http://confocal-microscopy-list.275.s1.nabble.com/CLARITY-objectives-tp7581548p7581562.html
Clarity for the brain tissue we are working with (white matter). We may
need to play around with it a bit more. I'll check out your reference!
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>
> Craig,
>
> There is another new technique for clearing brain tissue called SeeDB (DB =
> deep brain). I tried it with mouse thymus (my interest) and it worked
> better than my un-optimized Clarity did. And it was much quicker, cheaper
> and easier. It does not, however, work for immunolocalization which Clarity
> does.
>
> Here is the paper describing the SeeDB method. In
> the supplementary information there is a table comparing methods for
> clearing brain tissue (but not including Clarity).
> 1) Meng-Tsen Ke, Satoshi Fujimoto, Takeshi Imai. "SeeDB: a simple and
> morphology-preserving optical clearing agent for neuronal circuit
> reconstruction". Nature Neuroscience 16, 1154-1161 (2013) doi:
> 10.1038/nn.3447
>
>
> Paul Herzmark
> Specialist
>
[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 Sun, Feb 16, 2014 at 12:05 AM, Craig Brideau <
[hidden email]
> >wrote:
>
> > *****
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> >
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> >
> > It's interesting you can skip the electrophoresis if you are willing to
> > wait long enough. That puts it more on par with Scale. I wonder how
> they
> > compare head to head this way?
> >
> >
> > On Fri, Feb 14, 2014 at 6:27 PM, Paul Herzmark <
[hidden email]>
> > wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > > *****
> > >
> > > 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:
> > >
> > > > *****
> > > > To join, leave or search the confocal microscopy listserv, go to:
> > > >
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]
> > > > >
> > > >
> > >
> >
>