capture first followed by UV and capture order cutting Performing an IR Capture first can be beneficial such as when using HS Caps which are farther from the surface of the tissue or when performing live cell applications using frame membrane slides After the IR laser has adhered the cells of interest to the CapSure cap the UV laser can be fired to cut around the areas of interest For some applications you may want to fire the UV laser first You can choose the order in the Arcturus software ArcturusX Laser Capture Microdissection System User Guide 75 o Chapter 6 Microdissecting Cells and Tissue To set the order of cutting and capture Setting properties for cut and capture 76 1 To set properties
49. Prefix and the SuffixNumber for each image of the slide before microdissection 8 In the LabelSpecimenAfter field enter the text that you want to add after the AutodocPrefix and the SuffixNumber for each image of the slide after microdissection For example for the second image of a slide before LCM with the AutoDocPrefix LeafStudy the LabelSpecimenBefore Before and the SuffixNumber 10 the file is named LeafStudyBefore10 ArcturusX Laser Capture Microdissection System User Guide 35 Chapter 3 Starting the System and Loading Samples 36 Arcturus T Laser Capture Microdissection System User Guide CHAPTER 4 Inspecting Slides Chapter contents Summary of chapter topics 37 Using the Inspect tool Skersio a ea e aa E o8a4 37 Working with the Bright Field lamp 40 Performing phase contrast and DIC imaging 43 Working with fluorescence 47 Summary of chapter topics This chapter explains how to move around a slide to view the sample and how to work with the microscope and the fluorescence lamp It also explains how to capture static images GED and movies Using the Inspect tools After you have loaded the slides and caps you use the tools in the Inspect tools pane to view the slides and to identify the cells you want to microdissect These tools allo
50. System User Guide Using formalin fixed paraffin embedded tissue samples Using other types of samples Chapter 2 Preparing Samples Formalin fixed paraffin embedded tissue can also be microdissected for downstream applications To see suggested protocols based on the experience of other customers see www appliedbiosystems com For gene expression profiling studies using FFPE tissue we recommend using the Paradise PLUS Reagent System This system provides all of the reagents for sample preparation RNA extraction and isolation reverse transcription and linear amplification of the RNA For optimal sample preparation of FFPE tissue samples and downstream processing we recommend the reagent systems listed in Table 7 Table 7 Sample preparation kits for formalin fixed paraffin embedded tissue samples Reagent System Part Number Paradise PLUS Reagent System 2 rounds of KIT0312 amplification Kit Paradise PLUS Whole Transcript Reverse KITO315 Transcription WT RT Kit Note For a complete list of Arcturus microgenomics reagents see Appendix E Laser Capture Microdissection has been used for a variety of research applications besides tissue microdissection including forensics live cells neurons live plant tissue and single chromosomes You can download application notes and protocols related to the use of other types of samples from www appliedbiosystems com You can also call technical support Call 1 800
51. These kits provide detailed step by step protocols for extracting DNA and RNA from frozen cells e PicoPure RNA Isolation Kit 40 purifications cat KIT0204 e PicoPure RNA Isolation Kit 200 purifications cat KIT0214 e PicoPure DNA Extraction Kit cat KIT0103 The Arcturus Paradise PLUS Reagent System also from Life Technologies is designed specifically for extracting RNA from formalin fixed paraffin embedded tissue For more information about related instruments and Arcturus Microgenomics reagents kits see www appliedbiosystems com ArcturusX Laser Capture Microdissection System User Guide 79 Chapter 7 Extracting Cells and Tissue Extracting tissue from CapSure Macro LCM Caps Following microdissection you can place CapSure Macro LCM Caps directly onto a 0 5 mL microcentrifuge tube containing extraction buffer For best results Life Technologies recommends MicroAmp Autoclaved Thin Walled Reaction tubes available from Applied Biosystems PN N8010611 To extract tissue using the PicoPure RNA Isolation Kit or the PicoPure DNA Extraction Kit you can refer to the appropriate PicoPure User Guide or you can follow these instructions 1 Place at least 40 uL of extraction buffer into a 0 5 mL microcentrifuge tube Note Less than 40 uL of buffer may not provide enough volume to cover the surface of the CapSure Macro LCM Cap 2 Use the CapSure insertion tool to insert the CapSure Macro LCM Cap into the microce
52. To maneuver the stage use the trackball provided with the Arcturus Instrument Use the buttons on the trackball to change the objective Note If you have 60x or 100x objectives installed on the Arcturus Instrument make sure that you have proper stage clearance before changing objectives using the trackball buttons Arcturus Laser Capture Microdissection System User Guide 19 Chapter 1 System Overview 20 Arcturus T Laser Capture Microdissection System User Guide CHAPTER 2 Preparing Samples Chapter contents Summary of chapter topics 21 Choosing slides and Petri dishes 21 Preparing tissue samples 22 Summary of chapter topics This chapter explains which types of slides you can use for laser microdissection and which type to select based on your application It also explains how to prepare tissue for laser microdissection with the suggested reagent kits Choosing slides and Petri dishes With the Arcturus Instrument you can use glass slides PEN membrane glass slides PEN membrane frame slides and Petri dishes Here are our recommendations e For applications that use the UV cutting laser use either PEN membrane frame or PEN membrane glass slides e For live cell applications use an untreated PEN membrane frame slide or the Arcturus Live Cell Growth Chamber and Microdissec
53. Troubleshooting Chapter contents Summary of chapter topics 83 Cleaning the ArcturusXT Instrument 83 Replacing user serviceable parts 83 Troubleshooting tips 86 Summary of chapter topics This chapter contains instructions for cleaning the Arcturus T Instrument information about parts that you can service yourself and tips for troubleshooting IMPORTANT Do not attempt to remove the covers from the instrument except as specified for the user serviceable parts If the instrument requires service contact your Life Technologies service representative Cleaning the Arcturus Instrument To keep the instrument clean Clean the outside of the instrument using a damp cloth Do not use any solvents or abrasives Clean the work surface as necessary by wiping it with a cloth moistened with ethanol Replacing user serviceable parts The Arcturus Instrument has only four parts that you can service yourself Bright field illumination lamp Interchangeable fluorescence filter cube if your instrument has one Fluorescent lamp if your instrument has one Fuse Instructions for replacing these components are provided here For other service needs contact your Life Technologies service representative Arcturus Laser Capture Microdissection System User Guid
54. ace a cap on the slide The instrument places a cap at the center of the field of view disignated by the red box in the slide overview image The cap location is outlined in green The entire area inside the circle is available for capture 2 Locate the laser s as appropriate a See Locating the UV cutting laser on page 73 b See Locating the IR capture laser on page 74 3 Move the stage so the cap is on an area away from the tissue but still within the cap 4 Click the IR Laser Test Fire button to test the capture laser 5 Inspect the laser spot You should see a dark ring with a clear center as shown in Figure 48 on page 62 If you do not see this open the Microdissect Options dialog box adjust the Power and duration slider in the IR Spot Sizes tab Microdisecting the Sample 1 Click the UV Cut button to perform all cuts 2 Click the IR Capture button to perform all captures Note If you are using DIC the DIC prism slider will need to be removed before proceeding Follow the instructions provided in the pop up window 3 Inspect the cap and slide See Inspecting microdissected material on page 71 Repeating If you want to repeat the microdissection if no new drawing items have been placed microdissection and the cap has not been moved from its original position 1 Click the UV Cut button to repeat all cuts 2 Click the IR Capture button to repeat all captures 3 Ifthe microdissection was not co
55. ale Environmental symbols on instruments The following symbol applies to all Applied Biosystems electrical and electronic products placed on the European market after August 13 2005 Symbol Description Do not dispose of this product as unsorted municipal waste Follow E local municipal waste ordinances for proper disposal provisions to reduce T the environmental impact of waste electrical and electronic equipment WEEE t European Union customers Call your local Applied Biosystems Customer Service office for equipment pick up and recycling See www appliedbiosystems com for a list of customer service offices in the European Union Arcturus T Laser Capture Microdissection System User Guide Safety labels on instruments General instrument safety Appendix C Safety Please note the warning labels and symbols on the instrument They are shown here CAUTION CLASS 3B INVISIBLE LASER RADIATION WHEN OPEN AND INTERLOCKS DEFEATED AVOID EXPOSURE TO THE BEAM NOTE LASERS ARE DISABLED UNLESS THE ILLUMINATION ARM IS FORWARD AND THE CAP OR LASER INTERLOCK OVERRIDE IS ENABLED Attention Laser invisible classe 3B Exposition la radiation si ouvert et couplages neutralises vitez l exposition au rayon CAUTION CLASS 3B INVISIBLE LASER RADIATION WHEN OPEN AVOID EXPOSURE TO THE BEAM Attention Laser invisible classe 3B Exposition a la radiation si ouvert Evitez l exposition au rayon
56. ane or on the IR Spot Size in the Select tools pane Working with caps This section explains how to view caps in the QC area how to view and update cap properties and how to view the Cap Interaction History Viewing a cap In To view a cap in the QC area the QC area 1 Place the cursor to the right of the slide overview image right click on the Cap button for the cap of interest in the QC cap position see Figure 56 2 Select View Cap at QC Station from the pop up menu The stage will move the cap over the objective You can view the cap and any microdissected material on the cap You can also capture static images of the cap Note The highest objective allowable at the QC position is 20X If you want to inspect the material on the cap with a higher objective move the cap to a clean place on the slide and follow standard protocols for inspection Arcturus Laser Capture Microdissection System User Guide 77 6 Chapter 6 Microdissecting Cells and Tissue Viewing and updating cap properties Viewing the cap interaction history You can view the cap type in the cap and slide handling area to the left of the slide overview image If you did not select the Load Caps Slides correct cap type in the Load Options dialog box when you ae BE loaded your caps you can change it here ML You can also add caps to the Load Caps area by clicking a cap in the Load area here see Figure 56 This shortcut lets
57. ap position minute when the file was created The file is saved to the location specified in the ReportSubfolder in the Load Options dialog box 2 Inspect the tissue on the cap 3 Optional You can capture a static image at this point The highest objective allowable at the QC position is 20X If you want to inspect the cap with a higher objective move the cap to a clean place on the slide and follow standard protocols for inspection 4 To inspect the slide click the slide overview image at the position you want to see in the main image window or click an item number in the Drawing Items list in the Select tools pane The stage will move as needed to display the item in the main image window 5 If any drawing item was not captured during microdissection a Move the cap from the QC station back to the slide by placing the cursor on the cap in the QC Caps area to the right of the overview image Right click and select Replace Cap on Slide from the pop up menu b Follow the steps outlined in Repeating microdissection on page 70 to recut the drawing items ArcturusX Laser Capture Microdissection System User Guide 71 6 Chapter 6 Microdissecting Cells and Tissue Unloading materials 72 To unload materials 1 Click Present Stage in the Setup tools pane The work surface is displayed Slide the cap insertion tool onto the work surface Make sure the open end of the insertion tool faces the cap in the QC stat
58. applied Dos stems by Life technologies ArcturusT Laser Capture Microdissection System Nikon Eclipse Ti E Microscope Base User Guide November 2010 For Research Use Only Not intended for any animal or human therapeutic or diagnostic use Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Nikon and Eclipse are trademarks of Nikon Corporation Pentium is a trademark of Intel Corporation Windows is a trademark of Microsoft Corporation Limited Use Label License The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purch
59. apture a video make sure AutoDocument is tumed off in the SetUp option dialog box If you have turned on the AutoDocument feature the video feature will not work as the live image feed is frozen when th ArcturusX Laser Capture Microdissection System User Guide 53 Chapter 4 Inspecting Slides 54 Arcturus T Laser Capture Microdissection System User Guide Chapter contents Summary of chapter topics Marking cells for microdissection Working with drawing items Measuring distances and objects Setting the IR Capture Spot size Working with overlays Working with Capture Groups Working with stored positions Summary of chapter topics This chapter explains how to indicate the cells and tissue you want to microdissect how to use the drawing tools how to measure a distance or object size in the main CHAPTER 5 Selecting Cells for Microdissection image window and how to set the size of the IR capture spots inside your samples It also explains how to use overlays capture groups and stored positions You will access all of these features from the Select tools pane see Figure 42 Freehand Drawing Select Objects s ___ BE Define Circle area Eraser Single IR Spot Ruler IR Spot Line IR Spot Sizes control Stored Posiitons controls autoScanxT 22 f Stored Positions lue pas pa Figure 42 The Select tools pane ArcturusX Laser Capture Microdissection System
60. ared from either frozen or formalin fixed paraffin embedded FFPE tissue Freezing tissue helps to ensure the integrity of the biological molecules within the cells Thus cells microdissected from frozen tissue sections provide material that is suitable for many downstream applications This is especially true for molecular biology applications requiring intact RNA While the integrity of the RNA from formalin fixed tissue may not be as optimal as that from frozen tissue using the recommended protocols and reagents will allow you to use these samples for molecular biology applications as well Life Technologies provides an application note describing the recommended protocol for working with frozen samples Application Note 1 Optimized Protocol for Preparing and Staining LCM Samples from Frozen Tissue and Extraction of High Quality RNA Use this link to access this information Arcturus Literature For optimal preparation and processing of frozen tissue samples we recommend the reagent kits listed in Table 6 Table 6 Reagent kits for processing frozen tissue samples Reagent Kit Part Number HistoGene LCM Frozen Section Staining Kit KITO401 HistoGene LCM Immunofluorescence Staining Kit KITO420 PicoPure RNA Isolation Kit KITO204 PicoPure DNA Extraction Kit KITO103 RiboAmp PLUS RNA Amplification Kit KITO521 RiboAmp PLUS High Sensitivity RNA Amplification Kit KITO525 Arcturus T Laser Capture Microdissection
