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How "deep" can you see in a brain slice?

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Petr Busek

How "deep" can you see in a brain slice?

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Dear all,
I am trying to view fluorescently labeled glioma cells invading into a 400um
thick brain slice on an Olypus FV300. Has anyone experience with this and
how "deep" it is reasonable to expect to see in the slice using a confocal
microscope? How can you maximize this depth? (selection of objectives,
processing of the slice....)
Thanks for any suggestions, Petr.

Petr Busek, MD, PhD
Charles University in Prague
First Faculty of Medicine
Laboratory of Cancer Cell Biology
Institute of Biochemistry and Experimental Oncology
U Nemocnice 5
128 53 Prague 2
Czech Republic
www.lf1.cuni.cz/lbnb
Fax +420 224 965 826

Hugh Newman

Re: How "deep" can you see in a brain slice?

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To join, leave or search the confocal microscopy listserv, go to:
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I'm no medical guy but I work for Japanese microscope company that will not be named.
Brain isn't very translucent so I wouldn't hold out much hope. I would say 100um tops, maybe even closer to 50um. After this your image will start to degrade quite a bit. Any deeper than that and you are going to want a 2P-Confocal. I've imaged colloidal suspensions that are ~100um thick, and they are very close to being index matched. At the maximum depth the image wasn't that pretty.

Maybe some more Neuro people will have a better answer for you.

Good luck

H

Hugh Newman

Graduate Researcher

Dept. Physics and Physical Oceanography

Memorial University

St. Johns, Newfoundland, Canada

> Date: Sun, 5 Feb 2012 13:53:33 -0600
> From: [hidden email]
> Subject: How "deep" can you see in a brain slice?
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
> I am trying to view fluorescently labeled glioma cells invading into a 400um
> thick brain slice on an Olypus FV300. Has anyone experience with this and
> how "deep" it is reasonable to expect to see in the slice using a confocal
> microscope? How can you maximize this depth? (selection of objectives,
> processing of the slice....)
> Thanks for any suggestions, Petr.
>
> Petr Busek, MD, PhD
> Charles University in Prague
> First Faculty of Medicine
> Laboratory of Cancer Cell Biology
> Institute of Biochemistry and Experimental Oncology
> U Nemocnice 5
> 128 53 Prague 2
> Czech Republic
> www.lf1.cuni.cz/lbnb
> Fax +420 224 965 826

ChrisWilms

Re: How "deep" can you see in a brain slice?

In reply to this post by Petr Busek

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

From my experience using a 488 nm laser line, you can expect to get
about 80 microns into the slice tissue.

For anything deeper, I would recommend moving to far red dyes and
excitation and beyond that 2P.

Best of luck,

Christian

> Dear all,
> I am trying to view fluorescently labeled glioma cells invading into
> a 400um
> thick brain slice on an Olypus FV300. Has anyone experience with
> this and
> how "deep" it is reasonable to expect to see in the slice using a
> confocal
> microscope? How can you maximize this depth? (selection of objectives,
> processing of the slice....)
> Thanks for any suggestions, Petr.

George McNamara

Re: How "deep" can you see in a brain slice?

In reply to this post by Petr Busek

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

Fixed and cleared: all the way:

Three-dimensional imaging of the unsectioned adult spinal cord to assess
axon regeneration and glial responses after injury. </pubmed/22198277>
Ertürk A, Mauch CP, Hellal F, Förstner F, Keck T, Becker K, Jährling N,
Steffens H, Richter M, Hübener M, Kramer E, Kirchhoff F, Dodt HU, Bradke
F. Nat Med. 2011 Dec 25;18(1):166-71. doi: 10.1038/nm.2600. PMID: 22198277

Scale: a chemical approach for fluorescence imaging and reconstruction
of transparent mouse brain. </pubmed/21878933> Hama H, Kurokawa H,
Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A,
Miyawaki A. Nat Neurosci. 2011 Aug 30;14(11):1481-8. doi:
10.1038/nn.2928. PMID: 21878933.

A colleague here at the U told me his lab had much better clearing and
imaging with the Erturk et al method than with the versions of Hama et
al's Scale that they tried (no, I do not know which many variants they
tried or how extensively they tested each). This colleague told me that
with the Erturk et al method they needed to image the same day (and the
sooner the better). The Erturk et al method uses tetrahydrofuran (THF)
to strip the lipids from the tissue, followed by immersion in benzyl
alcohol:benzyl benzoate (BABB). BABB has a long history of use in
optical clearing - see various papers by Bob Zucker, for examples:

Whole insect and mammalian embryo imaging with confocal microscopy:
morphology and apoptosis. </pubmed/17051584>* *Zucker RM. Cytometry A.
2006 Nov 1;69(11):1143-52. PMID: 17051584

Confocal laser scanning microscopy of whole mouse ovaries: excellent
morphology, apoptosis detection, and spectroscopy. </pubmed/16969804>*
*Zucker RM, Jeffay SC. Cytometry A. 2006 Aug 1;69(8):930-9. PMID: 16969804

I will hypothesize here that 2,2'-thiodiethanol (TDE) might be a better
ultimate destination after THF. For TDE see:

2,2'-thiodiethanol: a new water soluble mounting medium for high
resolution optical microscopy. </pubmed/17131355>* *Staudt T, Lang MC,
Medda R, Engelhardt J, Hell SW. Microsc Res Tech. 2007 Jan;70(1):1-9.
PMID: 17131355

See also Stan Vitha's post(s) here on transitioning specimens into TDE
and imaging.

