BakingTray
  • BakingTray Documentation
  • Getting Started
    • Hardware requirements
    • Known issues
    • Initial Installation
      • Software installation
      • Setting up ScanImage
    • Hardware setup
      • Motor Setup
        • PI stage setup
        • Calibrating a linear actuator
        • Verifying stage motions
      • Setting up a VT1000 vibratome
      • Setting up the laser
    • Finishing the install
      • Check the noise on your amplifiers
      • Starting BakingTray
      • Settings Files
      • Calibration
        • Basic calibrating procedures
        • Calibrating image size
        • Achieving high stitching accuracy
        • Fine-tuning positioning accuracy
        • Stitching tweak walkthrough
    • Stitching data
  • Users
    • Introduction
    • Sample preparation
    • User Guide
      • Starting BakingTray
      • Step 0: Loading the sample
      • Step 1: Setting imaging parameters
      • Step 2: Preparing the sample
      • Step 3: Selecting the imaging area
      • Step 4: Starting the acquisition
      • Step 5: Concluding the acquisition
      • Setting up checklist
      • Resuming an acquisition
      • Manual ROI acquisitions
    • Excitation choices
    • Choosing imaging settings
    • Troubleshooting
      • Hardware problems
      • Computer problems
      • Cutting problems
      • Imaging problems
    • Data structure
    • autoROI
  • Developers
    • Developers
      • Code overview
      • Developer notes
      • Motion control classes
      • The recipe file
      • Auto-ROI
      • Simulated mode
      • Contributing
    • FAQ
    • Gallery
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  2. User Guide

Setting up checklist

PreviousStep 5: Concluding the acquisitionNextResuming an acquisition

Last updated 1 year ago

Common steps

  • Select Sample -> New from the menu.

  • Turn on the laser, open the shutter, and .

  • 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

select the appropriate wavelength for your fluorophores