61. aser No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing dlifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 This product is covered under one or more of U S Patent Nos 5 843 644 5 843 657 6 010 888 6 204 030 6 251 467 6 251 516 6 569 639 2010 Life Technologies Corporation All rights reserved Part Number 0112 0153 Rev C 11 2010 Contents About This Guide 2 12 4003 teste 9 Purp os of this quide 2 22 fes mme Sesion ea IDa TAa ha the as 9 Prerequisites 2 25 pute ame Tente whee E EA bn en Bea ie ue hee ER a 9 Safety informations ele ee a Lanterne ane one np nent Re tete 9 Safety alert Words 4 esi 2 nn nn Renan ann e A eda eded glad abate Seat dns 9 Safety labels on instruments 10 CHAPTER 1 System Overview 11 About the Arcturus Instrument 11 About laser capture microdissection 11 Types of cut a
62. asures should include the following e Do not insert reflective surfaces into the beam path e Wear protective eye wear that blocks 349 nm and 810 nm radiation with optical density gt 2 5 e Post the following warning outside of the room when the instrument is being operated with the interlock overridden CAUTION CLASS 3B INVISIBLE LASER RADIATION AVOID EXPOSURE TO THE BEAM 810 nm 100 mW 349 nm 60 uJ variable pulse frequency 10 5000 Hz Arcturus Laser Capture Microdissection System User Guide 105 Appendix C Safety Workstation safety Safety and electromagnetic compatibility EMC standards 106 Additional laser safety information Refer to the user documentation provided with the laser for additional information on government and industry safety regulations AA WARNING LASER HAZARD Lasers can burn the retina causing permanent blind spots Never look directly into the laser beam Remove jewelry and other items that can reflect the beam into your eyes Do not remove the instrument top or front panels Wear proper eye protection and post a laser warning sign at the entrance to the laboratory if the top or front panels are removed for service Ps WARNING LASER BURN HAZARD An overheated laser can cause severe burns if it comes in contact with the skin Correct ergonomic configuration of your workstation can reduce or prevent effects such as fatigue pain and strain Minimize or eliminate these effects by
63. at its published rates Applied Biosystems also provides service agreements for post warranty coverage Applied Biosystems reserves the right to use new repaired or refurbished instruments or components for warranty and post warranty service agreement replacements Repair or replacement of products or components that are under warranty does not extend the original warranty period Applied Biosystems warrants that all optional accessories supplied with its Arcturus Laser Capture Microdissection LCM System such as peripherals printers and special monitors will be free of defects in materials and workmanship for a period of ninety 90 days from the date the warranty begins Applied Biosystems will repair or replace at its discretion defective accessories during this warranty period After this warranty period Applied Biosystems will pass on to the buyer to the extent that it is permitted to do so the warranty of the original manufacturer for such accessories With the exception of consumable and maintenance items replaceable products or components used on or in the instrument are themselves warranted to be free of defects in materials and workmanship for a period of ninety 90 days Applied Biosystems warrants that chemicals and other consumable products will be free of defects in materials and workmanship when received by the buyer but not thereafter unless otherwise specified in documentation accompanying the product Arcturus
64. cipants should coordinate lift and move intentions with each other before lifting and carrying Instead of lifting the object from the packing box carefully tilt the box on its side and hold it stationary while someone slides the contents out of the box Arcturus Laser Capture Microdissection System User Guide 103 Appendix C Safety Physical hazard safety Electrical safety 104 Operating the instrument Ensure that anyone who operates the instrument has e Received instructions in both general safety practices for laboratories and specific safety practices for the instrument e Read and understood all applicable Safety Data Sheets SDSs Cleaning or decontaminating the instrument AN CAUTION Before using a cleaning or decontamination method other than those recommended by the manufacturer verify with the manufacturer that the proposed method will not damage the equipment Ultraviolet Light WARNING ULTRAVIOLET LIGHT HAZARD Looking directly at a UV light source can cause serious eye damage Never look directly at a UV light source and always prevent others from UV exposure Follow the manufacturer s recommendations for appropriate protective eyewear and clothing Moving Parts x WARNING PHYSICAL INJURY HAZARD Moving parts can crush and cut Keep hands clear of moving parts while operating the instrument Disconnect power before servicing the instrument A WARNING PHYSICAL INJURY HAZARD Do not operate the in
65. ck the Copy button and save the csv file to the appropriate location Click OK Spots Cut rea Captured 6 37 590 5 51 158 2 4 873 73 1 1 1 99 Figure 44 Drawing item information Note This information is also available in the information box located to the right of the slide overview Click on a drawing item in the Select tool pane to display the information To set options for the Eraser Freehand Drawing and Defined Circle Area tools 1 Click the Information button i in the upper right corner of the Select tools pane to open the Select Options dialog box Click the Tools Options tab In the Tools Options tab set the following options as desired e Select the Eraser size Click the button corresponding to the size for the Eraser tool e Select the Snap to End Size Click the button to indicate the distance for the two ends of a shape drawn by the Freehand tool to automatically snap together and close the drawing item Figure 45 Tools options tab Arcturus Laser Capture Microdissection System User Guide Chapter 5 Selecting Cells for Microdissection e Define a Custom Circle Check Use Custom Circle to set the Circle tool to draw a circle of a fixed diameter In the Diameter field enter the diameter for the custom circle in microns To draw a custom circle tap the Circle tool and then tap the main image window where the circle should be placed 4 Click OK to c
66. ction explains how to prepare for and perform DIC imaging interference contrast DIC imaging For optimal DIC image quality ensure that all components are properly aligned Prior to starting an experiment using DIC 1 Click on the DIC icon in the Inspect tool pane The diffuser will move to the Out position The diffuser must be in the Out position for proper DIC imaging Getting set up for DIC imaging 2 Ensure that both the field aperture and the condenser aperture are fully open 3 Remove the DIC slider from beneath the objective see Figure 32 Figure 32 DIC slider prism _ 4 QED ct the objective nosepiece fluorescence turret 5 Make sure the analyzer is set at O as shown in Figure 33 It should be locked in place Note If you have purchased the DIC Analyzer Cube in conjunction with the motorized fluorescence filter turret when you click on the DIC button the DIC Analyzer Cube moves into Figure 33 DIC analyzer position automatically If you are using the DIC Analyzer Cube you do not need to use the sliding analyzer ArcturusX Laser Capture Microdissection System User Guide 45 Chapter 4 Inspecting Slides 9 above the condenser turret and set the index mark to 0 as shown in Figure 34 Loosen the condenser turret set screw and rotate until the darkest image lowest brightness is achieved This is the extinction point Tighten the screws to set the condenser to this
67. d behind the front cover of the UV laser housing on the left side of the instrument Note Instruments with the Enhanced UV Laser do not have neutral density filters They have software controlled UV Laser Current and UV Pulse Frequency control Tissue is thick or fibrous Reduce the UV cutting speed or for systems with the Enhanced UV Laser increase UV Laser Current located in the Microdissect tool pane UV Cutting is inefficient Cutting speed Reduce the UV cutting speed using the slide bar located in the Microdissect Tool pane Double UV Cut line appears DIC prism slider is in place Remove the DIC prism slider from beneath the objective prior to firing the UV laser Note The DIC prisms sliders should only be in place during DIC visualization For optimal visualization using brightfield fluorescence or phase contrast and for optimal UV cutting ensure that the sliders prisms have been removed from the objectives Table 12 Possible problems with image quality SYMPTOM POSSIBLE CAUSE REMEDY Long lag time when updating the live image Light intensity or camera gain settings are not optimized 1 Check the Brightness setting located in the Microscope tool panel It should be gt 0 200s 2 Open the Select dialog box and re adjust intensity or camera gain to achieve an appropriate brightness setting Arcturus T Laser Capture Microdissection System User Gu
68. d on 2 Open the Inspect Options dialog box e k The Fluorescence tab is TI E selected automatically Microscope EEL lamp RSR Lock 3 Make sure that the intensity SS U E Unlock c g Camera gain 1023 y amera gain unchecked see Figure 36 Fluorescence uorescence Settings lamp Intensity 100 4 Manually rotate the intensity ee fluorescence filter Timed q a turret to the desired Gain for Etre 600 filter cube timed exposure OK Cancel Figure 36 Fluorescence tab Note If you have the motorized fluorescence filter turret in place click on the appropriate filter mel w o button see Figure 37 at the bottom of the Inspect pane see Figure 21 on page 37 to rotate the filter Figure 37 Filter buttons turret active 5 Locate the fluorescence signal a Set the Fluorescence Lamp Intensity to 100 see Figure 36 b Set the Camera Gain to the maximum setting see Figure 36 c exposure time in the Inspect panel until a fluorescence signal is seen see Figure 38 Brighiness d Focus the image e If needed check the Microscope Lamp on see Figure 36 domes and adjust the intensity to allow minimal brightfield light 6 Once the sample has been located and focused make these adjustments to optimize the image Figure 38 Brightness a CR Figure 36 to thelowest pere possible value to still allow sufficient signal Higher
69. d tap on multiple objects tap on one of the selected drawing items and right click Then choose Delete Objects s from the pop up menu Arcturus Laser Capture Microdissection System User Guide Chapter 5 Selecting Cells for Microdissection To delete all drawing items from the active capture group e Press Ctrl A on the keyboard and then press Ctrl X on the keyboard or e Click on the Delete All button located in the Select tool panel below the drawing items list Deleting IR Capture To delete IR capture spots from a drawing item Spots from a 1 Place the cursor on an IR Capture Spot drawing item and right click the IR spot 2 In the pop up menu select either Delete Spot to delete the selected IR capture i pe MN Delete Spots In Object spot or Delete Spots in Object to delete NE Lom ony all the IR capture spots in the drawing AAO Move Obiect s To item 2 ti io SEL IG The specified IR Capture Spots are Figure 43 Deleting IR Capture Spots deleted Changing the To change from LCM IR Capture only to UV Cut and IR Capture microdissection 1 Place the cursor on the drawing item and right click properties of drawing items 2 Select LCM Only in the pop up window e The check mark will disappear next to LCM Only e The LCM spots will remain in the object but the perimeter line will change to the UVCutColor designated in the Capture Group settings window e The IR and UV lasers will both fire for this object Not
70. e To reduce the number of LCM spots in the object right click within the object and select Delete Spots in Object and then manually place as many LCM spots as desired to attach the area to the CapSure cap To change from UV Cut and IR Capture to LCM Only 1 Place the cursor on the drawing item and right click 2 Select LCM Only in the pop up window e A check mark will appear next to LCM Only e The object will be filled in with LCM spots and the outline of the object will change to the IRSpotColor designated in the Capture Group settings window e Only the IR laser will fire for this object TTM Arcturus T Laser Capture Microdissection System User Guide 59 Chapter 5 Selecting Cells for Microdissection Viewing information about a drawing item Setting drawing tools options 60 You can see the properties such as the number of IR capture spots or capture area for any of the drawing items you have drawn To view information about drawing items 1 Click the Information button i in the upper right corner of the Select tools pane to open the Select Options dialog box see Figure 44 Click the Drawing Items tab to view the properties of interest There is a row for each drawing item in the selected capture group You can view the area of the item its status and other information To view information for another capture group select the button for that group To export this data chart cli
71. e 83 8 Chapter 8 Maintenance and Troubleshooting Replacing the bright field illumination lamp Replacing fluorescence filter cubes 84 Depending upon the model of your instrument there are different instructions for replacing the bright field illumination lamp Replacing the 100 W halogen lamp You will need the replacement lamp a 2 mm hex driver a slotted screw driver clean powder free gloves and the instructions from the microscope manufacturer for this procedure Follow the Nikon microscope instructions provided with the ArcturusXT Instrument to replace the lamp Replacing the high intensity LED You will need the replacement LED a 2 Phillips screwdriver a 2 5 mm hex key and clean powder free gloves for this procedure To replace the bright field illumination lamp 1 If necessary exit the Arcturus software 2 Turn the instrument off and unplug it When you are facing the instrument the switch is located on the left side The plug is on the back of the instrument 3 Remove the top cover of the instrument There are four screws on the top of the cover and one screw on the left side Unscrew the cover and lift it up and off Place the cover in a safe location Use the hex key to unscrew the two screws holding the LED Unplug the LED cable it has a white connector and remove the LED Replace the LED connecting the cable and replacing the two screws Replace the top cover and then tighten the screws
