LMD6500 User Manual - Microscopy Core Facility
Contents
1. orange color when the laser is cutting 8 5 You can choose Move and Cut to cut specimen manually using mouse If you use Move and Cut always go back to Draw and Cut when you are done 8 6 The most common dissection mode draw cut select it in the cut shape s manual While in Single Shapes mode you can draw a new shape and the previous shape will be automatically erased While in Multiple Shapes mode every time a shape is created it s saved in the Shape list These shapes will be recut whenever Start Cut is selected 8 7 You can save your shapes as well as the shape list data To save the shape list information go to File and select Save Shape list Data 8 8 You can use the specimen overview option to easily navigate specific areas of your sample detail See 7 Obtaining an Overview of the Specimen 8 8 You can save image by selecting Save Image As in the File menu 9 Viewing the cut specimen in the collector cup 9 1 Click the Collector button in the toolbar 9 2 The stage will move and the collection cup will come into view 9 3 Use the joystick to navigate and find the tissue that was collected in the cap 9 4 Return to the specimen image of previous cutting position by pressing Specimen on the toolbar 10 Shut Down Procedure 10 1 Activate lefthand Unload slide and Unload PCR Tube holders and remove the slide and PCR tube holder 102. button in the software toolbar 2 2 Place tube holder 0 2 or 0 5 mL into stage support with caps facing upwards there is only 1 way it will fit 2 3 Click OK in the loading window 2 4 The center position is kept empty for viewing the sample 3 Calibrate the Reference Point RP for the tube holder tray Purpose To calibrate collection device and preparation of caps You should set the Reference Point the first time you use the unit 3 1 If there is a sample slide on the specimen holder clicking on the leftmost Unload icon controls specimen holder The Change Specimen window will appear Remove the slide and the specimen holder Click Continue 3 2 Clicking the rightmost Unload icon the Change Collector Device window will appear Place the collection device on the stage and make sure it s properly going straight back Click on the Move to Reference Point button in the change Collector Device window 3 3 The microscope should automatically switch to 5x and the collection device should move to the reference point RP The RP should be in the center of the field of view You may need to focus the Utah State University Microscopy Core Facility camera to clearly view the RP If it does not use the arrows in the window to move the RP to the center of our field view 3 4 Once the RP has properly positioned click OK to save the settings 3 5 Make sure No Cap
3. 2 Make sure you re in Specimen mode 10 3 Select the 5x lens in Microscope control 10 4 Quit software by File gt Exit 10 5 Turn off Laser by turning key switch to vertical position 10 6 Shut down computer 10 7 Turn off fluoresence power if fluorence is used 10 8 Turn off microscope controller box
4. is selected under Collector Device Tube Caps 4 Loading Specimen Slide 4 1 To put a slide on the stage or exchange slides press the lefthand Unload button in the toolbar The stage then move to a lower position for slide change Withdraw specimen holder by pulling towards yourself 4 2 Optional Remove the laser safety shield 4 3 Slide is oriented with the section and film facing downwards sections are below the film 4 4 Insert the slide onto specimen holder then slide the holder back into position on the stage 4 5 Click on Continue 4 6 Replace the laser safety shield Laser will not fire unless the safety shield is correctly in place 5 Setting Laser Parameters 5 1 To change laser setting select the Control option in the Laser menu or click the Laser icon 5 2 Power adjust the power of the laser to the cutting beam 5 3 Aperture adjust thickness of the laser 5 4 Speed how quickly the line is cut 5 5 Specimen Balance changes the current setting s to provide more or less cutting These values varies between objectives Value should be between 5 and 20 5 6 Factory Settings Returns the settings to the factory defaults for the selected objective lens 5 7 Offset focus offset for the lens set at the factory for each lens 6 Laser Calibration Purpose To ensure the figure drawn by the mouse on the screen is identical to the shape cut by the laser 6 1 Open the Laser and Mi
5. Utah State University Microscopy Core Facility Leica Laser Microdissection Scope LMD 6500 Manual Purpose LMD is a UV laser based microdissection system combines with an automated upright microscope LMD uses laser ablation technology to cut out single cells or cell clusters directed along the contours of the area of interest The area cut by the laser are transferred to PCR test tubes by gravity alone without contamination 1 Start Up Procedure Wear powder free nitrile gloves while using the LMD 6500 1 1 Turn on computer then turn on microscope controller box 1 2 Logon to computer by clicking the user icon type in user as the password 1 3 Click the desktop Leica Laser Microdissection icon to start the software 1 4 If you plan to use fluorescence during your laser microdissection session then turn on the fluorescence illuminator power supply white box on the right of microscope 1 5 Turn the key to the On position to start up the laser only when you are about ready to cut the sample The Laser Power Supply Silver Gray box labeled CryLaS needs to fully warm up from a cold start When the key is first turned on the LED display will show a single orange LED This is the laser Warm up mode After a few minutes you will see the two green LED s when laser is in the Standby Mode Make sure the Laser Safety Shield is properly in place 2 Loading Specimen Tubes 2 1 Click on the rightmost Unload
6. cCtrl windows You can use these to calibrate adjust laser setting change objectives and adjust illumination 6 2 Navigate using the joystick y axis is on the top x axis is on the bottom Utah State University Microscopy Core Facility 6 3 Switch to the cutting objective of your choice 5x 10x or 40x using left buttons on the joystick the touch pad Objective icon or the MicCtrl icon in the software 6 4 Find and focus on an empty area of a slide using the joystick 6 5 On the right hand panel of the software draw shape s choose Single Shapes mode Select the Calibrate option in the Laser menu or using Laser icon and confirm message on the screen with Yes 6 6 The laser cuts a cross in all 4 corners of the current view Follow the pop up instruction move the mouse cursor to the center of each cross and confirm by clicking on it Select the Start cut key and ensure that laser precisely follows your drawing 6 7 Conclude calibration procedure by clicking OK in the dialog box 6 8 To validate laser setting make sure the Close Line option is not selected and draw S shaped line using Line key in the toolbar 7 Obtaining an Overview of the Specimen 7 1 Bring your specimen into focus with a low magnification objective 7 2 Select Option gt Specimen Overview from menu bar 7 3 A window will open select Create Specimen Overview 7 4 Position the up
7. per left corner of the specimen in the eyepieces click Save top left position 7 5 Position the lower right corner of the specimen in the eyepieces click Save bottom right position 7 6 Press Scan at the bottom of setup window the system starts to create a slide overview 7 7 Once your image has been scanned you can activate the yellow box by double clicking on it and change the magnification The activated the box will then turn green and you have the ability to move 7 8 If you click Save Overview Image the montage will be saved 8 Cutting and Collecting Area of Interest 8 1 Locate your area of interest Select a cap A B C D by clicking the corresponding circular red marking at the Collector Device Tube Caps window The selected cap turns green You can change the names of the caps by clicking on the letter You can also assign each cap its own corresponding color by selecting the color field just below the cap to change the color 8 2 Draw a shape around target area by pressing Line Ellipse or Rect icon in the left toolbar 8 3 If you are using the Line tool the Close line option can be activated if the drawn contour needs to be automatically closed The drawing line is closed upon release of the mouse key Utah State University Microscopy Core Facility 8 4 Select Draw Cut cutting mode and cut specimen by pressing the Start Cut key The 2 green light will be
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