For fresh tissue - that is, hemisectioned mouse brain: sac the mouse,
flush the RBCs, take out the brain, slice in half (along a line that
will bisect the glioma mass that you introduced by stereotaxic
injection, being careful not to have cells up the needle track), bring
to the confocal - a user of mine in L.A. on a Leica SP1 confocal, 10x
objective lens (probably 0.4 NA), went 800 um. On a City of Hope
LSM510/MP, I helped image hemisectioned mouse brains previously
implanted with GFP+ neural stem cells (Argon ion laser) plus DAPI
(Coherent Chameleon laser, probably 750 nm excitation) several hundred
micrometers deep. Again, one of the keys is to flush out the blood cells
from the mouse vasculature - they scatter a lot more than mouse brain
tissue. I have never been involved with brain slices - hopefully those
protocols flush the blood cells after sac'ing the mouse.

George

On 2/5/2012 2:53 PM, Petr Busek wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
> I am trying to view fluorescently labeled glioma cells invading into a 400um
> thick brain slice on an Olypus FV300. Has anyone experience with this and
> how "deep" it is reasonable to expect to see in the slice using a confocal
> microscope? How can you maximize this depth? (selection of objectives,
> processing of the slice....)
> Thanks for any suggestions, Petr.
>
> Petr Busek, MD, PhD
> Charles University in Prague
> First Faculty of Medicine
> Laboratory of Cancer Cell Biology
> Institute of Biochemistry and Experimental Oncology
> U Nemocnice 5
> 128 53 Prague 2
> Czech Republic
> www.lf1.cuni.cz/lbnb
> Fax +420 224 965 826
>
>

--

George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

Craig Brideau

Re: How "deep" can you see in a brain slice?

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

The clearing method gives some really impressive results from the examples
I've seen. You would want to make sure to have a long-working distance
lens on hand to take full advantage of it though, yes? What sort of
aberrations would you get imaging deeply? The clearing takes care of all
the scatter, which is the biggest problem, but wouldn't the tissue still
have some refractive index boundaries?

Craig

2012/2/5 George McNamara <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
>
> Fixed and cleared: all the way:
>
> Three-dimensional imaging of the unsectioned adult spinal cord to assess
> axon regeneration and glial responses after injury. </pubmed/22198277>
> Ertürk A, Mauch CP, Hellal F, Förstner F, Keck T, Becker K, Jährling N,
> Steffens H, Richter M, Hübener M, Kramer E, Kirchhoff F, Dodt HU, Bradke F.
> Nat Med. 2011 Dec 25;18(1):166-71. doi: 10.1038/nm.2600. PMID: 22198277
>
> Scale: a chemical approach for fluorescence imaging and reconstruction of
> transparent mouse brain. </pubmed/21878933> Hama H, Kurokawa H, Kawano H,
> Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A. Nat
> Neurosci. 2011 Aug 30;14(11):1481-8. doi: 10.1038/nn.2928. PMID: 21878933.
>
> A colleague here at the U told me his lab had much better clearing and
> imaging with the Erturk et al method than with the versions of Hama et al's
> Scale that they tried (no, I do not know which many variants they tried or
> how extensively they tested each). This colleague told me that with the
> Erturk et al method they needed to image the same day (and the sooner the
> better). The Erturk et al method uses tetrahydrofuran (THF) to strip the
> lipids from the tissue, followed by immersion in benzyl alcohol:benzyl
> benzoate (BABB). BABB has a long history of use in optical clearing - see
> various papers by Bob Zucker, for examples:
>
> Whole insect and mammalian embryo imaging with confocal microscopy:
> morphology and apoptosis. </pubmed/17051584>* *Zucker RM. Cytometry A. 2006
> Nov 1;69(11):1143-52. PMID: 17051584
>
> Confocal laser scanning microscopy of whole mouse ovaries: excellent
> morphology, apoptosis detection, and spectroscopy. </pubmed/16969804>*
> *Zucker RM, Jeffay SC. Cytometry A. 2006 Aug 1;69(8):930-9. PMID: 16969804
>
> I will hypothesize here that 2,2'-thiodiethanol (TDE) might be a better
> ultimate destination after THF. For TDE see:
>
> 2,2'-thiodiethanol: a new water soluble mounting medium for high
> resolution optical microscopy. </pubmed/17131355>* *Staudt T, Lang MC,
> Medda R, Engelhardt J, Hell SW. Microsc Res Tech. 2007 Jan;70(1):1-9. PMID:
> 17131355
>
> See also Stan Vitha's post(s) here on transitioning specimens into TDE and
> imaging.
>
>
> For fresh tissue - that is, hemisectioned mouse brain: sac the mouse,
> flush the RBCs, take out the brain, slice in half (along a line that will
> bisect the glioma mass that you introduced by stereotaxic injection, being
> careful not to have cells up the needle track), bring to the confocal - a
> user of mine in L.A. on a Leica SP1 confocal, 10x objective lens (probably
> 0.4 NA), went 800 um. On a City of Hope LSM510/MP, I helped image
> hemisectioned mouse brains previously implanted with GFP+ neural stem cells
> (Argon ion laser) plus DAPI (Coherent Chameleon laser, probably 750 nm
> excitation) several hundred micrometers deep. Again, one of the keys is to
> flush out the blood cells from the mouse vasculature - they scatter a lot
> more than mouse brain tissue. I have never been involved with brain slices
> - hopefully those protocols flush the blood cells after sac'ing the mouse.
>
> George
>
>
>
>
>
> On 2/5/2012 2:53 PM, Petr Busek wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> *****
>>
>> Dear all,
>> I am trying to view fluorescently labeled glioma cells invading into a
>> 400um
>> thick brain slice on an Olypus FV300. Has anyone experience with this and
>> how "deep" it is reasonable to expect to see in the slice using a confocal
>> microscope? How can you maximize this depth? (selection of objectives,
>> processing of the slice....)
>> Thanks for any suggestions, Petr.
>>
>> Petr Busek, MD, PhD
>> Charles University in Prague
>> First Faculty of Medicine
>> Laboratory of Cancer Cell Biology
>> Institute of Biochemistry and Experimental Oncology
>> U Nemocnice 5
>> 128 53 Prague 2
>> Czech Republic
>> www.lf1.cuni.cz/lbnb
>> Fax +420 224 965 826
>>
>>
>>
>
>
> --
>
>
> George McNamara, PhD
> Analytical Imaging Core Facility
> University of Miami
>

Barbara Foster

Re: How "deep" can you see in a brain slice?