72. e Arcturus Instrument The ArcturusXT Instrument has a manual fluorescence filter turret that you rotate by hand Moving the nosepiece using the Focus knob Use the Focus knob to move the nosepiece up and down for focus adjustment If you use the Focus knob while using the Arcturus Instrument the software will track the position The numbers displayed in the Arcturus software interface will continue to match the numbers displayed in the Status Display window in the front operation panel Returning to the original position with the Refocus button Press the Refocus button to move the nosepiece and objective back to its original position after you have pressed the Escape button After you have pressed the Refocus button you can use the Focus knob for manual focus control Moving to the retracted position with the Escape button Press the Escape button to move the nosepiece and objective to the retracted position which is approximately 2 mm below the reference position When you press the Escape button the current position is recorded so that the nosepiece can return to this position when you press the Refocus button Arcturus Laser Capture Microdissection System User Guide Chapter 3 Starting the System and Loading Samples Starting the Arcturus operating software To follow these instructions you can use the interactive pen display supplied with the Arcturus Instrument or you can use the mouse To begin laser microdissection
73. e LCM Cap area However there may be applications for which the cap is not desired or required In such instances it is necessary to bypass the instrument laser safety settings In order to bypass the laser safety mechanism the laser bypass key must be in place in the back of the instrument You must then depress the Laser Bypass button see Figure 50 on page 67 activating the UV laser For a full description of laser status indicators as observed by color changes of the Laser Bypass button see Appendix D Arcturus T Laser Capture Microdissection System User Guide CHAPTER 7 Extracting Cells and Tissue Chapter contents Summary of chapter topics 79 Choosing an extraction kit 79 Extracting tissue from CapSure Macro LCM Caps 80 Extracting tissue from CapSure HS Caps 80 Summary of chapter topics After you have microdissected cells you can extract the biomolecules from the samples Depending on the type of CapSure Cap you used the procedure varies This chapter contains suggestions for appropriate extraction kits and instructions for extracting microdissected tissue from the CapSure Macro LCM Caps and the CapSure HS LCM Caps Choosing an extraction kit Life Technologies offers two extraction kits specifically designed to work with the CapSure LCM Sample Preparation System
74. e Overview Image 34 Refocus button 30 repetitive motion safety 106 replacing fluorescent filters 84 reporting damages 119 returning damaged items 119 returns 119 RiboAmp PLUS RNA Amplification Kit 115 S safety before operating the instrument 103 electrical 104 ergonomic 106 Arcturus Laser Capture Microdissection System User Guide instrument operation 104 laser 105 moving and lifting computers and monitors 103 moving and lifting instrument 103 moving parts 104 physical hazard 104 repetitive motion 106 standards 106 ultraviolet light 104 workstation 106 safety labels on instruments 10 103 safety standards 106 SavelmageOverlay field 34 Select tools 55 SlideName field 32 SlideNotes field 32 stand alone microscope 19 standards EMC 106 safety 106 static images 52 Status Display window 25 StudyFolder field 33 stylus 14 SuffixNumber field 35 T technical support for computers with altered configuration 117 tiled images 51 tool tips 14 training information on 122 Triple Dichroic Filter Set 95 Turbo Labeling Kits 115 U ultraviolet light safety 104 UV Cube 95 UV Cutting Laser specifications 91 UV Cutting Laser IR 73 V version number 17 VideoSubfolder field 33 viewing a cap 77 Arcturus Laser Capture Microdissection System User Guide Index W WARNING description 9 warranty damages claims returns 119 exceptions 118 for computers with altered configuration 117 period 117 wor
75. e ea ge ees 73 Locating the UV cutting laser 73 Locating the IR capture laser 74 Selecting preferences for cut and capture 75 Changing the cut and capture order 75 Setting properties for cut and capture 76 Working WIth Caps erep Tan en a ere eel te A de ARR 77 Viewing a cap In the QC area 77 Viewing and updating cap properties 78 Viewing the cap interaction history 78 Using the Laser Bypass feature 78 Arcturus Laser Capture Microdissection System User Guide 5 Contents CHAPTER 7 CHAPTER 8 APPENDIX A APPENDIX B Extracting Cells and Tissue 79 Summary of chapter topics 79 Choosing an extraction kit 2 79 Extracting tissue from CapSure Macro LCM Caps 80 Extracting tissue from CapSure HS Caps 80 Performing LCM captures with CapSure HS Caps 80 Extracting tissue 4 25209 an Anne ceive neta Re bytes AS ceva nt 81 Maintenance and Troub
76. e from the File menu Command Submenu Description Open Image Opens a saved image file The image appears in a new window Markup Opens a saved markup file Save Image Saves the image currently visible in the main image window to a file Markup Saves the current drawing item as an overlay Exit Closes the software Arcturus Laser Capture Microdissection System User Guide 17 Chapter 1 System Overview Edit menu commands and submenus Key commands 18 Table 2 Functions available from the Edit menu Command Submenu Description Paste Selected Pastes the currently selected drawing items Object s Delete Deletes the currently selected drawing items Selected Object s Select Objects In Current Selects all drawing items in the current capture group Capture Group In Capture Group Allows you to choose the A B C or D capture group Selects all drawing items in the specified capture group In Al Capture Selects all drawing items irrespective of their capture Groups group Move Selected A Moves the selected objects to the capture group in the Object s to B submenu Group C D View menu commands and submenus Table 3 Functions available from the View menu Command Description Scale Displays a scale bar on the main image Change to Moves the tools pane from the right side of the software to the left side Left hand Orientati
77. e laser operation e The system must be installed and maintained by an Applied Biosystems Technical Representative e Allinstrument panels must be in place on the instrument while the instrument is operating When all panels are installed there is no detectable radiation present If any panel is removed when the laser is operating during service with safety interlocks disabled you may be exposed to laser emissions e Do not remove safety labels or disable safety interlocks Safety interlock system The Arcturus LCM System incorporates an interlock system that enables laser operation only when the cap is in place the interlock switches are not defeated or bypassed and the illumination tower is not tilted Do not modify or override the tilt interlock It is possible to override the cap interlock system and operate the lasers when the cap is not in place To do this the interlock override key must be inserted in the instrument s control unit and the laser must be enabled using the software controls With the override key in place it is possible for a reflective surface to be introduced in the space between the objective and illumination tower which can deflect the laser beam out of the instrument and allow human exposure to hazardous laser radiation WARNING Contact technical support for information on using the interlock override key Users should not override the interlock without adequate training to ensure safe operation Safety me
78. eded by tapping or clicking 10x the label corresponding to the chosen objective The selected objective is red see Figure 22 Slider Bar 4x Note If you drag the slider bar up between objectives E the Arcturus Instrument will zoom the image 2x digitally oi Figure 22 Objective control The brightness knob on the left operation panel of the x microscope is disabled when the Arcturus T software is Brightness running You control brightness using the software buttons shown in Figure 23 g 864s To adjust the brightness as needed to illuminate the sample e Click or tap the up arrow to increase the brightness e Click or tap the down arrow to decrease the brightness Figure 23 Brightness Note If you press the stylus down on the arrow buttons dnt ol the software adjusts the brightness in larger steps e Click the Autobrightness button in the middle to adjust the brightness automatically To set a different level of brightness for the Autobrightness button see Changing the autobrightness settings on page 42 camera If the value in the brightness control i he time needed for the main image window to refresh after you move the stage may be slow If this is the case adjust the Intensity and Camera Gain in the Illumination tab in the Inspect Options dialog box see Figure 26 on page 40 so that you can set the brightness value lower Note The brightness value corresponds to the shutter 7 vie time of the Arcturus Las
79. ent To obtain a copy of this procedure for your records call Life Technologies technical support at 1 800 831 6844 and select option 5 Installing software upgrades Each version of the software is accompanied by detailed instructions for updating an existing installation Follow the update instructions distributed with the software The latest version of Arcturus T operating software is available on the Life Technologies website at www appliedbiosystems com ArcturusX Laser Capture Microdissection System User Guide 99 Appendix B Installation Instructions 100 Arcturus T Laser Capture Microdissection System User Guide APPENDIX C Safety Instrumentation Safety Symbols on Electrical symbols on instruments Instruments The table below describes the electrical symbols that may be displayed on Applied Biosystems instruments Symbol Description Indicates the On position of the main power switch Indicates the Off position of the main power switch Indicates a standby switch by which the instrument is switched on to the Standby condition Hazardous voltage may be present if this switch is on standby Indicates the On Off position of a push push main power switch Indicates a terminal that may be connected to the signal ground reference of another instrument This is not a protected ground terminal Indicates a protective grounding terminal that must be connected to earth ground before any other e
80. er Capture Microdissection System User Guide Chapter 4 Inspecting Slides Focusing the image Use the Focus controls to focus the live image Focus e Click or tap the up arrow to move the objective closer to the slide e Click or tap the down arrow to move the objective farther 1334 from the slide microns Note If you press the stylus down on the arrow buttons the software adjusts the focus in larger steps Figure 24 Focus controls e Click the Autofocus button in the middle to adjust the focus automatically If the Z Reset button located on the front panel of the microscope has been pushed the focus position indicated on the LED readout will not match the focus position on the Arcturus T software To have the numbers coincide once again you must turn off the Eclipse Ti E microscope then turn it on again and restart the Arcturus software You can set up the instrument to Illumination Fluorescence Focus Magnification automatically focus the live image each time you change the objective To set up automatic fo cusing When Tracking Auto Focus is ON Auto Focus will run automatically after changing objectives and before acquiring slide overview 1 Click the Information i button in the upper right corner of the Tracking Autofocus On Inspect tools pane see Figure 21 on page 37 to open the Inspect Options dialog box 2 Click the Focus tab see Figure 25 3 Check Tracking Autofocus On
81. ered while using the Arcturus Instrument Arcturus PicoPure RNA 12682 Provides instructions for using the Arcturus Isolation Kit User Guide OOARC PicoPure RNA Isolation Kit Rev A Arcturus HistoGene 12294 00 Provides instructions for using the Arcturus Frozen Section Staining Kit Rev C HistoGene Frozen Section Staining Kit User Guide Arcturus HistoGene 12653 00 Provides instructions for using the Arcturus Immunofluorescence Rev C HistoGene Immunofluorescence Staining Kit Staining Kit User Guide Note To open the documentation included on the Installation CD use the Adobe Reader software available from www adobe com How to obtain support For the latest services and support information for all locations go to www appliedbiosystems com At the Applied Biosystems website you can e Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Order Applied Biosystems user documents SDSs certificates of analysis and other related documents e Download PDF documents e Obtain information about customer training e Download software updates and patches 122 Arcturus T Laser Capture Microdissection System User Guide A Annotatelmage field 34 AutoDocPrefix field 35 AutoDocument field 35 AutoDocument Filename Settings 34 base station 93 Base U
82. ew the phase plate Annular diaphragm image black ring through the microscope oculars If needed focus the ring by rotating the screw located on the eyepiece turret to the right of the Bertrand lens selection see Figure 30 Contrast ring image Rotate into the light path Figure 30 Phase ring focus the appropriate phase and annular diaphragm annulus plate to match the objective in use Phase plates are located in the condenser turret Note If you have the motorized condenser the appropriate annulus plate will automatically move into position based on the selected objective when you click the Ph icon These are the plates and objectives e PhL 4x e Ph1 10x 20x e Ph2 40x 60x Arcturus Laser Capture Microdissection System User Guide Chapter 4 Inspecting Slides 6 If necessary center the annular diaphragm Using 2 mm hex head screwdrivers adjust the two set screws associated with phase insert until the phase annulus coincides with the phase plate see Figure 31 7 Focus the image and adjust the brightness for the optimal image Figure 31 Annular Diaphragm adjustment 8 To return to brightfield illumination click on the Ph icon The Diffuser will return to the original position 9 For bright field imaging rotate the condenser to the A position Note If you have a motorized condenser the phase annulus will automatically return to the A position Using differential This se
83. f the spot a In the main window tap the Ruler tool in the Select tools pane b Click the left side of the spot created by the IR capture laser then drag the stylus to the other side of the spot until the system draws a line and displays a label showing the diameter of the spot c Click the Move Stage tool in the Select tools pane to turn off the Ruler tool If the diameter is not of the desired size adjust both the laser power and duration settings simultaneously with the Power and duration slider To make the spot larger move the slider to the right e To make the spot smaller move the slider to the left e You can also enter values for both the power and duration setting fields manually Once you have achieved the desired spot size enter the diameter for the IR capture spot in the Spot Diameter um field If necessary repeat the procedure for the other spot sizes Click OK to save the adjusted IR capture spot settings Arcturus Laser Capture Microdissection System User Guide Chapter 5 Selecting Cells for Microdissection IMPORTANT If you click the X to close the dialog box the new settings will not be saved The Default button resets the values for all of the IR capture spots not just the currently selected spot Working with overlays You can save drawing items to a separate file then open the file and use it to capture identical regions from the same slide The drawn object that is laid over
