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.