In reply to this post by Hugh Newman

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

Hi,

I was shocked when I visited Neuroscience this past Fall and saw what
they were doing with special clearing agents and special objectives
with multiphoton. The new limit is.... (ready?) at least 4 mm (no,
that is not microns) and I think that there may be some new
combinations which go as far as 8mm. This will be one of the topics
for the Editor's Page for the April issue of American Lab. I will be
writing that article this next week and can get you the current stats then.

Good hunting!
Barbara Foster, President and Sr. Consultant

Microscopy/Microscopy Education
P: (972)924-5310
W: www.MicroscopyEducation.com

We are now scheduling courses through June 2012

At 12:08 PM 2/5/2012, you wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>
>I'm no medical guy but I work for Japanese microscope company that
>will not be named.
>Brain isn't very translucent so I wouldn't hold out much hope. I
>would say 100um tops, maybe even closer to 50um. After this your
>image will start to degrade quite a bit. Any deeper than that and
>you are going to want a 2P-Confocal. I've imaged colloidal
>suspensions that are ~100um thick, and they are very close to being
>index matched. At the maximum depth the image wasn't that pretty.
>
>Maybe some more Neuro people will have a better answer for you.
>
>Good luck
>
>H
>
>Hugh Newman
>
>Graduate Researcher
>
>Dept. Physics and Physical Oceanography
>
>Memorial University
>
>St. Johns, Newfoundland, Canada
>
>
>
> > Date: Sun, 5 Feb 2012 13:53:33 -0600
> > From: [hidden email]
> > Subject: How "deep" can you see in a brain slice?
> > To: [hidden email]
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear all,
> > I am trying to view fluorescently labeled glioma cells invading
> into a 400um
> > thick brain slice on an Olypus FV300. Has anyone experience with this and
> > how "deep" it is reasonable to expect to see in the slice using a confocal
> > microscope? How can you maximize this depth? (selection of objectives,
> > processing of the slice....)
> > Thanks for any suggestions, Petr.
> >
> > Petr Busek, MD, PhD
> > Charles University in Prague
> > First Faculty of Medicine
> > Laboratory of Cancer Cell Biology
> > Institute of Biochemistry and Experimental Oncology
> > U Nemocnice 5
> > 128 53 Prague 2
> > Czech Republic
> > www.lf1.cuni.cz/lbnb
> > Fax +420 224 965 826
>

Deanne Veronica Catmull

Re: How "deep" can you see in a brain slice?

In reply to this post by Craig Brideau

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

Is it possible to section the brain prior to imaging? If you can make consistent sections of particular regions you will get far better results rather than imaging it whole. There are many issues with imaging the sample whole including laser penetration as the sample is quite opaque, working distance limits depending on your desired optical magnification requirements and blurring via spherical aberration if the sample is not 100% flat. You may be better off going 2P if sectioning is not an option.

Cheers,
Deanne.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Monday, 6 February 2012 2:22 PM
To: [hidden email]
Subject: Re: How "deep" can you see in a brain slice?

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

The clearing method gives some really impressive results from the examples
I've seen. You would want to make sure to have a long-working distance
lens on hand to take full advantage of it though, yes? What sort of
aberrations would you get imaging deeply? The clearing takes care of all
the scatter, which is the biggest problem, but wouldn't the tissue still
have some refractive index boundaries?

Craig

2012/2/5 George McNamara <[hidden email]>

Mark Cannell

Re: How "deep" can you see in a brain slice?

In reply to this post by George McNamara

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

I must admit to being a bit surprised that the Nature methods paper did not cite the prior use of THF in clearing insects... This was in 1958!

