BakingTray
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Setting up checklist

Common steps

  • Select Sample -> New from the menu.
  • Turn on the laser and open the shutter
  • Embed the sample
  • Load sample into microscope.
  • Set sample directory and sample name.
  • Set imaging parameters in main BakingTray window.
  • Set channels to acquire in ScanImage CHANNELS window.
  • Raise sample and set blade position.
  • Take the first slice: ensure blade ends up about 3 mm beyond the edge of the agar.
  • Trim off excess agar and approach imaging depth.
  • Hit the Auto-Trim button to go from thicker trimming section to the final slice thickness and speed used for imaging.
  • Center sample under objective. Open laser shutter. Turn off room lights.
  • Find the sample surface, take a slice, then re-measure surface to check for stability.

autoROI

  • Press the ROI button in the preview window and use the slide schematic to draw a box where you expect the samples to be.
  • Take a preview scan to ensure that samples don't enter the green border area. This can be at lower power for now. If necessary, increase imaged area. You don't need to re-take the preview scan right now.
  • Increase frame averaging to tweak bidirectional scanning. (You can middle-click on the preview image to move the stages to that location in the sample)
  • Set laser power and power increase with depth at an area without too much white matter.
  • Reset frame averaging and take another preview at the final power and PMT settings. Hit AutoThresh.
  • BAKE
  • After acquisition you should crop the stitched images from even single samples or you will end up with larger datasets than you expect.

Non-autoROI

  • Find sample ventral midline and use this to set initial imaging area.
  • Take a preview scan to ensure that samples are correctly framed. Re-frame as needed.
  • Increase frame averaging to tweak bidirectional scanning. (You can middle-click on he preview image to move the stages to that location in the sample).
  • Set laser power and power increase with depth at an area without too much white matter. Ensure PMT gains are reasonable.
  • BAKE
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