http://confocal-microscopy-list.275.s1.nabble.com/CLARITY-objectives-tp7581548p7581561.html
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
Here is the paper describing the SeeDB method. In
1) Meng-Tsen Ke, Satoshi Fujimoto, Takeshi Imai. "SeeDB: a simple and
reconstruction". Nature Neuroscience 16, 1154-1161 (2013) doi:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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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:
> >
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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:
> >
> > > *****
> > > 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]
> > > >
> > >
> >
>