Tetrahydrofuran and its Use in Insect Histology

T. N. Salthouse. The Canadian Entomologist, 1958, 90:555-557, 10.4039/Ent90555-9
Regards Mark

On 5/02/2012, at 11:38 PM, George McNamara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Fixed and cleared: all the way:
>
> Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. </pubmed/22198277> Ertürk A, Mauch CP, Hellal F, Förstner F, Keck T, Becker K, Jährling N, Steffens H, Richter M, Hübener M, Kramer E, Kirchhoff F, Dodt HU, Bradke F. Nat Med. 2011 Dec 25;18(1):166-71. doi: 10.1038/nm.2600. PMID: 22198277
>
> Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. </pubmed/21878933> Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A. Nat Neurosci. 2011 Aug 30;14(11):1481-8. doi: 10.1038/nn.2928. PMID: 21878933.
>
> A colleague here at the U told me his lab had much better clearing and imaging with the Erturk et al method than with the versions of Hama et al's Scale that they tried (no, I do not know which many variants they tried or how extensively they tested each). This colleague told me that with the Erturk et al method they needed to image the same day (and the sooner the better). The Erturk et al method uses tetrahydrofuran (THF) to strip the lipids from the tissue, followed by immersion in benzyl alcohol:benzyl benzoate (BABB). BABB has a long history of use in optical clearing - see various papers by Bob Zucker, for examples:
>
> Whole insect and mammalian embryo imaging with confocal microscopy: morphology and apoptosis. </pubmed/17051584>* *Zucker RM. Cytometry A. 2006 Nov 1;69(11):1143-52. PMID: 17051584
>
> Confocal laser scanning microscopy of whole mouse ovaries: excellent morphology, apoptosis detection, and spectroscopy. </pubmed/16969804>* *Zucker RM, Jeffay SC. Cytometry A. 2006 Aug 1;69(8):930-9. PMID: 16969804
>
> I will hypothesize here that 2,2'-thiodiethanol (TDE) might be a better ultimate destination after THF. For TDE see:
>
> 2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. </pubmed/17131355>* *Staudt T, Lang MC, Medda R, Engelhardt J, Hell SW. Microsc Res Tech. 2007 Jan;70(1):1-9. PMID: 17131355
>
> See also Stan Vitha's post(s) here on transitioning specimens into TDE and imaging.
>
>
> For fresh tissue - that is, hemisectioned mouse brain: sac the mouse, flush the RBCs, take out the brain, slice in half (along a line that will bisect the glioma mass that you introduced by stereotaxic injection, being careful not to have cells up the needle track), bring to the confocal - a user of mine in L.A. on a Leica SP1 confocal, 10x objective lens (probably 0.4 NA), went 800 um. On a City of Hope LSM510/MP, I helped image hemisectioned mouse brains previously implanted with GFP+ neural stem cells (Argon ion laser) plus DAPI (Coherent Chameleon laser, probably 750 nm excitation) several hundred micrometers deep. Again, one of the keys is to flush out the blood cells from the mouse vasculature - they scatter a lot more than mouse brain tissue. I have never been involved with brain slices - hopefully those protocols flush the blood cells after sac'ing the mouse.
>
> George
>
>
>
>
> On 2/5/2012 2:53 PM, Petr Busek wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear all,
>> I am trying to view fluorescently labeled glioma cells invading into a 400um
>> thick brain slice on an Olypus FV300. Has anyone experience with this and
>> how "deep" it is reasonable to expect to see in the slice using a confocal
>> microscope? How can you maximize this depth? (selection of objectives,
>> processing of the slice....)
>> Thanks for any suggestions, Petr.
>>
>> Petr Busek, MD, PhD
>> Charles University in Prague
>> First Faculty of Medicine
>> Laboratory of Cancer Cell Biology
>> Institute of Biochemistry and Experimental Oncology
>> U Nemocnice 5
>> 128 53 Prague 2
>> Czech Republic
>> www.lf1.cuni.cz/lbnb
>> Fax +420 224 965 826
>>
>>
>
>
> --
>
>
> George McNamara, PhD
> Analytical Imaging Core Facility
> University of Miami

ChrisWilms

Re: How "deep" can you see in a brain slice?

In reply to this post by Craig Brideau

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

I do believe that refractive index mismatch becomes a problem when
clearing. There are objectives available specifically for BABB, which
would make it the ideal clearing tool. If index matching the objective
to your clearing agent isn't an option, then I believe the clearest
imaging can be achieved by matching the embedding medium to immersion
oil. A while back we tested multiple mounting media from Citifluor (no
commercial interests on my end and I am sure alternatives exist) that
had the same refractive index as immersion oil. The tissue slices were
nearly perfectly clear to the eye and imaging was much better than
when using BABB or TDE (we could clearly image dendritic spines in
densely stained brain slices).

