HOW TO MAKE MOSAIC SEQUENTIAL SCANS IN MULTIPLE LEVELS WITH THE LEICA SP2?

(Notes:

the software does not allow to make only one level mosaic in sequential mode; sorry, choose at least two z-levels

See pictures at the end)

  1. Switch on the pc, microscope, and ventilation unit with the 3 red knobs (right to left). Take off the cover from the microscope and switch on the fluorescence mercury lamp.
  2. In Windows start screen, open your domain (GI / IMM / DCN/ IWWR).
  3. Set the 10x objective up and when Windows is loaded start the LCS software (icon on desktop). (If a message appears: Attention microscopy stage cannot reach lower limit/…/ turn the focus knob a little).
  4. The stage has two ranges: with the word “scan” positioned horizontally,close to the red dot is the small range,3/4 rotated is large range, for example for multi-wells plates.
  5. Tilt the arm of the condenser to the rear position so that there is more space below the condenser.
  6. Answer yes to the questionon protection for stage calibration; the motorized stages moves to set its reference position.
  7. When stage calibration is ready, choose the desired objective(with appropriate immersion medium), insert the sample in the holder (cover slip down) and focus (visual).
  8. Switch required lasers on (take normally into account that at least 30 min warming up is required)
  9. Go to scan
  10. If you want re-attribute (right mouse click on low bars)the function for the adjustment of offset,and gain as well as the sensitivityto black knobs. Note that by default the most right black knob has been attributed the function z-wide field, which is the coarse z focus. The knob z-position is for the fine z-scanner. Do not overwrite main entry settings (DCN, IWWR, IMM).
  11. Choose the mode (XYZ), format (number of pixels), speed of scans (Hz), scan field rotation (usually default 0), if desired check UV on (for imaging with the 405 nm laser 20x for the 10x and 20x lenses, 63x oil for 40x and 63x oil, and 63x glycerol for the corresponding objective),set pinhole opening, and for xyz stacks choose z-wide under the icon z-Scan.
  12. Move the stage to the expected brightest area within the region of interest. Adjust the zoom factor and define for each channel the acquisition range (wavelengths) to avoid overlapping. Using Continuous imaging for a first evaluation, set for each channel laser power, gain and offset and save each setting with an own name.After finding the brightest optical section in a rapid z-focus through the sample, refine for each channel the values for laser power, gain, offset, lut,and save parameter settings again (overwrite) for each channel. Comment: In general record an as broad as possible dynamic range but compromise on laser power to avoid bleaching, and on gain which amplifies noise. Avoid saturation and adjust the basic offset just around pixel value 1 using the Q-lut [level 1 green, saturation blue]). Keep in mind that some other fields may be even brighter (thus going off scale) than the one used for this adjustment.
  13. Make one image at random, just to allow the software to save today’s images as a “name.lei” file in the appropriate directory on the data disc.
  14. Using Continuous scanning choose (rapidly again, to avoid bleaching) the desired highest z-level to start (click Begin) the z stack and the one to finish (click End). Determine the number of z-stacks to record in total (Series and Sect. buttons) and the number of averages (frame averages).
  15. EVEN IF ONLY 1 LEVEL IS DESIRED, CHOOSE AT LEAST 2 LEVELS, OTHERWISE THE PROGRAM GETS STUCK.
  16. Move to motorized stage with the joystick to the first area in the sample where to start the mosaic (check with Continuous scan or in visual with transmission illumination).
  17. Open Stage and choose Tile scan. Click Make position: position 1 is retained.
  18. Move to last area to image with mosaic, in Scan (as opposed to visual),click Make position: position 2 is retained.
  19. At least Raw data should be clicked on and under options chooseat least -10% overlapping. Still in Stage click Calculate Map: a yellowrectangle/square representing the total covered area of the mosaic is drawn automatically. The number offrames in horizontal and vertical direction (xy) is noted in Scan fields of map. Keep the Stage function (window) open!
  20. In sequential scan drag the previously recorded single channel parameter settings. IMPORTANT: choose Frame average (not line average: this causes a program crash). Keep the Sequential scan widow open.
  21. Switch tothe still open Stage Window and click the grey Start button. The scan starts. Progress can be monitored in the left panel of the software and in the Stage field (green color for scanned fields, yellow for still to be done)
  22. When ready SAVE.

Short check list of items to check for optimal adjustment:

  • Stage activated
  • Type of z-stage
  • Objective
  • Focus and positioning
  • Lasers settings
  • Channel settings
  • Zoom
  • Pinhole
  • Speed
  • Format
  • Rotation
  • UV
  • Offset
  • Gain
  • LUT
  • Begin and end in z levels and # z scans (at least 2)
  • Position 1 and position 2 of stage
  • Map overlapping and raw
  • Sequential
  • Number of frame averages