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Hi,
I made a modest compilation of Clarity methods and articles in this Web
page.
http://www.cbm.uam.es/mkfactory.esdomain/webs/CBMSO/plt_Servicio_Pagina.aspx
?IdServicio=19&IdObjeto=400
If somebody misses something or find some errors, please tell me to
correct/include them.
Good luck.
Carlos Sánchez Martín
Servicio de Microscopía Óptica y Confocal (SMOC) (Lab. 310)
(Optical and Confocal Microscopy Facility)
Centro de Biología Molecular Severo Ochoa (UAM-CSIC)
C/Nicolás Cabrera, 1. Universidad Autónoma de Madrid
Cantoblanco. E-28049. Madrid. Spain
Tlf. 34-91 196 4613/4643
Fax. 34-91 196 4420
E-mail: [log in to unmask]@cbm.csic.es
Blog: http://csanchezmad.wordpress.com
Web: http://www.cbm.uam.es/confocal
Red Española de Microscopía Óptica Avanzada (REMOA)
(Spanish Network of Advanced Optical Microscopy)
http://remoa.wikispaces.com
http://remoa.net
Plataforma de Microscopía para Biociencias
Red de laboratorios de la Comunidad de Madrid
http://www.madrimasd.org/Laboratorios/plataformas-red/ficha.asp?IdPreli=3
-----Mensaje original-----
De: Confocal Microscopy List [mailto:[log in to unmask]] En
nombre de Paul Herzmark
Enviado el: martes, 18 de febrero de 2014 18:31
Para: [log in to unmask]
Asunto: Re: CLARITY objectives
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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
[log in to unmask]
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
<[log in to unmask]>wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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 <[log in to unmask]>
> 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
> > [log in to unmask]
> >
> > 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
> > <[log in to unmask]
> > >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 <
> > > [log in to unmask]> 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: [log in to unmask]
> > > >
> > >
> >
>
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