AMF

SWC Advanced Microscopy

1 minute read

This post describes an easy set up procedure for brains: you won’t need to keep jumping over to the scope and sighting with the laser or even set the laser to a visible wavelength.

The procedure works for us because we always set up the mouse brain in the same way: bulb down, ventral surface facing the blade, and starting to image mid-way through the cerebellum. So long as the brain is close to vertical, we always use the same number of tiles in X and Y. Therefore we know in advance the the size of the image field (multiply the step size in X and Y by the number of tiles in those directions). The procedure is as follows:

  • Cut down to the cerebellum and ensure cuts are consistent.
  • Pan around the sample whilst imaging (hitting 2-D scan) and find the ventral surface. This is easy because the pons is prominent.
  • Find the mid-line, which is also pretty easy. Place the midline in the middle of the tile.
  • Place the ventral edge of the brain (interface of brain and agar) wherever you prefer based on where you want the ventral surface of the brain to begin in your stitched image stacks. We position it such that the ventral surface will end up near edge of the final stitched stack.
  • You know the size of the full, stitched, field of view. Move the X stage to the right by the full width of the stitched field. Move the Y stage away from the doors by half the length of the full stitched field of view. This is the front-left position.
  • You can now start the acquisition.

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