Hope this helps,

Christian

> The clearing method gives some really impressive results from the
> examples
> I've seen. You would want to make sure to have a long-working
> distance
> lens on hand to take full advantage of it though, yes? What sort of
> aberrations would you get imaging deeply? The clearing takes care
> of all
> the scatter, which is the biggest problem, but wouldn't the tissue
> still
> have some refractive index boundaries?
>
> Craig
>
>
>
> 2012/2/5 George McNamara <[hidden email]>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >
>> *****
>>
>>
>> Fixed and cleared: all the way:
>>
>> Three-dimensional imaging of the unsectioned adult spinal cord to
>> assess
>> axon regeneration and glial responses after injury. </pubmed/
>> 22198277>
>> Ertürk A, Mauch CP, Hellal F, Förstner F, Keck T, Becker K,
>> Jährling N,
>> Steffens H, Richter M, Hübener M, Kramer E, Kirchhoff F, Dodt HU,
>> Bradke F.
>> Nat Med. 2011 Dec 25;18(1):166-71. doi: 10.1038/nm.2600. PMID:
>> 22198277
>>
>> Scale: a chemical approach for fluorescence imaging and
>> reconstruction of
>> transparent mouse brain. </pubmed/21878933> Hama H, Kurokawa H,
>> Kawano H,
>> Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A.
>> Nat
>> Neurosci. 2011 Aug 30;14(11):1481-8. doi: 10.1038/nn.2928. PMID:
>> 21878933.
>>
>> A colleague here at the U told me his lab had much better clearing
>> and
>> imaging with the Erturk et al method than with the versions of Hama
>> et al's
>> Scale that they tried (no, I do not know which many variants they
>> tried or
>> how extensively they tested each). This colleague told me that with
>> the
>> Erturk et al method they needed to image the same day (and the
>> sooner the
>> better). The Erturk et al method uses tetrahydrofuran (THF) to
>> strip the
>> lipids from the tissue, followed by immersion in benzyl
>> alcohol:benzyl
>> benzoate (BABB). BABB has a long history of use in optical clearing
>> - see
>> various papers by Bob Zucker, for examples:
>>
>> Whole insect and mammalian embryo imaging with confocal microscopy:
>> morphology and apoptosis. </pubmed/17051584>* *Zucker RM. Cytometry
>> A. 2006
>> Nov 1;69(11):1143-52. PMID: 17051584
>>
>> Confocal laser scanning microscopy of whole mouse ovaries: excellent
>> morphology, apoptosis detection, and spectroscopy. </pubmed/
>> 16969804>*
>> *Zucker RM, Jeffay SC. Cytometry A. 2006 Aug 1;69(8):930-9. PMID:
>> 16969804
>>
>> I will hypothesize here that 2,2'-thiodiethanol (TDE) might be a
>> better
>> ultimate destination after THF. For TDE see:
>>
>> 2,2'-thiodiethanol: a new water soluble mounting medium for high
>> resolution optical microscopy. </pubmed/17131355>* *Staudt T, Lang
>> MC,
>> Medda R, Engelhardt J, Hell SW. Microsc Res Tech. 2007 Jan;70(1):
>> 1-9. PMID:
>> 17131355
>>
>> See also Stan Vitha's post(s) here on transitioning specimens into
>> TDE and
>> imaging.
>>
>>
>> For fresh tissue - that is, hemisectioned mouse brain: sac the mouse,
>> flush the RBCs, take out the brain, slice in half (along a line
>> that will
>> bisect the glioma mass that you introduced by stereotaxic
>> injection, being
>> careful not to have cells up the needle track), bring to the
>> confocal - a
>> user of mine in L.A. on a Leica SP1 confocal, 10x objective lens
>> (probably
>> 0.4 NA), went 800 um. On a City of Hope LSM510/MP, I helped image
>> hemisectioned mouse brains previously implanted with GFP+ neural
>> stem cells
>> (Argon ion laser) plus DAPI (Coherent Chameleon laser, probably 750
>> nm
>> excitation) several hundred micrometers deep. Again, one of the
>> keys is to
>> flush out the blood cells from the mouse vasculature - they scatter
>> a lot
>> more than mouse brain tissue. I have never been involved with brain
>> slices
>> - hopefully those protocols flush the blood cells after sac'ing the
>> mouse.
>>
>> George
>>
>>
>>
>>
>>
>> On 2/5/2012 2:53 PM, Petr Busek wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> >
>>> *****
>>>
>>> Dear all,
>>> I am trying to view fluorescently labeled glioma cells invading
>>> into a
>>> 400um
>>> thick brain slice on an Olypus FV300. Has anyone experience with
>>> this and
>>> how "deep" it is reasonable to expect to see in the slice using a
>>> confocal
>>> microscope? How can you maximize this depth? (selection of
>>> objectives,
>>> processing of the slice....)
>>> Thanks for any suggestions, Petr.
>>>
>>> Petr Busek, MD, PhD
>>> Charles University in Prague
>>> First Faculty of Medicine
>>> Laboratory of Cancer Cell Biology
>>> Institute of Biochemistry and Experimental Oncology
>>> U Nemocnice 5
>>> 128 53 Prague 2
>>> Czech Republic
>>> www.lf1.cuni.cz/lbnb
>>> Fax +420 224 965 826
>>>
>>>
>>>
>>
>>
>> --
>>
>>
>> George McNamara, PhD
>> Analytical Imaging Core Facility
>> University of Miami
>>

Pertti Panula

Re: How "deep" can you see in a brain slice?

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

Hi all,

In our hands, the absolutely best images are obtained (from zebrafish
brain) using high-quality long working distance objectives, glycerol
optics and careful treatment of the samples while embedding slowly in
glycerol. In our hands, oil immersion optics have never matched the
results obtained with this method.
I can send more detailed protocols off list if needed.

