Posted by
Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/CLARITY-objectives-tp7581548p7581549.html
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I've been working on comparing some of the different clearing techniques.
Clarity was quite difficult, and we're still working out the bugs in the
technique. If you don't have the tissue lined up well you can fry it if it
brushes the electrodes, and it can also get fairly warm. The electrodes are
also quite expensive, being made of platinum, so if you DO burn one it is
quite an expensive mistake! It also took quite a while for it to really
work, so you have to be patient to get the tissue really clear.
In terms of imaging lenses, can you float or pin the section under water
and then use a water dipping lens?
Craig
On Fri, Feb 14, 2014 at 10:22 AM, Feinstein, Timothy <
[hidden email]> wrote:
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>
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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
> Phone: 616-234-5819 | Email:
[hidden email]
>