84. fluorescently Sheet Fluorescence Rev A labeled samples using the Arcturus Imaging Optimization Ti Instrument Arcturus Instrument Tip 4458769 Provides tips for setting up and viewing phase Sheet Phase Contrast and Rev A contrast and DIC images DIC Arcturus Instrument 4458770 Provides tips and recommendations for Troubleshooting Guide Rev A handling problems encountered while using the Arcturus Instrument Arcturus Laser Capture Microdissection System User Guide 121 Documentation and Support Document par Description number Reagents Arcturus RiboAmp HS 12672 00 Provides instructions for using the Arcturus PLUS Kit User Guide Rev E RiboAmp HS PLUS Kit Arcturus Paradise PLUS 12872 00 Provides instructions for using the Arcturus Reagent System Kit User Rev D Paradise PLUS Reagent System Kit Guide Arcturus Turbo Labeling 14827 00 Provides instructions for using the Arcturus Kit User Guide Rev B Turbo Labeling Kit with Biotin Cyr 3 and Cy 5 dyes Arcturus Paradise Whole 14360 00 Provides instructions for using the Arcturus Transcript RT WT RT Rev C Paradise PLUS WT RT Kit Reagent System Kit User Guide Arcturus PicoPure DNA 12637 00 Provides instructions for using the Arcturus Extraction Kit User Guide Rev D PicoPure DNA Extraction Kit Arcturus Troubleshooting 4458770 Provides tips and recommendations for Guide Rev A handling problems encount
85. for cut and capture 1 Click the Information button in the upper right corner of the Microdissection tools pane Click IR Capture first or UV Cut first to set the order see Figure 54 Click OK at the bottom of the dialog box to close the box and save your changes Click the Information button i in the upper right corner of the Microdissection tools pane Click the Select Settings tab see Figure 55 If needed click IR SpotSpacing and enter a value U Current This value is the spacing between IR capture spots as a percentage of the spot diameter Figure 55 Select Settings tab If IRSpotSpacing is e lt 100 the IR capture spots will overlap Note With overlap there is tighter IR capture of the cells If your cells are loosely aggregated less overlap might be preferable e 100 all the IR capture spots will be adjacent without overlap e gt 100 there will be spaces between the IR capture spots Note Changes to IR SpotSpacing will only take effect on new drawing items Changes are not retroactive on existing drawing items If needed modify the UV Settings e IR SpotsPerCutLength is the number of IR spots provided per each UV cut length and between tabs Note Tabs are short regions that are not cut and which prevent the tissue from curling off the slide before the capture laser can attach the tissue to the CapSure Caps The use of tabs is important only when you are performing the UV c
86. g box and follow the steps below to enter information about your slides see Figure 17 1 Check each slide that you are loading or if you are loading all slides click Load All Slides The slots are identified from top to bottom A C based on the slot location on the stage Slide C is the slide closest to the front of the instrument Click Load with Overviews at the top to instruct the instrument to automatically create the slide overview image when you close this dialog box Figure 17 Load options slides For each slide choose the type of slide Glass Membrane or Framed Optional Enter a name to identify each slide in the SlideName field This name is shown on the static image when you have selected Yes in the Annotated Image field in the Image Settings tab Optional Enter any comments for each slide in the SlideNotes field These comments are saved to the cap interaction history file Note You can also edit the SlideName and the SlideNotes in the information area to the right of the slide overview image Arcturus T Laser Capture Microdissection System User Guide Selecting load options for caps Selecting file path options To enter information about the caps 1 2 1 Chapter 3 Starting the System and Loading Samples Click the Caps tab Check each cap that is loaded or click Load All Caps to check all of the check boxes at once see Figure 18 Click HS or Macro to identify the
87. g guide KIT0401 72 slides HistoGene LCM Immunofluorescence Staining Kit The HistoGene LCM Immunofluorescence Staining Kit is designed to enable retrieval of high quality RNA from immunofluorescently stained frozen tissue It enables convenient and reliable staining dehydration and LCM of tissue sections The kit s protocols are streamlined and optimized for efficient LCM capture while maintaining RNA quality for downstream applications that require intact RNA such as microarray analysis and RTPCR KIT0420 32 slides PicoPure RNA Isolation Kit The PicoPure RNA Isolation Kit is used for the extraction and isolation of total RNA from small samples particularly LCM cells The PicoPure RNA Kit comes with optimized buffers MiraCol Purification Columns and an easy to use protocol to maximize recovery of high quality total cellular RNA ready for amplification with the RiboAmp Plus RNA Amplification Kits KIT0204 40 isolations ArcturusX Laser Capture Microdissection System User Guide 113 AppendixE Arcturus Reagent Kits PicoPure DNA Extraction Kit The PicoPure DNA Extraction Kit is optimized to maximize the recovery of genomic DNA from 10 or more cells captured by LCM The kit comes with reagents and protocols tested to ensure complete extraction of DNA from LCM samples prepared with any standard tissue preparation procedure DNA prepared using the kit is PCR ready and needs no additional purification to perf
88. g with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following e U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov e Occupational Safety and Health Standards Bloodborn Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Note Additional information about biohazard guidelines is available at www cdc gov 108 Arcturus T Laser Capture Microdissection System User Guide APPENDIX D Laser Safety Scenarios For safety reasons the lasers are disabled when they are outside of the CapSure LCM Cap area However there may be applications for which the cap is not required In such instances you can bypass the instrument laser safety settings In order to bypass the laser safety mechanism the laser bypass key must be in place in the back of the instrument You must then depress the Laser Bypass button activating the UV laser The laser bypass key should be in place when the instrument is received If the bypass key is not present contact customer support The table below details possible status scenarios showing action combinations and the resulting system responses For example Line 4 ind
89. gure 12 Nikon Eclipse Ti E front operation panel Changing image output with Optical Path Selector buttons Press the Optical Path Selector buttons to change the ouput for the image For details refer to Table 8 Table 8 Optical Path Selector options Optical Light Use on path re Arcturus Details of Suggested Use ae Distribution Position Instrument EYE Eyepiece Standard When you want to use the microscope in manual mode port 100 microscopy without initiating the Arcturus operating software select the EYE position You can also switch to the EYE position if you want to view the sample through the eyepieces during an LCM session but in this position you will not see the live video image on the monitor Note To use the EYE position you must also have the optional Microscope Binoculars PN 0200 6228 L100 Left side Microdissection The live video camera for the Arcturus Instrument is port 100 installed in the left microscope port To view the image using the operating software you must select either the L100 or L80 optical paths The Arcturus operating software will default to the L100 position when initiated 26 Arcturus Laser Capture Microdissection System User Guide Chapter 3 Starting the System and Loading Samples Optical Use on path Light Arcturus T Details of Suggested Use pus Distribution Position Instrument L80 Left Side Microdissection The live video camera for the Arcturus T Instrument i
90. he Autobrightness button 1 Adjust the brightness in the main image window as desired 2 Click the Information button in the upper right corner of the Inspect tools pane to open the Inspect Options dialog box 3 Click the Illumination tab see Figure 26 4 Click Save AutoBrightness then click OK to close the dialog box and save your changes The next time you click the Autobrightness button in the Inspect tools pane the illumination is set to this value Returning to the original setting To return to the original autobrightness setting 1 Click the Information button i in the upper right corner of the Inspect tools pane to open the Inspect Options dialog 2 Click the Illumination tab a The next time you tap or click the Autobrightness button in the Inspect tools pane the illumination is set to the default value 42 Arcturus T Laser Capture Microdissection System User Guide Chapter 4 Inspecting Slides Performing phase contrast and DIC imaging This section explains how to work with both Phase Contrast imaging and Differential Interference Contrast DIC imaging Using phase Figure 28 shows the phase contrast components for a T DH diailluminator 100W and focus knob Condenser Condenser j focus knob centering knobs Figure 28 Phase contrast components Note Proper set up requires the Arcturus T Binoculars option PN 0200 6228 Getting set up for phase contrast imaging For optimal phase co
91. ical clearance of 32 in Requirements 80 cm Altitude For use up to 6600 ft 2000 m Computer specifications The following specifications apply to the computer used with the ArcturusXT Instrument e 2 8 GHz Pentium 4 processor minimum e 2 GB RAM minimum 40 GB hard drive minimum e Windows XP Professional operating system with SP 2 e Read write DVD drive e Interactive pen display 17 diagonal LCD 1280 x 1024 SXGA 92 Arcturus T Laser Capture Microdissection System User Guide Appendix A System Specifications Available instrument configurations You can choose the options for your Arcturus T Instrument These options are pictured below and described in the following pages In Figure 60 the photo on the left is an Arcturus Instrument with an LED illuminiation tower the instrument on the right has a Nikon illumination tower lt Wire Arcturus Figure 60 Arcturus Instrument with LED tower and with Nikon tower Base station There are three base station configurations for the ArcturusXT Instrument Each instrument includes IR laser capture an operating system and software three objectives 2X 10X 40X and a standard 0 7 MP video camera You may select from three illumination options to complete a base system configuration Note The catalog numbers listed here are not included on the website as these items cannot be ordered online Illumination tower Table 17 Illumination tower options
92. icates that when the laser bypass button has been pressed and a CapSure cap is in place but the laser bypass key has not been inserted the laser status is Standby You must insert the key into position for the laser to be ready to fire Table 19 Laser Safety Actions Laser Bypass Button Pressed State Laser Bypass Key in Place CapSur Cap in Laser To Set Color Pop Cut zap n State State Poser up Capture Other Tool Tip Messages Stand Stand U Stop tt Laser disabled by by Red because cap is out of beam path and override is off ON 0 Stand U x Stop tt Laser disabled by Red because cap is out of beam path and override is off popup Lasers have been disabled because cap is out of beam path and override is off Change cap placement or enable override to continue x Stand Stand U Stop Clean tt Laser ready to fire by by Yellow obj Cap placed in beam status path xX ON Power U tt Laser is firing Cap p Green placed in beam path Standby Stand U Stop Clean tt Laser disabled by Orange obj because cap is out of status beam path and override is off Arcturus Laser Capture Microdissection System User Guide 109 Appendix D Laser Safety Scenarios Laser Laser Bypass Bypass Button Key in Pressed Place CapSur Cap in Place Laser State State To State Set Laser Color
93. ide 87 8 Chapter 8 Maintenance and Troubleshooting Solving problems with fluorescence 88 SYMPTOM POSSIBLE CAUSE REMEDY Brightfield image is fully or partially blocked Apertures Field or Condenser 1 If you have the Nikon phase contrast illumination tower check the field and condenser apertures to make sure they are both fully open 2 Check that the filter sliders found in the illumination tower are not partially pushed in Field aperture is not centered Center the field aperture Fluorescent cube turret Check to ensure that the position is fully clicked in Magnification Tube Check to ensure that the position is fully clicked in Poor image quality at 40x and 60x DIC sliders Remove the DIC sliders located below each objective Note Replace any empty slot with a cover to prevent dust from entering Background of live image is not clear or white White balance is off Re establish white balance in camera properties dialog box located under View in the pull down menu Fluorescence cube in place Check the fluorescence turret If using brightfield illumination it should be in a position not containing a filter cube Slide overview image too dark or too bright Settings are not optimized 1 Change magnification to 2x and adjust the brightness setting 2 Right click in the slide overview area and select Remember Settings in the po
94. image for all slides loaded Note If you did not select Load with Overviews the slide overview image area will be blank To acquire and display the slide overview right click in the slide overview area and click Reacquire Overview Image 34 Arcturus T Laser Capture Microdissection System User Guide Chapter 3 Starting the System and Loading Samples Saving images automatically You can choose to create and save images automatically Three images are saved e The main image before microdissection e The main image after microdissection e The cap after microdissection You can refer to these images to see how effective capture was and or to see the context of the microdissected tissue To save images automatically 1 Open the Load Options dialog box 2 Click the Image Settings tab see Figure 20 on page 34 3 In the AutoDocPrefix field enter the prefix that you want to use at the beginning of the name of each image file saved 4 Inthe AutoDocument field click the field to display the drop down list and select Yes 5 In the SuffixNumber field enter the number for the first image This number is the last part of the file name for all images It increases by one each time microdissection occurs 6 In the LabelCapAfter field enter the text that you want to add after the AutodocPrefix and the SuffixNumber of each image of the cap 7 In the LabelSpecimenBefore field enter the text that you want to add after the Autodoc
95. informational text for every option in an Options dialog box To view it click the item of interest The descriptive text appears in the pane below see Figure 7 Figure 7 Options dialog box Selecting from a drop down menu For some options in a dialog box you can make choices from a drop down list These lists are indicated by an arrow on the right side of the field see Figure 8 Expanding the list of options If you do not see any options in a dialog box the options may be minimized To expand the list of options click the plus sign in the upper left corner In the example shown in Figure 9 notice where the white cursor arrow is pointing This list has just been expanded Figure 9 Expanding the list of options 16 Arcturus Laser Capture Microdissection System User Guide Finding the version number Using menus and commands Chapter 1 System Overview Entering text in dialog boxes For some options in a dialog box you can enter text in a field To do this you click the stylus or the mouse in the field of interest and then use the computer keyboard to type text see Figure 10 Figure 10 Typing text in dialog boxes To find the version number open the About Arcturus XT window see Figure 11 Figure 11 The About Arcturus window The tables in this section explain the functions available from the menus and submenus File menu commands and submenus Table 1 Functions availabl