Best regards

Pertti Panula

Quoting "Christian Wilms" <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I do believe that refractive index mismatch becomes a problem when
> clearing. There are objectives available specifically for BABB,
> which would make it the ideal clearing tool. If index matching the
> objective to your clearing agent isn't an option, then I believe the
> clearest imaging can be achieved by matching the embedding medium to
> immersion oil. A while back we tested multiple mounting media from
> Citifluor (no commercial interests on my end and I am sure
> alternatives exist) that had the same refractive index as immersion
> oil. The tissue slices were nearly perfectly clear to the eye and
> imaging was much better than when using BABB or TDE (we could
> clearly image dendritic spines in densely stained brain slices).
>
> Hope this helps,
>
> Christian
>
>
>> The clearing method gives some really impressive results from the examples
>> I've seen. You would want to make sure to have a long-working distance
>> lens on hand to take full advantage of it though, yes? What sort of
>> aberrations would you get imaging deeply? The clearing takes care of all
>> the scatter, which is the biggest problem, but wouldn't the tissue still
>> have some refractive index boundaries?
>>
>> Craig
>>
>>
>>
>> 2012/2/5 George McNamara <[hidden email]>
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>>> *****
>>>
>>>
>>> Fixed and cleared: all the way:
>>>
>>> Three-dimensional imaging of the unsectioned adult spinal cord to assess
>>> axon regeneration and glial responses after injury. </pubmed/22198277>
>>> Ertürk A, Mauch CP, Hellal F, Förstner F, Keck T, Becker K, Jährling N,
>>> Steffens H, Richter M, Hübener M, Kramer E, Kirchhoff F, Dodt HU, Bradke F.
>>> Nat Med. 2011 Dec 25;18(1):166-71. doi: 10.1038/nm.2600. PMID: 22198277
>>>
>>> Scale: a chemical approach for fluorescence imaging and reconstruction of
>>> transparent mouse brain. </pubmed/21878933> Hama H, Kurokawa H, Kawano H,
>>> Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A. Nat
>>> Neurosci. 2011 Aug 30;14(11):1481-8. doi: 10.1038/nn.2928. PMID: 21878933.
>>>
>>> A colleague here at the U told me his lab had much better clearing and
>>> imaging with the Erturk et al method than with the versions of Hama et al's
>>> Scale that they tried (no, I do not know which many variants they tried or
>>> how extensively they tested each). This colleague told me that with the
>>> Erturk et al method they needed to image the same day (and the sooner the
>>> better). The Erturk et al method uses tetrahydrofuran (THF) to strip the
>>> lipids from the tissue, followed by immersion in benzyl alcohol:benzyl
>>> benzoate (BABB). BABB has a long history of use in optical clearing - see
>>> various papers by Bob Zucker, for examples:
>>>
>>> Whole insect and mammalian embryo imaging with confocal microscopy:
>>> morphology and apoptosis. </pubmed/17051584>* *Zucker RM. Cytometry A. 2006
>>> Nov 1;69(11):1143-52. PMID: 17051584
>>>
>>> Confocal laser scanning microscopy of whole mouse ovaries: excellent
>>> morphology, apoptosis detection, and spectroscopy. </pubmed/16969804>*
>>> *Zucker RM, Jeffay SC. Cytometry A. 2006 Aug 1;69(8):930-9. PMID: 16969804
>>>
>>> I will hypothesize here that 2,2'-thiodiethanol (TDE) might be a better
>>> ultimate destination after THF. For TDE see:
>>>
>>> 2,2'-thiodiethanol: a new water soluble mounting medium for high
>>> resolution optical microscopy. </pubmed/17131355>* *Staudt T, Lang MC,
>>> Medda R, Engelhardt J, Hell SW. Microsc Res Tech. 2007 Jan;70(1):1-9. PMID:
>>> 17131355
>>>
>>> See also Stan Vitha's post(s) here on transitioning specimens into TDE and
>>> imaging.
>>>
>>>
>>> For fresh tissue - that is, hemisectioned mouse brain: sac the mouse,
>>> flush the RBCs, take out the brain, slice in half (along a line that will
>>> bisect the glioma mass that you introduced by stereotaxic injection, being
>>> careful not to have cells up the needle track), bring to the confocal - a
>>> user of mine in L.A. on a Leica SP1 confocal, 10x objective lens (probably
>>> 0.4 NA), went 800 um. On a City of Hope LSM510/MP, I helped image
>>> hemisectioned mouse brains previously implanted with GFP+ neural stem cells
>>> (Argon ion laser) plus DAPI (Coherent Chameleon laser, probably 750 nm
>>> excitation) several hundred micrometers deep. Again, one of the keys is to
>>> flush out the blood cells from the mouse vasculature - they scatter a lot
>>> more than mouse brain tissue. I have never been involved with brain slices
>>> - hopefully those protocols flush the blood cells after sac'ing the mouse.
>>>
>>> George
>>>
>>>
>>>
>>>
>>>
>>> On 2/5/2012 2:53 PM, Petr Busek wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>>>> *****
>>>>
>>>> Dear all,
>>>> I am trying to view fluorescently labeled glioma cells invading into a
>>>> 400um
>>>> thick brain slice on an Olypus FV300. Has anyone experience with this and
>>>> how "deep" it is reasonable to expect to see in the slice using a confocal
>>>> microscope? How can you maximize this depth? (selection of objectives,
>>>> processing of the slice....)
>>>> Thanks for any suggestions, Petr.
>>>>
>>>> Petr Busek, MD, PhD
>>>> Charles University in Prague
>>>> First Faculty of Medicine
>>>> Laboratory of Cancer Cell Biology
>>>> Institute of Biochemistry and Experimental Oncology
>>>> U Nemocnice 5
>>>> 128 53 Prague 2
>>>> Czech Republic
>>>> www.lf1.cuni.cz/lbnb
>>>> Fax +420 224 965 826
>>>>
>>>>
>>>>
>>>
>>>
>>> --
>>>
>>>
>>> George McNamara, PhD
>>> Analytical Imaging Core Facility
>>> University of Miami
>>>
>

--
Pertti Panula
Professor, Research Director
Neuroscience Center and
Institute of Biomedicine, Faculty of Medicine
POB 63, 00014 Univ Helsinki
Finland (Street Address: Haartmaninkatu 8, 00290 Helsinki)
Tel +358-9-191 25263
Fax +358-9-191 25261
Mob +358-40-5922 323

rfayek

Re: How "deep" can you see in a brain slice?

In reply to this post by Petr Busek

* Disclaimer - Huron Technologies manufactures and sells the TISSUEscope confocal laser scanner.

The TISSUEscope (http://www.huron-technologies.com/products/tissuescope.html) with its 3mm working distance has been used successfully to scan confocal fluorescence slices of cleared tissue sections more than 500um thick. It will be tried with thicker cleared tissue sections (1mm or more) when they become available. I'd be interested to know if you have such tissue sections.

Reda

Claudia Florindo

Re: How "deep" can you see in a brain slice?

In reply to this post by Hugh Newman

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

Dear Hugh

I have a user on my microscopy facility that has been using a combination of
2 protocols (using the scale reagent for making the samples transparent) and
a very strong permeabilization in order to make the antibody goo deep inside
the tissue.

The papers that he used where:

Nature Neuroscience --> v14n11pg1482

Microscopy and analysis --> Jan2011 pag5

I hope it helps

Good lick
Claudia.

______________________________________________

Claudia Florindo, PhD, Microscopy Unit Manager
[hidden email] /00351 289 244 489 ext:7489
Dept. de Ciências Biomédicas. e Medicina,
Uni. Algarve, Campus de Gambelas Ed. 8, Lab 1.18
8005-139 Faro, Portugal

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Hugh Newman
Sent: domingo, 5 de Fevereiro de 2012 20:14
To: [hidden email]
Subject: Re: How "deep" can you see in a brain slice?

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

I'm no medical guy but I work for Japanese microscope company that will not
be named.
Brain isn't very translucent so I wouldn't hold out much hope. I would say
100um tops, maybe even closer to 50um. After this your image will start to
degrade quite a bit. Any deeper than that and you are going to want a
2P-Confocal. I've imaged colloidal suspensions that are ~100um thick, and
they are very close to being index matched. At the maximum depth the image
wasn't that pretty.

Maybe some more Neuro people will have a better answer for you.

