http://confocal-microscopy-list.275.s1.nabble.com/CLARITY-objectives-tp7581548p7581553.html
wait long enough. That puts it more on par with Scale. I wonder how they
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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.org>
> And 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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> >
> > 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:
> > >
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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]
> > >
> >
>