Posted by
Paul Herzmark on
URL: http://confocal-microscopy-list.275.s1.nabble.com/CLARITY-objectives-tp7581548p7581550.html
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Hi all,
I just came from a CLARITY workshop at the Karl Deisseroth's lab at
Stanford. That is where the technique was developed. Here are a couple of
points I learned.
To avoid burning your sample keep it away from the electrodes with some
plastic mesh.
Deisseeroth's lab is using a long working distance, high NA water immersion
lens that Olympus has custom built for them. Unfortunately we mortals don't
yet have access to that lens. In my lab we use a 20X 0.95NA 2 mm wd lens
from a two photon microscope. They are long working distance, high NA,
water dipping lenses. They aren't designed to have an intervening coverslip
and they are not designed for a sample immersed in FocusClear, as the
Clarity samples are. You will undoubtedly get spherical aberration but it
probably work better than your other options. Otherwise you can use
something like a 4X air lens with a long working distance to get deep. They
also use that in the Deisseroth lab, but only for the big picture.
One of the most important things I learned at the workshop is that for the
best results don't do the electrophoresis step. Just soak the tissue in the
SDS clearing solution for a long time (e.g. 2 months for a whole mouse
brain).
Look here for all kinds of CLARITY advice:
http://clarityresourcecenter.orgAnd good luck with your experiments!
Paul Herzmark
Microscopist to the stars
[hidden email]
Department of Molecular and Cellular Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA 94720-3200
(510) 643-9603
(510) 643-9500 fax
On Fri, Feb 14, 2014 at 3:02 PM, Craig Brideau <
[hidden email]>wrote:
> *****
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>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> 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:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > *****
> >
> > 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]
> >
>