Good luck

H

Hugh Newman

Graduate Researcher

Dept. Physics and Physical Oceanography

Memorial University

St. Johns, Newfoundland, Canada

400um

> thick brain slice on an Olypus FV300. Has anyone experience with this and
> how "deep" it is reasonable to expect to see in the slice using a confocal
> microscope? How can you maximize this depth? (selection of objectives,
> processing of the slice....)
> Thanks for any suggestions, Petr.
>
> Petr Busek, MD, PhD
> Charles University in Prague
> First Faculty of Medicine
> Laboratory of Cancer Cell Biology
> Institute of Biochemistry and Experimental Oncology
> U Nemocnice 5
> 128 53 Prague 2
> Czech Republic
> www.lf1.cuni.cz/lbnb
> Fax +420 224 965 826

Mark Cannell

Re: How "deep" can you see in a brain slice?

In reply to this post by Mark Cannell

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

Following a PM, here are some more references that come up in the obvious Pub Med search:

"Clearing tetrahydrofuran"

Am J Clin Pathol. 1959 Apr;31(4):357-61.
Tetrahydrofuran (THF) for routine dehydration, clearing, and infiltration.
HAUST MD.

Arch Dermatol. 1960 Nov;82:798-803.
Use of tetrahydrofuran for routine and rapid dehydration and clearing.
MALKINSON FD, POTTER B.

Still dismayed at the lack of references to prior work -or did they pluck this chemical out of thin air?

Regards Mark

On 6/02/2012, at 11:59 AM, Mark Cannell wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I must admit to being a bit surprised that the Nature methods paper did not cite the prior use of THF in clearing insects... This was in 1958!
>
> Tetrahydrofuran and its Use in Insect Histology
>
> T. N. Salthouse. The Canadian Entomologist, 1958, 90:555-557, 10.4039/Ent90555-9
> Regards Mark
>
>
> On 5/02/2012, at 11:38 PM, George McNamara wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>>
>> Fixed and cleared: all the way:
>>
>> Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. </pubmed/22198277> Ertürk A, Mauch CP, Hellal F, Förstner F, Keck T, Becker K, Jährling N, Steffens H, Richter M, Hübener M, Kramer E, Kirchhoff F, Dodt HU, Bradke F. Nat Med. 2011 Dec 25;18(1):166-71. doi: 10.1038/nm.2600. PMID: 22198277
>>
>> Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. </pubmed/21878933> Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A. Nat Neurosci. 2011 Aug 30;14(11):1481-8. doi: 10.1038/nn.2928. PMID: 21878933.
>>
>> A colleague here at the U told me his lab had much better clearing and imaging with the Erturk et al method than with the versions of Hama et al's Scale that they tried (no, I do not know which many variants they tried or how extensively they tested each). This colleague told me that with the Erturk et al method they needed to image the same day (and the sooner the better). The Erturk et al method uses tetrahydrofuran (THF) to strip the lipids from the tissue, followed by immersion in benzyl alcohol:benzyl benzoate (BABB). BABB has a long history of use in optical clearing - see various papers by Bob Zucker, for examples:
>>
>> Whole insect and mammalian embryo imaging with confocal microscopy: morphology and apoptosis. </pubmed/17051584>* *Zucker RM. Cytometry A. 2006 Nov 1;69(11):1143-52. PMID: 17051584
>>
>> Confocal laser scanning microscopy of whole mouse ovaries: excellent morphology, apoptosis detection, and spectroscopy. </pubmed/16969804>* *Zucker RM, Jeffay SC. Cytometry A. 2006 Aug 1;69(8):930-9. PMID: 16969804
>>
>> I will hypothesize here that 2,2'-thiodiethanol (TDE) might be a better ultimate destination after THF. For TDE see:
>>
>> 2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. </pubmed/17131355>* *Staudt T, Lang MC, Medda R, Engelhardt J, Hell SW. Microsc Res Tech. 2007 Jan;70(1):1-9. PMID: 17131355
>>
>> See also Stan Vitha's post(s) here on transitioning specimens into TDE and imaging.
>>
>>
>> For fresh tissue - that is, hemisectioned mouse brain: sac the mouse, flush the RBCs, take out the brain, slice in half (along a line that will bisect the glioma mass that you introduced by stereotaxic injection, being careful not to have cells up the needle track), bring to the confocal - a user of mine in L.A. on a Leica SP1 confocal, 10x objective lens (probably 0.4 NA), went 800 um. On a City of Hope LSM510/MP, I helped image hemisectioned mouse brains previously implanted with GFP+ neural stem cells (Argon ion laser) plus DAPI (Coherent Chameleon laser, probably 750 nm excitation) several hundred micrometers deep. Again, one of the keys is to flush out the blood cells from the mouse vasculature - they scatter a lot more than mouse brain tissue. I have never been involved with brain slices - hopefully those protocols flush the blood cells after sac'ing the mouse.
>>
>> George
>>
>>
>>
>>
>> On 2/5/2012 2:53 PM, Petr Busek wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear all,
>>> I am trying to view fluorescently labeled glioma cells invading into a 400um
>>> thick brain slice on an Olypus FV300. Has anyone experience with this and
>>> how "deep" it is reasonable to expect to see in the slice using a confocal
>>> microscope? How can you maximize this depth? (selection of objectives,
>>> processing of the slice....)
>>> Thanks for any suggestions, Petr.
>>>
>>> Petr Busek, MD, PhD
>>> Charles University in Prague
>>> First Faculty of Medicine
>>> Laboratory of Cancer Cell Biology
>>> Institute of Biochemistry and Experimental Oncology
>>> U Nemocnice 5
>>> 128 53 Prague 2
>>> Czech Republic
>>> www.lf1.cuni.cz/lbnb
>>> Fax +420 224 965 826
>>>
>>>
>>
>>
>> --
>>
>>
>> George McNamara, PhD
>> Analytical Imaging Core Facility
>> University of Miami

mcammer

Re: How "deep" can you see in a brain slice?

