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Hello all, 

Since you were so helpful the last time I asked about clarifying
techniques, I thought I would shoot out one more question.  CLARITY
involves embedding the tissue in a polyacrylamide matrix and then
extracting the non-proteins, and it necessarily ends with the clarified
brain under a glass coverslip.  This rules out dipping objectives and
seems like it would eliminate the relative advantage of an upright scope.
The problem is that most coverslip-compatible water objectives that I can
find do not have the working distance to reach very far into the brain.

So far our best pics have come from a 25x multi-immersion lens from Zeiss
with a WD of about 0.57 mm, but even with that we would hit the glass
before we get far enough to see beyond the closer parts of the cortex.
Air objectives reach a lot farther of course but diffraction goes from bad
to worse as you go deep, and from what I understand dipping objectives
would have problems with the coverslip.

At the moment we have thought about sectioning the brain into sagittal or
coronal halves in order to lay the most important stuff close to the
glass.  

For those of you working with clarified samples, what objectives have you
found most useful?  Many thanks,


TF

Timothy Feinstein, Ph.D. | Confocal Manager
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
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