Setting up checklist
- 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.
- 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.
- 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
Last modified 2mo ago