96. ing from misuse neglect or accident including without limitation operation with incompatible solvents or samples in the system operation outside of the environmental or use specifications or not in conformance with the instructions for the instrument system software or accessories improper or inadequate maintenance by the user installation of software or interfacing or use in combination with software or products not supplied or authorized by Applied Biosystems and modification or repair of the product not authorized by Applied Biosystems THE FOREGOING PROVISIONS SET FORTH APPLIED BIOSYSTEMS SOLE AND EXCLUSIVE REPRESENTATIONS WARRANTIES AND OBLIGATIONS WITH RESPECT TO ITS PRODUCTS AND APPLIED BIOSYSTEMS MAKES NO OTHER WARRANTY OF ANY KIND WHATSOEVER EXPRESSED OR IMPLIED INCLUDING WITHOUT LIMITATION WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE WHETHER ARISING FROM A STATUTE OR OTHERWISE IN LAW OR FROM A COURSE OF DEALING OR USAGE OF TRADE ALL OF WHICH ARE EXPRESSLY DISCLAIMED THE REMEDIES PROVIDED HEREIN ARE THE BUYER S SOLE AND EXCLUSIVE REMEDIES WITHOUT LIMITING THE GENERALITY OF THE FOREGOING IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE INCLUDING WITHOUT LIMITATION ANY TRADE PRACTICE UNFAIR COMPETITION OR OTHER STATUTE OF SIMILAR IMPORT OR ON ANY OTHER BASIS FOR DIRECT INDIRECT PUNITIVE INCIDENTAL MULTIPLE CONSEQUENTIAL OR SPECI
97. ion Slide the insertion tool towards the cap until the cap is engaged Remove the insertion tool from the QC position with the cap attached to it IMPORTANT Be sure you do not touch the polymer surface that holds the microdissected cells as you remove the caps from the unload stations Repeat steps 2 3 and 4 for each cap in the QC Cap area Press the tension button in to release the springs holding the slides in place and then lift each slide out of its slot Load new slides and caps to continue microdissection or close the Arcturus software to end your session If you plan to continue microdissection and need to load new caps you must reset the software To clear caps from the QC position do one of the following e Click the Reset button in the Load Options dialog box e Click on each Cap icon in the QC Caps area next to the Slide overview image When you click on the cap icon it will disappear indicating that you have removed the cap from the position on the stage off load area IMPORTANT Make sure that you remove any caps whose positions have been cleared in the QC caps area Failure to do so can result in a new cap running into the one remaining Microdissected cells are now available for extraction of nucleic acids and proteins The final step in microdissection is extracting biomolecules from the caps Arcturus T Laser Capture Microdissection System User Guide Chapter 6 Microdissecting Cells and Tissue 6 L
98. ion UY Cut Object s Paste Object s Making selections from the slide overview Een image Reacquire Overview Image Place Cap at Region Center Move Cap to QC Station Remember Settings To make a menu selection from the slide overview image place the stylus or cursor in the image press the lower button on the stylus or right click the mouse and make Figure 6 Menu from slide overview image your selection Note that in this menu the first selection has an additional set of options which you access by clicking on the arrow to the right Using the options Each of the four tool panes in the Arcturus software has an Options dialog box dialog boxes associated with it You can use this dialog box to set properties and perform actions associated with that set of tools You can open an Options dialog box in one of three ways e Select the name of the tool pane from the Options menu e Click the Information button i in the upper right corner of the tool selection pane see Figure 3 on page 14 e Right click anywhere in the tool pane You can close an Options dialog box in one of two ways e To save changes click OK at the bottom of the Options dialog box e To exit without saving changes click Cancel at the bottom of the Options dialog box or click the X in the upper right corner ArcturusX Laser Capture Microdissection System User Guide 15 Chapter 1 System Overview Viewing informational text There is
99. is in the upper left corner and the tool panel is on the right You can move the tool panel to the left side by clicking Left hand Orientation in the View menu L Cap and slide handling area Slide overview image and QC caps area Figure 2 The Arcturus Instrument primary screen ArcturusX Laser Capture Microdissection System User Guide 13 Chapter 1 System Overview At the bottom of the screen are the cap and slide handling areas the slide overview image and the QC caps area To move the stage to a particular region and to view that region in the main image you click the slide overview image in that region The information displayed includes the properties of the currently selected object such as a slide or a cap Viewing tool tips Most items in the ArcturusXT primary screen have a tool tip associated with them The tool tips give you information about the item Capture Drawing Items Groups F 2 Sf Delete All AutoScanxT To view tool tips hover the stylus or mouse over the item of interest on the screen In the example shown in Figure 3 placing the cursor over the hand displays a message indicating that this tool moves the stage Stored Positions Kd lt gt gt l xa Figure 3 Select tools pane Making selections Some commands are available from pop up menus in both the main image and the from pop up overview image In either image to view a pop up menu
100. jects in the main image window 3 When you are done marking cells for microdissection tap or click the Move Stage tool Figure 42 on page 55 to turn off the drawing tool For each item that you draw a number appears in the Drawing Items list located to the right of the Capture Groups list see Figure 42 on page 55 4 As needed move the stage to display other areas in the main image window so you can mark more cells for microdissection 5 If you want to designate cells and tissue for a second capture group EE 6 Once you have one or more drawing items drawn you can e Use the Eraser tool to remove any marks made by the drawing tools e Use the Select Object s tool to draw a rectangle that includes all drawing items that fall inside a region e Once selected you can move the drawing items copy and paste them delete them move them to a different capture group or view information about the them e Add more IR capture spots to any item Click the Single IR Spot tool and then tap or click inside any of the drawing items Arcturus Laser Capture Microdissection System User Guide 57 Chapter 5 Selecting Cells for Microdissection Working with drawing items Moving drawing items Moving drawing items to a different Capture Group Deleting drawing items 58 When working with drawing items all actions apply only to the active Capture Group To move a drawing item 1 Click the Select Object s
101. kstation safety 106 Z Z reset button 27 125 Part Number 0112 0153 Rev C 11 2010 0112 0153 C gt applied A binsystems by Life technologies Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 760 603 7200 www lifetechnologies com Technical Resources and Support For the latest technical resources and support information for all locations please refer to our Web site at www appliedbiosystems com
102. lectrical connections are made to the instrument Indicates a terminal that can receive or supply alternating current or voltage Indicates a terminal that can receive or supply alternating or direct current or voltage Ii D OGO Safety symbols on instruments The following table describes the safety symbols that may be displayed on Applied Biosystems instruments Each symbol may appear by itself or with text that explains the relevant hazard These safety symbols may also appear next to DANGERS WARNINGS and CAUTIONS that occur in the text of this and other product support documents ArcturusX Laser Capture Microdissection System User Guide 101 102 Appendix C Safety Description Indicates that you should consult the manual for further information and to proceed with appropriate caution Indicates the presence of an electrical shock hazard and to proceed with appropriate caution Indicates the presence of a hot surface or other high temperature hazard and to proceed with appropriate caution Indicates the presence of a laser inside the instrument and to proceed with appropriate caution Indicates the presence of moving parts and to proceed with appropriate caution Indicates the presence of a biological hazard and to proceed with appropriate caution Indicates the presence of an ultraviolet light in the instrument and to proceed with appropriate caution Vaca alzalcalbc
103. leshooting 83 Summary of chapter topics 83 Cleaning the Arcturus Instrument 83 Replacing user serviceable parts 83 Replacing the bright field illumination lamp 84 Replacing fluorescence filter cubes 84 Replacing the fluorescence lamp 85 Replacing the TUSE ii Line heat oie ea So dt he dee hee ae 85 Troubleshooting tips fre ce 25934600408 et eae eeu gies d eee ees les 86 Solving problems with IR Laser Capture LCM 86 Solving problems with UV Laser cutting 87 Solving problems with image quality 87 Solving problems with fluorescence 88 Solving problems with phase contrast DIC 89 Solving general problems 90 System Specifications 91 Instrument specifications 91 Computer specifications 92 Available instrument configurations 93 SE ETES LE LOT 93 Illumination tower options
104. lose the dialog box and save your changes Measuring distances and objects You can measure distances or objects in the main image window using the Ruler tool To measure with the Ruler tool 1 Tap or click the Ruler tool Pi EE microns 2 Inthe main image window drag the stylus from one edge to the other across the distance or object you want to measure 3 The software draws a line and Figure 46 Ruler measurement displays a label showing the length of the line see Figure 46 4 To remove the measuring lines and labels tap or click the Move Stage tool Setting up the Test Fire In the IR Spot Sizes tab you can set the size for each of the three sizes of IR capture spots When you create a drawing item you choose the size of the capture spots associated with the drawing item If you know you are only going to use one of the three spot sizes follow the procedure below only for that size This procedure involves adjusting the laser power and duration so that the laser spot in the main image window is the same diameter as set in the Select Options dialog box If you do not set the spot size you risk incomplete capture of your tissue Note You should set the IR Capture Spot size before creating any drawing items The selected IR spot size will be placed in all drawn or marked items IR laser power and duration are stored with the drawing item and will be used for IR capture To get set up for the Test Fire Note Pri
105. mage consists of only the area visible in the main image window Adjust the image as needed using the Inspect tools Click the Camera button specified in the File Paths tab of the Load Options dialog box It is saved in the format specified in the Image Setting tab of the Load Options dialog box When you are done viewing and or working with the image tap the close box in the upper right corner of the static image window Opening an image To open an image 1 2 Choose Open Image from the File menu In the resulting dialog box tap or click the name of the image you want to open and tap or click Open The image opens in a window When you have finished viewing the image tap the close box in the upper right corner of the window Arcturus T Laser Capture Microdissection System User Guide Chapter 4 Inspecting Slides Ca ptu ring saving This section explains how to capture save and view a video recording and viewing videos Capturing and saving a video To capture and save a video 1 To begin recording a video click the Video Recorder Ed x button in the Inspect tools panel Figure 41 Video ae CUT 2 To stop the recording click the Video recorder button again to stop recording The camera icon turns black when recording stops The file is named with the date and a number Viewing a video To view a video use Windows Media Player or a similar program that can handle avi files Note To c
106. mize risk of fire ensure the illumination tower cable is connected before the control unit is powered on AVERTISSEMENT Pour r duire le risque de feu assurez le cable de tour d illumination est reli avant que l Unit de commande soit mise en marche IMPORTANT To prevent damage to the instrument turn power OFF before connecting or disconnecting cables ATTENTION Pour emp cher endommager l instrument coupez le courant OFF avant de relier ou d brancher des c bles Do not remove or modify any of the Arcturus Instrument optical components or subassemblies Any modifications to the Arcturus Instrument may void the system warranty The ArcturusXT Instrument is for indoor use only Arcturus Laser Capture Microdissection System User Guide 107 Appendix C Safety Biological Hazard Safety WARNING Biological samples such as tissues body fluids infectious agents Ad and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before workin
107. mplete or if new drawing items were placed after performing microdissection highlight the desired drawing items to be repeated and select one of the following from the Microdissect menu e UV Cut gt Selected Items or Current Capture Group e IR Capture gt Selected Items or Current Capture Group e IR Capture and UV Cut gt Selected Items or Current Capture Group Note An asterix appears next to a drawing item number in the Drawing Items list once IR Capture or UV cut have been performed 70 Arcturus T Laser Capture Microdissection System User Guide Chapter 6 Microdissecting Cells and Tissue 6 Inspecting microdissected material After you have completed the microdissection you can inspect the slide and the cap to verify that the collection was successful If microdissection was incomplete you can repeat the cut and or capture steps To inspect the microdissected material 1 Click the Move Cap to QC station button in the Microdissect tool pane Alternatively right click on the slide overview and select Move Cap to QC station from the pop up menu cos The cap is moved to the QC position and the stage moves the O QC position over the objective The cap is shown in the main O image window O The system automatically creates the cap interaction history O file when the cap is moved to the QC position This file is named CapReport YYMMDD HHMM htm where Figure 51 QC YYMMDD is the year month day and HHMM is the hour c
