# 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](/users/choosing-wavelength.md).
* 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


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