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Acquisition problems and solutions

Rob Campbell edited this page Apr 4, 2019 · 5 revisions

Funny gradient across sample

You see this odd gradient in your images and the gradient is worse in upper optical planes. It may be disappear completely in deeper optical planes.

What is happening

The issue stems from the blade not being flat with respect to the Y stage. The Y stage is that which moves the sample along the direction parallel to the blade. In the above image, the blade is cutting ventral to dorsal. The blade is deeper on the right of the sample than the left. The image looks a little dimmer on the right because the imaging plane is partly out of the brain.

Temporary fix

Start the first plane deeper into the sample to avoid the problem area.

Full solution for super-users

Correct the blade angle by doing a roll adjustment. i.e. don't alter the pitch of the balde (it's angle of attack into the tissue). The blade angle can be corrected by imaging a fluorecent block of agar (dope it with marker pen fluid) and tweak the angle until two points about 15 mm apart are within 10 microns of each other after cutting.

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