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Photostimulate ROIs.#

  • Powerful1 405 nm laser
  • Simultaneous image acquisition during photostimulation is possible
  • ND Sequence Acquisition allows for complex experiments with ND Acquisition

Calibration#

The Galvo XY unit needs to be calibrated before use, but you can save/load calibration files for future use. Different calibration files are needed between single-cam and dual cam modes.

Currently, we should have calibration files for the 60x (single-cam) and 100x (single-cam and dual cam) objectives (at 37C). These calibration files should be accurate for the lower mag objectives, but since no one's FRAPing with them, it's been low on my priority list to check.

How to calibrate (text)#

  1. Load a slide with a fluorophore able to be bleached or activated with a 405nm laser.
    • For 60x/100x objectives, reasonably confluent cell monolayers with autofluorescence works.
  2. Select a FRAP OC (or move the upper turret such that the 405-FRAP filtercube is in place)
    • Change the bit-depth to 16-bit to avoid oversaturation.
    • Set exposure time to something short like 50ms
  3. Uncheck the camera ROI
    • N.B.: The calibration must be done without any camera ROI selected.
  4. Open the "Galvy XY" window and set laser power to something like 1.5%
  5. Open Galvo XY settings by clicking on the gear icon
  6. Set "Volts low" to -1.5 and "Volts high" to 1.5
  7. Press "Calibrate"
    • Use "Toggle (Reflect laser)" instead of "Manual (Bleach point)"
    • Check the box for "Invert Y Axis" while making sure that "Invert X Axis" is unchecked
  8. Choose spots for the laser to go to and then tell the software where the center of the spot is.
    • Click on a spot in any quadrant while remaining somewhat close to the center until you get a sense for how far away you can go
      • If your chosen spot is within the camera ROI, you should see a bright spot in the Live view
    • Move the red cross to the center of the focused laser point.
      • Set the LUT settings appropriately--the buttons for autoscaling LUT may come in handy.
      • Make sure you zoom in to be sure you're choosing the right pixel.
    • When satisfied that the red cross is in the center, press the + symbol within the Galvo XY to add
    • Repeat until the usable field of view is well sampled. Aim for at least 12 throughout the rectangular FOV.
  9. Hit "Next" when satisfied and test by drawing an ROI and stimulating it.

How to calibrate (video)#

Calibration Video

  1. 120mW measured at the fiber tip before entering the Yokogawa CSU-X1 unit ↩