108. n automated approach to laser microdissection of individual cells or multi cellular structures from slides containing tissue sections or cytological samples The Arcturus LCM System consists of the Arcturus Instrument a computer and the Arcturus operating software See Appendix A for detailed specifications for the instrument While the ArcturusXT Instrument is intended for laser capture microdissection LCM you can also use it for standard microscopy applications under certain circumstances For more information see Using the ArcturusXT Instrument as a stand alone microscope on page 19 About laser capture microdissection Laser capture microdissection LCM is a method of procuring specific cell populations from specimen preparations using a low power infrared IR laser to activate a special thermoplastic film over the cells or tissue of interest The activated transfer film adheres to the cells that are located within the laser beam diameter The laser does not affect the tissue sample the quality of nucleic acids and proteins within the sample and the cell morphology are not compromised When you use the Arcturus Instrument specially designed CapSure HS LCM Caps or CapSure Macro Caps coated with thermoplastic film are placed on the region of interest The instrument directs the laser through the cap to activate the film onto the selected cells The cells adhere to the CapSure cap surface when it is lifted from the tissue sec
109. n the main image e Fill IR Spo If this is set to Yes the IR Capture spot will be filled with the IR SpotColor 5 Repeat steps 3 and 4 for any other capture groups 6 Click OK to close the dialog box and save your changes 64 Arcturus T Laser Capture Microdissection System User Guide Chapter 5 Selecting Cells for Microdissection Note These settings are also available in the Information area to the right of the slide overview image Click on a capture group in the Select tools pane to display the information and make any desired changes Sometimes you find a location on the slide that is interesting but you are unsure if you want to mark it up for microdissection In these instances you can store positions and then come back and review them before you mark them up for final microdissection To use stored positions e To add a position move the stage to a desired location on the slide and tap on the button to add that location as a stored position The stored position will be given a number starting with 1 and increasing with each next stored position e To review stored positions click on the forward and back arrows to scroll through the stored locations The stage will move to the location of the stored position and bring it to the center of the live image e To delete the displayed stored position click on the minus symbol e To delete all stored positions click on the X All button ArcturusX Laser
110. nd capture 0 12 Outline of the microdissection process 12 Using the Arcturus operating software 13 The primaryiscreen ie sereine haies ot Wesco eatin ne a 13 VIEWING tol HIPS a e g cee eee glee one goles Medias Pe hates Egle eet eat a et 14 Making selections from pop up menus 14 Using the options dialog boxes 15 Finding the version number 17 Using menus and commands 17 KeVCOMMANAS 2 Son ne A ie he CN De mar de di en de te Ae D 18 Using the Arcturus Instrument as a stand alone microscope 19 Getting into and out of manual mode 19 Using manual mode 19 CHAPTER 2 Preparing Samples 23 5 assis dates Niue 21 Summary of chapter topics 21 Choosing slides and Petri dishes 21 Acquiring slides and Petri dishes 21 For information about using these products 22 Preparing tissue samples 24e dust was Divas aa tte roi tee 22 Using frozen tissue samples
111. nd complies with ICES 001 Issue 3 Industrial Scientific and Medical Radio Frequency Generators Arcturus Laser Capture Microdissection System User Guide Appendix C Safety European Safety and EMC standards This instrument meets European requirements for safety Low Voltage Directive 73 23 EEC This instrument has been tested to and complies with standards e EN 61010 1 2001 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements e EN 60825 1 Radiation Safety of Laser Products Equipment Classification Requirements and User s Guide This instrument meets European requirements for emission and immunity EMC Directive 89 336 EEC This instrument has been tested to and complies with standard EN 61326 Group 1 Class B Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Australian EMC standards This instrument has been tested to and complies with standard AS NZS 2064 Limits and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radio frequency Equipment Product specific Please review the following precautions carefully to ensure safe and effective use of warnings the Arcturus Laser Capture Microdissection LCM System which consists of the Arcturus Instrument a computer and the Arcturus operating software WARNING To mini
112. nit dimensions 91 binoculars 94 Bright Field lamp 40 specifications 91 Brightness button 26 C cap interaction history file 78 caps extracting material from 81 Caps tab 33 CapSure HS Cap 80 CapSure Macro LCM Caps 80 CAUTION description 9 claims processing 119 computer configuration requirement 117 specifications 92 technical support for altered configuration 117 connections between system components 98 cut and capture 12 D damage reporting 119 damaged items returning 119 DANGER description 9 DIC Analyzer Cube 45 Differential Interference Contrast DIC imaging 43 Display buttons 25 Arcturus Laser Capture Microdissection System User Guide Index E electrical safety 104 electrical supply specifications 91 electromagnetic compatibility standards See EMC standards EMC standards 106 entering text 17 ergonomics safety 106 Escape button 30 EXFO fluorescence 47 extinction point 46 ExtracSure device 80 extraction kits 79 F File Paths tab 33 fluorescence filter turret 30 fluorescence filters replacing 84 specifications 91 Fluorescent lamp specifications 91 Focus knob 28 30 Focus Selection switch 28 29 formalin fixed paraffin embedded FFPE tissue 22 fuse specifications 91 H HistoGene LCM Frozen Section Staining Kit 113 HistoGene LCM Immunofluorescence Staining Kit 113 Illumination tab 40 illumination tower options 93 Image Settings tab 34 123 Index ImageFile Extensi
113. nt Improper lifting can cause painful and permanent back injury Keep the following points in mind while lifting Make sure that you have a secure comfortable grip when lifting Make sure that the path is clear of obstructions Do not lift an object and twist your torso at the same time Keep your spine in a good neutral position while lifting with your legs Share the load Use the lifting handles that were in place when the instrument was shipped and lift with four people Preparing for installation The location for the instrument must meet the following requirements Stable laboratory bench capable of supporting 400 Ib 200 kg Work surface 36 in x 72 in 92 cm x 180 cm with 32 in 80 cm vertical clearance Electrical Supply 100 240 VAC 50 60 Hz 500 W voltage fluctuations not to exceed 10 of nominal supply voltage Up to six power receptacles depending on system configuration The base system IR laser only requires four power receptacles 1 Arcturus T 2 Eclipse Ti E 3 Computer 4 Monitor Temperature 18 30 C relative humidity lt 60 Indoors pollution degree 2 Installation Category II Altitude requirements specified in Appendix A Arcturus Laser Capture Microdissection System User Guide 97 Appendix B Installation Instructions General unpacking and installation instructions CAUTION Failure to correctly install the instrument can result in damage that ZN is not covered by the war
114. ntrast image quality ensure that all components are properly aligned Prior to starting an experiment using Phase Contrast 1 If necessary center the field aperture IMPORTANT The condenser cannot be focused completely To focus the condenser you would need to drop it lower than is allowable due to the IR laser assembly 2 Click on the Ph icon in the Inspect tools pane see Figure 21 on page 37 The diffuser will move to the Out position 3 Select the 10X objective and focus the live image 4 Close down the field aperture diaphragm located in the illumination tower see Figure 28 until the field diaphragm appears in the field of view The diaphragm edges will not be in focus ArcturusX Laser Capture Microdissection System User Guide 43 Chapter 4 Inspecting Slides 5 Turn the two condenser centering screws to move the field diaphragm to the center of the field of view see Figure 29 Field diaphragm image Field of view OO Figure 29 Condenser centering screws and condenser allignment Working with phase contrast imaging 1 44 If you haven t done so already click on the Ph icon in the Inspect tools pane see Figure 21 on page 37 The diffuser must be in the Out position for proper phase contrast and DIC imaging Ensure that both the field aperture and the condenser aperature are fully open Rotate the Bertrand Lens B on the binoculars located in the eyepiece turret of the binoculars Vi
115. ntrifuge tube 3 Press down firmly on the insertion tool to ensure the Macro LCM Cap is tightly and evenly sealed with the microcentrifuge tube 4 Invert the tube so that all the extraction buffer comes in contact with the microdissected cells on the cap surface If necessary flick the microcentrifuge tube lightly 5 Incubate the sample as described in the appropriate extraction procedure 6 Place the tube into a microcentrifuge and briefly spin 5 10 seconds to bring the buffer to the bottom of the tube Extracting tissue from CapSure HS Caps Performing LCM captures with CapSure HS Caps 80 Following microdissection place the ExtracSure Extraction Devices onto the CapSure HS Caps containing the microdissected cells The ExtracSure Device seals around the perimeter of the cap surface and covers the circular ridge that was in contact with the sample during LCM With the ExtracSure Device you can incubate the cells in a small volume of extraction buffer Due to the way in which the ExtracSure device fits onto the CapSure HS Cap you should perform LCM captures in the center of the CapSure HS Cap within the black capture ring see Figure 58 h we Tr gt ont s m we Le Y 2 ur or Veale a a ine em eh o rnn y lt 5 d CapSure HS Cap ExtraSure device Capture HS Cap sealed with ExtracSure Figure 58 CapSure HS Cap and ExtracSure device Arcturus T Laser Capture Microdissection Sys
116. o defaults 4 drop down menu Color 2 Move the stage to a an j position on the slide Saturation E D 194 that has both tissue and White Balance 7 Auto One Push blank slide areas visible White Balance Blue E F amp in the field of view White Balance Red QI RU 3 Adjust the focus and brightness as needed ee Brightness 0 4 Check the White Gait ef 2 D at el Balance Auto check E et box Exposure 1 104sec Auto Auto Reference Parameter 800 5 Uncheck the White Balance Auto check Cancel box Figure 27 Camera properties 6 Click the 7 Once the system has completed the auto adjustment click Apply and then OK to close the window 8 If further adjustment is needed repeat these steps Note If the automatic routine is insufficient for specific requirements you can also adjust the White Balance Blue and Red settings manually by moving each slider until you achieve the desired color balance ArcturusX Laser Capture Microdissection System User Guide 41 Chapter 4 Inspecting Slides Changing the The ArcturusXT software automatically sets the brightness when you tap or click the autobrightness Autobrightness button on the Inspect tools pane see Figure 21 on page 37 If the settings default autobrightness is not appropriate for your sample you can adjust the value manually and save it as the default Saving the current setting To save the current brightness as the default for t
117. ocating the lasers This section explains how to locate both the the UV Cutting Laser IR and the IR Capture Laser Locating the UV To ensure that the laser will cut accurately locate the UV cutting laser for each cutting laser objective at the beginning of each session If you notice that the UV laser is not firing at the desired location you should repeat this procedure To locate the UV laser 1 Click the Information button i in the upper right corner of the Microdissection tools pane to open the Microdissect Options dialog box 2 Click the UV Locate tab see Figure 52 3 Follow the steps in the dialog box The check mark indicates the current step in the procedure 4 Click the Locate UV button The Arcturus Instrument fires the cutting laser 5 In the main image window place the cursor in the center of the UV laser spot and click the spot 6 Click OK in the dialog box Figure 52 UV Locate tab 7 Click OK to close the dialog box A green circle will appear on the live image at the location of the UV laser Arcturus Laser Capture Microdissection System User Guide 73 o Chapter 6 Microdissecting Cells and Tissue Locating the IR capture laser To locate the IR capture laser from the Microdissect options dialog box 1 Open the Microdissect Options dialog box by clicking the Information i button in the upper right corner of the Microdissection tools pane 2 Click the IR Loca
118. on Camera Opens the Camera Properties dialog box and allows you to set up the video Properties camera Zoom Not available The table below provides some keyboard shortcuts that will speed up your processing Table 4 Keyboard shortcuts Pane Key Command Description Primary Screen Ctrl A Selects all drawing items in the main image window Ctrl V Pastes selected drawing items in the main image window Ctrl X Deletes selected drawing items Arcturus Laser Capture Microdissection System User Guide Chapter 1 System Overview Using the Arcturus Instrument as a stand alone microscope Although the Arcturus Instrument is intended for laser capture microdissection LCM you can also use it for standard microscopy applications if it is equipped with the optional binocular eyepiece PN 0200 6228 Getting into andout To use the Arcturus Instrument as a stand alone microscope you must be in manual of manual mode mode The Arcturus Instrument is in manual mode when it is first turned on and it remains in this state until you initialize the Arcturus operating software To return to manual mode after you have initialized the software you can shut down the software shut down the instrument and then restart the instrument and the software Using manual While you are in manual mode use the lamp intensity knob to adjust the lamp mode brightness and the focus control knobs to manipulate the focus
119. on field 34 IMPORTANT description 9 Information button i 15 informational text 16 Inspect tools 37 instrument carrying instructions 97 instrument operation safety 104 IR Capture Laser 73 specifications 91 K keyboard shortcuts 18 L LabelCapAfter field 35 LabelSpecimenAfter field 35 LabelSpecimenBefore field 35 Large Format Slide Stage Insert 95 Laser Bypass 78 laser capture microdissection 11 laser classification 105 laser safety 105 requirements 105 left hand orientation 13 Load All Caps 33 Load All Slides 32 Load with Overviews 32 34 M Magnification Selection knob 27 main image window 13 manual mode 19 Microdissect options pane 74 Microdissect tools pane 67 microdissection process 12 microscope stage specifications 91 mouse 14 moving and lifting safety computers and monitors 103 instrument 103 moving parts safety 104 124 N Nikon Eclipse Ti E microscope base 9 Nikon illumination tower 93 Nikon TE2000 based Instrument 9 0 Objective switch 28 objective upgrades 94 operating humidity 91 operating temperature 91 operation panel front 26 left 28 right 29 Optical Path Selector buttons 26 options dialog boxes 15 P Paradise PLUS FFPE Kits 114 Paradise PLUS FFPE WT RT Kit 114 Petri Dish Stage Insert 95 Petri dishes 21 Phase Contrast imaging 43 photoablation 12 physical hazard safety 104 PicoPure DNA Extraction Kit 114 PicoPure RNA Isolation Kit 113 primary screen 13 R Reacquir
