Confocal Imaging

Contents:

 

Starting the Confocal Microscope

  • Switch on lamp
    • -white switch
    • -black button

wait until light turns orange

  • Switch on computer
    • Press enter (no password)
  • Switch on microscope (biorad mrc scum)
  • Switch on laser (turn key) but only if you are taking photos. The laser is above the computer.
  • On the microscope, turn dial on the side to A for viewing down the microscope, and to C for taking photos.
  • Blue light picks up green fluorescence. Green light picks up red fluorescence.
  • Hanging cord moves the stage left and right and up and down.
  • You can change the objectives, usually use 20x or 10x.
  • On the computer, use the program ‘laser sharp 2000’ (not the emulation program)
  • Login/Pass Required

 

Taking Photos

  • Switch dial on microscope to C. Turn off fluorescence.
  • Set laser speed to slow (move down)
  • Leave the zoom at about 1.
  • Change the objective to what’s on the microscope.
  • Select direct scanning for the laser. (use kalman when merging).
  • Select the focus motor, either manually or on the screen to make the picture bright.

 

  • Adjust the krypton/argon to low; between 10-30.
  • Adjust the iris fairly low, but raise if needed (iris is PMT bar).
  • Adjust the gain fairly high. If its too high, you’ll get background, but usually ok to have it right up.
  • Adjust the offset by changing the colour of the image to SETCOL. You want the black spaces to appear black etc.

Make these adjustments for each colour (red, blue, green). Remember to keep the focus motor the same between colours.

  • Up the top of the screen, press the button with the 3 boxes and tick each colour to optimise the picture.
  • The * button takes each picture, press it again to stop.
  • The venn diagram merges the pictures, remember to kalman 1-3 times.
  • Switch back to ‘direct’ before you take the picture for a different sample.

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