CLARITY objectives

[Feinstein, Timothy]

*****
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: [phone] | Email: [hidden email]

[Craig Brideau]

*****
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: [phone] | Email: [hidden email]
>

[Paul Herzmark]

*****
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
[phone]
[phone] 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: [phone] | Email: [hidden email]
> >
>

[Craig Brideau]

*****
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 <[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
> [phone]
> [phone] 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: [phone] | Email: [hidden email]
> > >
> >
>

[Volodymyr Nechyporuk-Zloy]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Timothy,

There is an online PhD thesis dedicated to STED nanoscopy of the living brain
http://ediss.uni-goettingen.de/handle/11858/00-1735-0000-000D-F0B1-E
It has a chapter dedicated to different aberrations in brain imaging which
can be useful. Few objectives were tested as well.

Cheers,
Volodymyr

[Paul Herzmark]

*****
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
[hidden email]

Department of Molecular and Cellular Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA  94720-3200
[phone]
[phone] fax


On Sun, Feb 16, 2014 at 12:05 AM, 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
> *****
>
> 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
> > [phone]
> > [phone] 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: [phone] | Email: [hidden email]
> > > >
> > >
> >
>

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

We actually tried SeeDB but found it doesn't work as well as Scale or
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!

Thanks,

Craig



On Tue, Feb 18, 2014 at 10:30 AM, 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
> *****
>
> 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
> [phone]
> [phone] fax
>
>
> On Sun, Feb 16, 2014 at 12:05 AM, 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
> > *****
> >
> > 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
> > > [phone]
> > > [phone] 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: [phone] | Email: [hidden email]
> > > > >
> > > >
> > >
> >
>

[jmvdwin]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi,

You may also consider the "ClearT" formulation
http://www.ncbi.nlm.nih.gov/pubmed/23444362
Kuwajima T, Sitko AA, Bhansali P, Jurgens C,
Guido W, Mason C. ClearT: a detergent- and
solvent-free clearing method for neuronal and
non-neuronal tissue. Development 2013;140:1364-1368.

In my - fairly limited - experience, FPs
endogenous fluorescence runs down pretty quickly
(in a matter of hours or a couple of days
depending of the FP), both in ClearT and in SeeDB.
Imaging promptly after completion of the clearing
procedure is therefor advisable.
The same holds true for organic clearing agents,
e.g. BABB - Erturk A Nat Protoc 2012;7:1983-1995.
- or benzyl ether - Becker K PLoS ONE
2012;7:e33916.) while FP intrinsic fluorescence
is retained for a while in Scale solution.

I have not tried the Clarity approach yet...

Cheers.

JM

At 19:33 18/02/2014, you wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>We actually tried SeeDB but found it doesn't work as well as Scale or
>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!
>
>Thanks,
>
>Craig
>
>
>
>On Tue, Feb 18, 2014 at 10:30 AM, 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
> > *****
> >
> > 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
> > [phone]
> > [phone] fax
> >
> >
> > On Sun, Feb 16, 2014 at 12:05 AM, 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
> > > *****
> > >
> > > 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
> > > > [phone]
> > > > [phone] 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: [phone] | Email: [hidden email]
> > > > > >
> > > > >
> > > >
> > >
> >

Jean-Marie.

Jean-Marie Vanderwinden, M.D., Ph.D.
Directeur de Recherche F.N.R.S. / Research
Director, National Fund for Scientific Research (Belgium)

Université Libre de Bruxelles (ULB)

ULB Neuroscience Institute - Neurophysiology lab
http://www.ulb.ac.be/medecine/neurophy/

Light Microscopy Facility http://limif.ulb.ac.be/

Postal address:
Laboratoire de Neurophysiologie (Prof. S.N. Schiffmann),
Faculté de Médecine, Campus Erasme, CP 601,
Université Libre de Bruxelles,
808 route de Lennik, B-1070 Brussels, Belgium.

(: Secretary: +(32).2.555.42.30, Direct: +(32).2.555.69.88
7: +(32).2.555.41.21,
. [hidden email]

Skype: Jean Marie Vanderwinden
12voip / netappel: jeanmarievanderwinden  

[SANCHEZ MARTIN, CARLOS]

*****
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.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. [phone]
E-mail: [hidden email]/[hidden email]
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:[hidden email]] En
nombre de Paul Herzmark
Enviado el: martes, 18 de febrero de 2014 18:31
Para: [hidden email]
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
[hidden email]

Department of Molecular and Cellular Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA  94720-3200
[phone]
[phone] fax


On Sun, Feb 16, 2014 at 12:05 AM, 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
> *****
>
> 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
> > [phone]
> > [phone] 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: [phone] | Email: [hidden email]
> > > >
> > >
> >
>

[Feinstein, Timothy]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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: [phone] | Email: [hidden email]







On 2/19/14, 2:53 AM, "Carlos Sanchez Martin" <[hidden email]>
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. [phone]
>E-mail: [hidden email]/[hidden email]
>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:[hidden email]] En
>nombre de Paul Herzmark
>Enviado el: martes, 18 de febrero de 2014 18:31
>Para: [hidden email]
>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
>[hidden email]
>
>Department of Molecular and Cellular Biology
>479 Life Science Addition
>University of California, Berkeley
>Berkeley, CA  94720-3200
>[phone]
>[phone] fax
>
>
>On Sun, Feb 16, 2014 at 12:05 AM, 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
>> *****
>>
>> 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
>> > [phone]
>> > [phone] 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: [phone] | Email: [hidden email]
>> > > >
>> > >
>> >
>>