120. or to setting the IR Capture Spot size ensure that you have located the IR laser 1 Place a CapSure cap onto the slide and move to a clear non tissue area on the slide 2 Click the Information button i in the upper right corner of the Select tools pane to open the Select Options dialog box 3 Click the IR Spot Sizes tab ArcturusX Laser Capture Microdissection System User Guide 61 Chapter 5 Selecting Cells for Microdissection Checking the spot diameter 62 4 Click the button under Edit settings per 2 Select Options x spot sizes for the S1Z of spot small Tools Options IR Spot Sizes Capture Groups Drawing Items medium or large for which you want to en set the parameters see Figure 47 E Check Auto move stage to move the paneter un 50 120 190 stage each time the laser is test fired Power 45 82 39 Click Test IR Shot Be The software will fire the IR capture U laser vom SET Default Test IR Shot v Auto move stage OK Cancel wetted IR Capture Spot Figure 47 IR Spot Sizes tab Ensure that you obtain a properly You should see a dark ring with a clear center as shown in Figure 48 If you do not see this adjust the Power and duration slider and then tap the IR Laser Test Fire button again Figure 48 Properly wetted spots To check the diameter of the properly wetted spot Measure the diameter o
121. orm amplification KIT0103 150 HS cap extractions 30 Macro cap extractions or 10 tissue scrapes Paradise PLUS FFPE Kits The Paradise PLUS Reagent System is designed to enable gene expression studies using formalin fixed paraffin embedded FFPE tissue samples Components include sample preparation and staining reagents RNA extraction and isolation reagents RNA amplification reagents and a comprehensive user guide KIT0312 12 samples KIT0312B 12 samples with biotin labeling KIT0312C 12 samples with Cy3 labeling KIT0312D 12 samples with Cy5 labeling Paradise PLUS FFPE WT RT Kit The Paradise PLUS Whole Transcript Reverse Transcription WT RT Reagent System Kit enables QRT PCR using formalin fixed paraffin embedded FFPE tissue samples The kit was developed specifically to overcome obstacles often associated with formalin fixed tissue such as chemical modification and RNA fragmentation The kit provides RNA isolation and reverse transcription reagents optimized for use with archived FFPE samples at small sample input amounts and delivers unparalleled yield fidelity and representation The kit was designed with exon spanning primers at varying distances from the 3 end of the transcript and allows the study of splice variants in archived or degraded samples The Paradise WT RT system also allows the use of gene specific primers for reverse transcription to suit specific assay requirements KIT0315 12 Sam
122. overs that require tool access No user serviceable parts are inside Refer servicing to Applied Biosystems qualified service personnel AVERTISSEMENT Pour viter les risques d lectrocution ne pas retirer les capots dont l ouverture n cessite l utilisation d outils Linstrument ne contient aucune pi ce r parable par l utilisateur Toute intervention doit tre effectu e par le personnel de service qualifi venant de chez Applied Biosystems CAUTION Class 2 1l visible and or invisible LED radiation present when open Do not stare directly into the beam or view directly with optical instruments ATTENTION Rayonnement visible ou invisible d un faisceau LED de Classe 2 11 en cas d ouverture Ne pas regarder le faisceau directement ou au travers d un instrument optique CAUTION Moving parts Crush pinch hazard ATTENTION Pi ces en mouvement risque de pincement et ou d crasement 10 Arcturus Laser Capture Microdissection System User Guide CHAPTER 1 System Overview Chapter contents About the ArcturusXT Instrument 11 About laser capture microdissection 11 Using the ArcturusXT operating software 13 Using the ArcturusXT Instrument as a stand alone microscope 19 About the Arcturus T Instrument The Arcturus Laser Capture Microdissection LCM System provides a
123. p up window 3 Select Yes to save for all future overviews 4 Select Require Overview Table 13 Possible problems with fluorescence SYMPTOM POSSIBLE CAUSE REMEDY Fluorescence Image is fully or partially blocked Fluorescent Field Aperture Fluorescence ND Filters 1 Ensure that the fluorescence field aperture is centered 2 Ensure that the Fluorescence ND filters are in the OUT positions 3 Ensure that shutter on the fluorescence turret is open O position Arcturus Laser Capture Microdissection System User Guide Solving problems with phase contrast DIC Chapter 8 Maintenance and Troubleshooting 8 SYMPTOM POSSIBLE CAUSE REMEDY Fluorescence signal is suboptimal long exposure time is required Fluorescent ND Filters ND fluorescent filters should be in the OUT positions DIC or Phase Optics The DIC analyzer and the polarizer should both be in the OUT positions The Condenser position should be set at A Fiber Optic Cable 1 Check the fluorescence adapter cone to ensure that it has been attached properly 2 Ensure that the fiber optic cable is fully inserted into the cone and the EXFO control box Table 14 Possible problems with phase contrast DIC SYMPTOM POSSIBLE CAUSE REMEDY The phase contrast image does not appear optimal The diffuser Ensure that the diffuser is in the OUT position in the Select Options
124. ples 114 Arcturus T Laser Capture Microdissection System User Guide AppendixE Arcturus Reagent Kits RiboAmp PLUS RNA Amplification Kits The RiboAmp PLUS RNA Amplification Kit enables the production of microgram quantities of antisense RNA aRNA from as little as picogram amounts of total cellular RNA Amplified RNA produced using the kit is suitable for labeling and use on expression microarrays The kit achieves 1 000 to 3 000 fold amplifications in one round of amplification and up to 1 000 000 fold in two rounds The kits include microarray labeling options for biotin fluorescent dyes and amino allyls Kits are available in two sensitivity options RiboAmp Plus 5 40 ng input and a high sensitivity version RiboAmp HS Plus 0 1 to 5 ng input KIT0521 RiboAmp PLUS 12 1 round amplifications or 6 2 round amplifications KIT0525 RiboAmp HS PLUS 6 2 round amplifications Turbo Labeling Kits The Turbo Labeling Kits provide a proprietary non enzymatic technology for the labeling of unmodified aRNA for gene expression profiling The unmodified aRNA is labeled post amplification thereby avoiding the need to incorporate modified nucleotides The use of natural nucleotides in the amplification step results in unmodified aRNA with higher yields and longer aRNA fragments thus providing better representation of the mRNA transcript for downstream analysis KIT0608 Biotin 12 samples KIT0609 Cy3 12 samples
125. position P Figure 34 DIC polarizer in position Place the DIC slider back into place in the objective Working with DIC imaging 1 ol 46 If you haven t done so already click on the DIC icon in the Inspect tool pane to move the diffuser to the Out position Ensure that the DIC slider is in place in the objective Rotate the condenser turret to the DIC N1 position Note If you have purchased the motorized condenser the condenser will automatically rotate into position when you click the DIC icon Focus the image and adjust the brightness setting ooo beneath the objective nosepiece fluorescent turret Note If you have purchased the DIC Analyzer Cube in conjunction with the motorized fluorescence filter turret when you click on the DIC button the DIC Analyzer Cube moves into position automatically If you are using the DIC Analyzer Cube you do not need to use the sliding analyzer Push in the polarizer located above the condenser turret and rotate until the best image is achieved Sharpen the image if necessary by adjusting closing down the condenser diaphragm Arcturus Laser Capture Microdissection System User Guide Chapter 4 Inspecting Slides Working with fluorescence This section explains how to set up and use fluorescence Before you begin To control the EXFO fluorescence illumination box through the Arcturus software turn on the EXFO box prior to initiating the software If you
126. ption of instrument specifications Item Description Electrical Supply 100 240 VAC 50 60 Hz 250 W Voltage fluctuations not to exceed 10 of nominal supply voltage Fuse 2A time delay 5 x 20 mm Capture Laser Solid state near IR laser state 810 nm UV Cutting Laser Diode pumped solid state UV laser 349 nm Adjustable UV Laser Current 0 100 Pulse Frequency 10 5000 Hz and Cutting Speed Microscope Stage Computer and trackball controlled Range 155 x 125 mm repeatability 2 um Bright Field Illumination Source 100 W halogen lamp or High intensity LED illumination system Fluorescent lamp EXFO X Cite 120 PC metal halide fluorescence illumination system Lamp life 2000 hours Filters Color Excitation Emission Red 570 630 nm gt 655 nm Green 503 548 nm gt 065 nm Blue 455 495 nm gt 910 nm Optional UV 340 390 nm gt 410 nm Optional Triple Dichroic Excitation 385 400 475 493 545 565 nm Emission 450 465 503 533 582 622 nm Operating Temperature 18 30 C Operating Humidity 60 relative humidity noncondensing Base Unit Dimensions Height 28 in 71 cm Width 22 in 56 cm Depth 30 in 76 cm Arcturus T Laser Capture Microdissection System User Guide 91 Appendix A System Specifications Item Description Weight 110 lb 50 kg Work Surface 36 in x 72 in 92 cm x 180 cm with vert
127. ranty A qualified Life Technologies service professional will install your Arcturus Instrument This will assure optimum performance and minimize risk of damage to the instrument Note Any damage encountered as a result of self installation may not be covered under instrument warranty or may result in voiding of the warranty Please contact Life Technologies for full details UV Laser Housing Controller Assembly Est Laser Head 2 Gy UV Control IR Laser D Nikon Control Interlock Bypass 14510 00 Laser Head 1 UV Control p Control TD TrackBall Aux 1 Trackball Mic Control UV Laser Touch Screen Monitor AC Inlet Host Computer Illumination Cap Robot AC Power Cord To 110 240 VAC Microscope Console Figure 61 Connections between components of the Arcturus T System 98 Arcturus Laser Capture Microdissection System User Guide AppendixB Installation Instructions Installation qualification Your Life Technologies service representative will complete the IQ checklist in the Arcturus TAdjustment Calibration and Test Procedure 14601 00 document To obtain a copy of this procedure for your records call Life Technologies technical support at 1 800 831 6844 and select option 5 Operational qualification Your Life Technologies service representative will complete the OQ checklist in the Arcturus Adjustment Calibration and Test Procedure 14601 00 docum
128. rol various microscope functions This section explains how to use these panels in general and also provides instructions for using them during an LCM session on the Arcturus Instrument Note For complete details on the Nikon Eclipse Ti E microscope please refer to the manufacturer s product user manual The front operation panel has a display area a magnification knob and several sets of buttons all of which are described below and shown in Figure 12 Viewing status from the Status Display window The status display window displays the microscope status including the z position You can use the Display buttons directly below the window to select the format of the display Selecting the display format with the Display buttons Press the up and down Display buttons to select the display format that you want to use The display formats are described in the Nikon user manual ArcturusX Laser Capture Microdissection System User Guide 25 Chapter 3 Starting the System and Loading Samples Controlling illumination with the Brightness button Press the Brightness button to change the illumination of the Status Display window and the LEDs on the operation panels Status Display window Brightness Z Reset button D O O z button Display l DISPLAY e Perfect buttons SOSA focus m O buttons Optical Path disabled or O O ro Magnification AE selection knob io 1 5X or 1X Fi
129. s Port 80 installed in the left microscope port To view the image using Eyepiece the operating software you must select either the L100 or Port 20 L80 optical paths The Arcturus operating software will default to the L100 position when initiated Note To use the L80 position you must also have the optional Microscope Binoculars PN 0200 6228 Use this selection to view the sample through the eyepieces while simultaneously displaying it through the operating software R100 Right Side High If you have installed the optional High Resolution Second Port 100 Resolution Camera for the Arcturus Instrument PN14379 00 it is Imaging installed in the right microscope port To use this optional camera for high resolution imaging select the R100 position Note The optional High Resolution Second Camera is intended only for imaging and is not available for microdissection Microdissection can only be performed using the integrated Arcturus Instrument camera mounted in the left port position L100 or L80 Reseting the Z position with the Z reset button Press the Z Reset butotn to reset the Z position on the Status Display window to zero The Z axis position display will be increased or decreased in conjunction with movement of the nosepiece with the zero position as the new reference point The Arcturus operating software recognizes the absolute Z position of the objective nosepiece If you press the Z Reset b
130. s or frame membrane slides the Freehand and Defined Circle Area tools mark the perimeter to define the UV cut line and IR capture spots will be placed automatically serving only to attach the sample to the capture To use LCM spots on a membrane slide glass or frame as for glass slides check the LCM Only button in the Select tool pane above the drawing tool icons Table 9 Drawing tools Tool Suggested Use Use the Freehand drawing tool to draw a free form shape indicating the cells to be microdissected a The software will automatically close the drawing item if the ends are close enough together and will draw IR capture spots inside the shape You can set the distance at which the software will automatically close the object in the Tools Options tab of the Select Options dialog box Use the Defined Circle Area tool to indicate circular areas for microdissection Z The software will draw IR capture spots inside the circle You can also set a fixed size for the Defined Circle Area tool in the Tools Options tab of the Select Options dialog box Use the IR Spot Line tool to indicate a line along which you want to place the IR capture spot Fz Arcturus T Laser Capture Microdissection System User Guide Chapter 5 Selecting Cells for Microdissection Tool Suggested Use Use the Eraser tool to remove any unwanted marks made by the drawing tools EE a Use the Ruler tool to measure distances or ob
131. strument without the arm shield in place Keep hands out of the deck area when the instrument is spotting WARNING ELECTRICAL SHOCK HAZARD Severe electrical shock can result from operating the instrument without its instrument panels in place Do not remove instrument panels High voltage contacts are exposed when instrument panels are removed from the instrument Fuses AN WARNING FIRE HAZARD For continued protection against the risk of fire replace fuses only with fuses of the type and rating specified for the instrument Power WARNING ELECTRICAL HAZARD Grounding circuit continuity is required for the safe operation of equipment Never operate equipment with the grounding conductor disconnected Plug the system into a properly grounded receptacle with adequate current capacity A WARNING ELECTRICAL HAZARD Use properly configured and approved line cords for the voltage supply in your facility Arcturus T Laser Capture Microdissection System User Guide Appendix C Safety Laser safety Laser classification The Arcturus Instrument is classified as a Class 1 laser device During normal operation non removable panels and safety interlocks limit access to laser radiation The ArcturusXT Instrument has one or more Class 3b lasers The infrared beam used for capture and the ultraviolet beam used for cutting are not visible Avoid direct skin and eye exposure to this laser radiation Laser safety requirements To ensure saf
