Setting up checklist

Common steps

• Select Sample -> New from the menu.

• Turn on the laser, open the shutter, and select the appropriate wavelength for your fluorophores.

• 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 if needed 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.

• Move objective down to imaging depth (e.g. 25 microns below sample surface).

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