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To join, leave or search the confocal microscopy listserv, go to:
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*****
Many thanks to everyone who answered both on and off the list. Your
feedback will help our researchers a ton.
I also want to congratulate the guys who developed SeeDB and ClearT for
coming up with unique names for their techniques. Searching for CLARITY
and Scale-related information with Google is a headache.
All the best,
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]
On 2/19/14, 2:53 AM, "Carlos Sanchez Martin" <[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,
>
>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.as
>px
>?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
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>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]
>> > > >
>> > >
>> >
>>
|