132. te tab and locate the IR Laser either manually or automatically To locate the IR laser manually a Click Locate IR The live image darkens and the IR laser guide light is visible Figure 53 IR Locate tab If necessary increase the Brightness settings located in the Inspect tools pane until the IR laser guide light is visible b To save the IR laser location place the cursor on the center of the IR laser guide light and click c Click the OK button next to the Locate IR button in the Microdissect Options dialog box A blue plus sign will appear on the live image at the new location of the IR laser 74 Arcturus T Laser Capture Microdissection System User Guide Chapter 6 Microdissecting Cells and Tissue 6 To locate the IR laser automatically d Click Auto Locate IR TheArcturus software turns on the IR capture laser at low power and tries to locate the laser If the laser is successfully located the system draws a blue plus sign on the main image where the laser is located e To verify the IR laser location click the IR Capture Test Spot icon and perform a test fire f Look at the main image and see if the laser spot is located within the circle e If the blue plus sign is within the LCM spot click OK at the bottom of the dialog to close the dialog box and and save the changes e If the blue plus sign does not fall in center of the LCM spot follow the steps to manually locate the capture laser Note
133. tem User Guide Extracting tissue To extract tissue from the CapSure HS Caps 1 Use clean tweezers to remove the cap from the cap insertion tool and place the cap with the sample facing up into the alignment tray Make sure the cap is properly seated in the alignment tray following the directions in the CapSure HS LCM Caps manual 2 Use clean tweezers to remove and position the ExtracSure Device over the cap The fill port on the ExtracSure Device should be facing up Device onto the cap The ExtracSure Device should fit securely into place At this point the ExtracSure Device should be firmly sealed to the CapSure HS Cap 4 Add extraction buffer to the device Do not remove the assembled ExtracSure and CapSure HS Device from the alignment tray until incubation is completed 4 3 Use tweezers to firmly push down the ExtracSure Chapter 7 Extracting Cells and Tissue Figure 59 CapSure HS Cap and 5 Place a 0 5 mL microcentrifuge tube over the fill port ExtracSure device in and allow the samples to incubate as described in the appropriate extraction procedure allignment tray 6 Place the tube into a microcentrifuge and spin briefly to bring the buffer to the bottom of the tube ArcturusX Laser Capture Microdissection System User Guide 81 Chapter 7 Extracting Cells and Tissue 82 Arcturus T Laser Capture Microdissection System User Guide CHAPTER 8 Maintenance and
134. tion Petri Dish e For applications that use only the IR capture laser use either plain glass or PEN membrane glass slides Acquiring slides PEN membrane slides and Arcturus Live Cell Microdissection dishes are available and Petri dishes for purchase from Life Technologies For part numbers see Table 5 Plain glass slides and large format slides 38 mm and 50 mm are available from major lab suppliers Table 5 PEN membrane slides Item Part Number PEN membrane frame slides LCM0521 50 slides PEN membrane glass slides LCM0522 50 slides PEN membrane frame slides for live cell microdissections LCM0530 5 slides LCM0531 25 slides Arcturus Live Cell Growth Chamber 5000300 6 dishes sterile Arcturus Microdissection Petri Dish 5000301 6 dishes sterile Arcturus Laser Capture Microdissection System User Guide 21 Chapter 2 Preparing Samples For information about using these products For more information about mounting tissue sections onto metal framed PEN membrane slides see Protocol 9 on the Life Technologies website For more information on live cell microdissection see Application Notes 11 and 13 Optimized Protocol for Laser Microdissection of Living In Vitro Cells on the Life Technologies website Use this link to access this information Arcturus T Literature Preparing tissue samples Using frozen tissue samples 22 For microdissection you can use tissue sections prep
135. tion while the surrounding tissue remains intact on the slide Contact with the microdissected material is maintained throughout the entire process You can then examine the captured material and place the cap directly into a microcentrifuge tube for extracting DNA RNA or protein ArcturusX Laser Capture Microdissection System User Guide 11 Chapter 1 System Overview Types of cut and capture Outline of the microdissection process 12 Photoablation the volatilization of tissue by light emitted from an ultraviolet UV laser can be used in conjunction with the IR capture laser In one application of photoablation a relatively wide moat can be ablated around the region of interest and then the remaining cells can be captured by the IR capture laser This minimizes contamination of the cells due to collateral pick up during the capture process This cut and capture method can be used for tissue mounted on regular glass slides An alternate cut and capture method can be used for tissue samples mounted on membrane such as 2 um thick polyethylene napthalate PEN either on glass or ina metal frame Here the UV cutting laser is used to cut a narrow outline around the region of interest after which the entire region within the outline is captured on the CapSure cap With this method a small number of IR capture points suffices to lift a region making it much faster than LCM alone for microdissecting larger areas
136. urus Laser Capture Microdissection System User Guide 9 About This Guide Safety labels on The following CAUTION WARNING and DANGER statements may be displayed on instruments Applied Biosystems instruments in combination with the safety symbols described in the preceding section Beale English Francais symbol ZN CAUTION Hazardous chemicals Read the SDSs before handling ATTENTION Produits chimiques dangereux Lire les fiches techniques de s ret de mat riels avant toute manipulation de produits CAUTION Hazardous waste Refer to SDS s and local regulations for handling and disposal ATTENTION D chets dangereux Lire les fiches techniques de s ret de mat riels et la r gulation locale associ es a la manipulation et l limination des d chets AA WARNING Hot lamp AVERTISSEMENT Lampe br lante WARNING Hot Do not remove lamp until 15 minutes after disconnecting supply AVERTISSEMENT Lampe br lante apr s avoir d connect le c ble d alimentation de l appareil attendre environ 15 minutes avant d effectuer un remplacement de la lampe WARNING Hot Replace lamp with an Applied Biosystems lamp AVERTISSEMENT Composants br lants Remplacer la lampe par une lampe Applied Biosystems CAUTION Hot surface ATTENTION Surface br lante DANGER High voltage DANGER Haute tension WARNING To reduce the chance of electrical shock do not remove c
137. us T Laser Capture Microdissection System User Guide Documentation and Support Related documentation The following documents are available for the Arcturus Laser Capture Microdissection LCM System and related products Document Patt Description number Instruments Arcturus Instrument Quick 5003009 Provides a concise set of instructions for using Reference Card Rev B the Arcturus Instrument Arcturus Instrument Large 5000313 Provides instructions for installing the optional Format Slide Stage Insert Rev C Large Format Slide Stage Insert This Installation Guide document is shipped with the product Arcturus Instrument Petri 5000554 Provides instructions for installing the optional Dish Stage Insert Installation Rev B Petri Dish Stage Insert This document is Guide shpped with the product Arcturus Instrument 4458765 Provides instructions for using the AutoScanXT AutoScanXT Software Rev A software This document is embedded in the Module User Manual online help for this product Arcturus Instrument 4458766 Provides instructions for installing the Software Installation Rev B Arcturus software This document Instructions QRC accompanies each new release of the software Arcturus Instrument Tip 4458768 Provides tips for microdissecting at 100x using Sheet Using the 100x Rev A the Arcturus Instrument Objective Arcturus Instrument Tip 4458767 Provides tips for working with
138. ut before the IR capture e Tab Length is the distance in microns of tab space in between each UV cut length e UV CutLength is the length of UV cut line in microns between tab insertion Arcturus Laser Capture Microdissection System User Guide Chapter 6 Microdissecting Cells and Tissue 6 e UVCuttingSpeed is the speed of the stage movement which affects the speed of UV cutting Note Increase this value to cut more quickly however if this value is too high UV cutting can be less accurate This value corresponds to the UV Cutting Speed slider in the Microdissect tool pane e UVCurrent is the current of the UV cutting laser Note Increase this value to apply more current to the line of ablation As this value increases the cutting width will increase This value corresponds to the UV Current slider in the Microdissect tool pane e UVPulseFrequency is the frequency repetition rate of the UV cutting laser Note Pulse frequency above 1000 Hz will attenuate the UV laser current 5 Click OK to close the dialog box and save your changes Note All the changes you make to the IR SpotsPerCutLength Tab Length UV Cut Length UVCuttingSpeed UVPower and UVPulseFrequency will be applied retroactively to all drawing items These settings are also available in the Information area to the right of the slide overview To display these settings in the Information area click in the UV Cutting Speed in the Microdissect tools p
139. utton the focus position on the Arcturus software interface will no longer match the Z position read out on the Status Display window If you want to realign the values between the Arcturus software interface and the Status Display window you must turn the instrument off and then on again and restart the operating software Disabling the Perfect Focus buttons PFS The Perfect Focus buttons marked PFS on the panel are disabled when you are using the ArcturusXT Instrument These controls are used for the Perfect Focus System option which is not available for the Arcturus Instrument Selecting the magnification level with the Magnification Selection knob Use the Magnification Selection knob to change between 1X and 1 5X objective magnification Apply this setting for all microscope output ports Arcturus Laser Capture Microdissection System User Guide 27 Chapter 3 Starting the System and Loading Samples Using the left operation panel Focus knob Dia illumination lamp brightness control knob manual mode only 28 The left operation panel contains a large focus knob a smaller brightness knob focus selection and objective switchs and a lamp ON OFF button These controls are described below and shown in Figure 13 Focus selection switch manual Coarse mode only Fine ExFine Objective ap a d Obj 4 i disabled 4 Dia ON OFF i 7 illumination lamp ON OFF button disabled
140. w you to adjust the microscope to control the fluorescence lamp and to work with images Information 1 5X Inspect b i le Focus utton Magnification 4 Focus controls Camera 100x button ses Tiled image 60x Re button E Brightness Video recorder Objective __ 40x meme controls Dutton controls Ph Phase contrast 20x Ge button Ooms DIC button 10x 7 Flourescence f button 2X Livelmage Mag 47 i butt HAAA D Figure 21 The Inspect tools pane Note Depending upon your instrument configuration you may see alternate objectives in the objective controls ArcturusX Laser Capture Microdissection System User Guide 37 Chapter 4 Inspecting Slides Viewing the slides Adjusting the brightness 38 To view the slides 1 Move the stage to display an area of interest in the main image in one of three ways e Move the trackball e Click the Move Stage tool in the Select tools pane and then press the stylus on the main image window Drag the stylus to move the stage e In the slide overview image tap the stylus at the location of interest The stage moves to that location and the main image updates centered on the location where you tapped 2 To view a different slide tap or click the Slide button for the slide of interest 40x The stage will move the selected slide over the objective and the slide overview will update to show 20x the new slide 3 Change the objective as ne
141. way from the tissue but still within the cap 3 Click the IR Laser Test Fire button to test the capture laser 4 Inspect the laser spot You should see a dark ring with a clear center as shown in Figure 48 on page 62 If you do not see this open the Microdissect Options dialog box adjust the Power and duration slider in the IR Spot Sizes tab Microdisecting the Sample Note The order of the two procedures cut and capture depends upon a setting in the Microdissect tab in the Microdissect Options dialog box The section below describes the default order which is IR capture followed by UV cutting When you are ready to microdissect 1 Click the Cut and Capture button see Figure 50 on page 67 68 Arcturus Laser Capture Microdissection System User Guide Capturing cells using the capture and cutting tools separately Chapter 6 Microdissecting Cells and Tissue 6 e Ifyou did not place a cap on the slide the instrument automatically picks up a cap from the load area and places it on the slide at the center of the main image window If you did place a cap on the slide the instrument picks up the cap and moves it to the area represented in the main image The cap location is outlined in green in the slide overview image e The instrument fires the IR capture laser at the position of each IR capture spot to fuse the cells to the cap e After IR capture is completed the instrument automatically continues with UV cutting
142. you O c ae load caps without opening the Load Options dialog box but to use this shortcut the corresponding QC Caps position must be Figure 56 Cap and empty slide handling area IMPORTANT Make sure that you remove any caps whose positions have been cleared in the CQ caps area Failure to do so can result in a new cap running into the one remaining You can also view Cap Information name and notes in the information area to the right of the slide overview a image You can edit the CapName or CapNotes here see er RETRA Figure 57 Editing here is the same as entering the information in the Load Options dialog box Cap 4 Information CapNotes The notes for this cap Figure 57 Cap information area The ArcturusXT software creates the cap interaction history file the first time a cap is off loaded It then updates this file throughout the session This file contains information about the caps the slides and the capture groups as well as the total area of microdissected tissue To view the cap interaction history 1 Click outside of the Arcturus application so that you can see the desktop 2 Navigate to the StudyFolder ReportSubfolder that you set in the Load Options dialog box 3 Double click on the CapReport YYMMDD HHMM html file The cap interaction history opens in an Internet Explorer window Using the Laser Bypass feature 78 For safety reasons the lasers are disabled when outside of the CapSur

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ArcturusXT™ Laser Capture Microdissection System User Guide