Zeiss PALM MicroBeam Laser Capture Microdissection Manual

Power-up protocol

  1. Main power switches
  2. (Optional) X-cite lamp for fluorescent samples
  3. Control box, turn the key from 'Off' to 'On'
  4. Computer

The Mercury lamp should always be first-on and last-off. This prevents any electrical surges caused by ignition damaging other equipment on the same circuit.

  • Log-in to the computer
  • Open the program: PALMRobo 4.5 Pro.

Loading sample(s) and finding the target region(s):

toolbar-annotated_cropped
    1. Either push back the light arm or click Icon_load_position in the toolbar to move the stage to load position. Secure the slide in the slide holder and make sure the sample side is up. The slide holder can hold 3 slides. When done, either return the light arm or click Icon_load_position again to return the stage to observation position.
    2. Use the joystick to move the stage and position the sample above the objective.
    3. On the touch screen on the microscope, choose an objective. The 20x, 40x, and the 63x objectives on the microscope have correction rings. When using a standard glass slide (1 mm thick), check and make sure the black line mark on the corrective ring is aligned with "1" on the lense barrel. For #1.5 glass coverslip, the mark should point at the dot.
    4. Position
      Magnification
      NA
      Correction Ring
      1
      5X
      0.25
      No
      2
      10X
      0.50
      No
      3
      20X
      0.40
      Yes
      4
      40X
      0.60
      Yes
      5
      63X
      0.75
      Yes
      6
      Empty
    5. The transmitted light (TL) on the microscope is automatically turned on, and the sample can be viewed both in the eyepieces and on the computer monitor. Light intensity can be adjusted with the wheel on the lower front of the microsocpe. Focus on the sample.
    6. In the program window, choose AxioCamICc1 camera and check the box next to 'Auto Live'.
    7. If a fluorescent sample will be observed, on the touch screen, choose the 'reflector' for blue (DAPI), green (FITC), or red (TR) fluorescence. Turn on the reflected light (RL), and in 'Light Path' choose 'Eyepiece'. The fluorescence camera should be selected.

      Microscope tools

      Alternatively, in the program window, find the 'Microscope' tools, click 'Reflected Light', select an 'Illumination'.

    8. Make the image on the monitor in focus and click 'Freeze' icon Icon-freeze in the tool bar. The shutter will be closed to minimize the bleaching of the fluorescence in the sample. Re-click the 'Freeze Mode' icon to inactivate the 'Freeze Mode'for further operations.

    The stage can be positioned through the following ways:

    1. Joystick
    2. Arrow keys on the keyboard
    3. Scroll bars in the image window (below and to the right of the image)
    4. Pointing the mouse cursor at the edge of the image
    5. Clicking the 'Stage Mode' icon and move the mouse over the image
    6. 'Navigator' function
    7. 'Centering' tool in the graphic toolbar and clicking a point that is desired to be centered.

    (optional) Calibrating the laser marker when laser cuts don't align with the regions drawn. (Also available on video here )

    1. Activate 'Stage Mode' by clicking on the icon Icon-Stage Mode, or by 'F7' key
    2. Move mouse over the image and find a relatively homogenous region, then take your hand off the mouse
    3. Using the foot pedal, fire the laser
    4. Exit the 'Stage Mode' by hitting the 'F7' key again
    5. In the 'Menu Bar', select 'Calibration', then 'Laser Marker'
    6. A circular target shows up. Move and center the target over the point where the laser has been fired at. Left click your mouse.
    7. Done!

    Loading collection tubes:

    1. Activate RoboMover by clicking the 'Capture Device' icon Icon-capture device. 'Capture Device' will be opened in a separate window.
    2. In the 'Capture Device' window, click 'Change Collector'
    3. The collector will be moved to loading position
    4. Load collection tube(s) (demo on-site)
    5. Click 'Scan Collector Type'
    6. A diagram of the collector appears, click on the cap of a tube in the diagram to move the tube in collection position. A collection tube can also be selected from the graphic toolbar or in the 'Element List' (see later).

    Cutting and catapulting

    Graphic Tools-annotated_cropped
    1. From the 'Graphic Tools' at the bottom of the window, select the type of laser operation for cutting and/or catapulting.
      For membrane-mounted specimen, 'RoboLPC' is a commonly used combination of cutting and subsequent catapulting; For glass-mounted samples, 'AutoLPC' or 'Close cut+AutoLPC' should be selected; For line-shaped structure, 'LineAutoLPC' is uaually used; 'Cut' or 'LPC' is for simple cutting or catapulting.
    2. Select the drawing tool from the graphic toolbar, and draw region(s) on the screen. If it is a big area, you can 'Creates Grid Rectangles' to divide the area into smaller ones.
    3. If multiple regions are defined, the regions (so called 'elements' in PALMRobo) can be easily managed in element list. Open the 'Element List' (Icon-Element List). All regions are organized in the list. Each element can be selected (also highlighted) by clicking on that row. Double-clicking on one element opens a window that laser operations can be further modifed for 'function type', 'objective','collection tube', etc. Operations on multiple elements (regions) can be exacuted if those elements are selected while holding down the 'Shift' key on the keyboard.
    4. The selected elements can be deleted by clicking the icon Icon-Element List_delete seleted in 'Element List', or simply using 'delete' key on the keyboard; Clicking Icon-delete allwill delete all elements (regions). Back in the Toolbar, clicking on icon Icon-delete last element deletes the last element (region).

    Check catapulting

    1. Click 'Cap Check' Icon-cap check to position the stage and the objective
    2. Raise the objective to focus and look for catapulted materials in the cap
    3. When done, click 'Point of Origin'(at the same position of 'Cap Check') to ruturn the stage to the original location

    Adjustment of laser power and focus (Also available on video here )

    It is highly recommended that you test the power AND focus of the laser for cutting and/or catapulting before you start working on your interested regions. In general, the catapulting power is higher than the cutting power and the laser has to be defocused for catapulting.

    1. Use graphic tool to draw a 'Z' line in a homogenous area. In 'Speed' tools, select 'Cutting'and a low speed.
    2. (To adjust laser power,) Start cutting and adjust the laser power by clicking on the arrows at the bottom of the 'Energy' column. Select the minimal laser power that generates a clear and fine cut.
    3. (To adjust laser focus,) start cutting and adjust the laser focus by clicking on the arrows at the bottom of the 'Focus' colomn. Use the focus that results in a clear and fine cut.
    4. Repeat the above steps if it is necessary.

    Snapshot of image

    Click 'Save Image' icon Icon-save iamgeand choose a location to save the images.

    To finish your session

    Check the online booking calendar to see if you should turn off the system or leave it on for the next user. Record your use of the system using the CoreResearch booking system

    1. Close the PALMRobo 4.5 Pro software
    2. If somebody is signed up in the next hour, please leave the whole system on (just need to log off the computer and stop here).
    3. If nobody else is signed up within one hour after you
      1. Shut down the computer from the start menu
      2. Turn off the controller box (Key from 'On' to 'Off')
      3. Record the X-cite hours and turn off X-cite light (if you have used it)
      4. Switch off the power strips
    4. Cover the microscope