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

And don't forget methyl salicylate, which also makes the confocal lab smell really fresh, for clearing tissues. We got some really nice deep images of developing mouse mammary glands using this.
________________________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: (212) 263-3208 Cell: (914) 309-3270

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mark Cannell
Sent: Monday, February 06, 2012 10:49 AM
To: [hidden email]
Subject: Re: How "deep" can you see in a brain slice?

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

Following a PM, here are some more references that come up in the obvious Pub Med search:

"Clearing tetrahydrofuran"

Am J Clin Pathol. 1959 Apr;31(4):357-61.
Tetrahydrofuran (THF) for routine dehydration, clearing, and infiltration.
HAUST MD.

Arch Dermatol. 1960 Nov;82:798-803.
Use of tetrahydrofuran for routine and rapid dehydration and clearing.
MALKINSON FD, POTTER B.

Still dismayed at the lack of references to prior work -or did they pluck this chemical out of thin air?

Regards Mark

On 6/02/2012, at 11:59 AM, Mark Cannell wrote:

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Stephen C. Kempf

Re: How "deep" can you see in a brain slice?

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

The following link might be of interest. I haven't tried this stuff, but it appears to make things very clear. If this was already posted to the list I apologize. I lost a few of the recent e-mails before reading them.

http://news.nationalgeographic.com/news/2011/09/110902-transparent-mouse-mice-embryos-brains-fluorescent-science/

http://microscope.olympus-global.com/uis2/en/scaleview/

Steve

______________________________________________________________________________
Stephen C. Kempf E-mail: [hidden email]<mailto:[hidden email]>
Associate Professor Tel: 334-844-3924
Department of Biological Sciences
331 Funchess Hall Fax: 334-844-9234
Auburn University, AL 36849
Lab Web site - http://gump.auburn.edu/kempflab/
Course web sites:
Vert. Dev. - http://www.auburn.edu/academic/classes/zy/vert_embryo/index_vert_embryo.html
Histology - http://www.auburn.edu/academic/classes/zy/hist0509/index_histology.html

On Feb 6, 2012, at 10:04 AM, Cammer, Michael wrote:

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

And don't forget methyl salicylate, which also makes the confocal lab smell really fresh, for clearing tissues. We got some really nice deep images of developing mouse mammary glands using this.
________________________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: (212) 263-3208 Cell: (914) 309-3270

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mark Cannell
Sent: Monday, February 06, 2012 10:49 AM
To: [hidden email]
Subject: Re: How "deep" can you see in a brain slice?

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

Following a PM, here are some more references that come up in the obvious Pub Med search:

"Clearing tetrahydrofuran"

Am J Clin Pathol. 1959 Apr;31(4):357-61.
Tetrahydrofuran (THF) for routine dehydration, clearing, and infiltration.
HAUST MD.

Arch Dermatol. 1960 Nov;82:798-803.
Use of tetrahydrofuran for routine and rapid dehydration and clearing.
MALKINSON FD, POTTER B.

Still dismayed at the lack of references to prior work -or did they pluck this chemical out of thin air?

Regards Mark

On 6/02/2012, at 11:59 AM, Mark Cannell wrote:

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

I must admit to being a bit surprised that the Nature methods paper did not cite the prior use of THF in clearing insects... This was in 1958!

Tetrahydrofuran and its Use in Insect Histology

T. N. Salthouse. The Canadian Entomologist, 1958, 90:555-557, 10.4039/Ent90555-9
Regards Mark

On 5/02/2012, at 11:38 PM, George McNamara wrote:

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

Fixed and cleared: all the way:

Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. </pubmed/22198277> Ertürk A, Mauch CP, Hellal F, Förstner F, Keck T, Becker K, Jährling N, Steffens H, Richter M, Hübener M, Kramer E, Kirchhoff F, Dodt HU, Bradke F. Nat Med. 2011 Dec 25;18(1):166-71. doi: 10.1038/nm.2600. PMID: 22198277

Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. </pubmed/21878933> Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A. Nat Neurosci. 2011 Aug 30;14(11):1481-8. doi: 10.1038/nn.2928. PMID: 21878933.

A colleague here at the U told me his lab had much better clearing and imaging with the Erturk et al method than with the versions of Hama et al's Scale that they tried (no, I do not know which many variants they tried or how extensively they tested each). This colleague told me that with the Erturk et al method they needed to image the same day (and the sooner the better). The Erturk et al method uses tetrahydrofuran (THF) to strip the lipids from the tissue, followed by immersion in benzyl alcohol:benzyl benzoate (BABB). BABB has a long history of use in optical clearing - see various papers by Bob Zucker, for examples:

Whole insect and mammalian embryo imaging with confocal microscopy: morphology and apoptosis. </pubmed/17051584>* *Zucker RM. Cytometry A. 2006 Nov 1;69(11):1143-52. PMID: 17051584

Confocal laser scanning microscopy of whole mouse ovaries: excellent morphology, apoptosis detection, and spectroscopy. </pubmed/16969804>* *Zucker RM, Jeffay SC. Cytometry A. 2006 Aug 1;69(8):930-9. PMID: 16969804

I will hypothesize here that 2,2'-thiodiethanol (TDE) might be a better ultimate destination after THF. For TDE see:

2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. </pubmed/17131355>* *Staudt T, Lang MC, Medda R, Engelhardt J, Hell SW. Microsc Res Tech. 2007 Jan;70(1):1-9. PMID: 17131355

See also Stan Vitha's post(s) here on transitioning specimens into TDE and imaging.

For fresh tissue - that is, hemisectioned mouse brain: sac the mouse, flush the RBCs, take out the brain, slice in half (along a line that will bisect the glioma mass that you introduced by stereotaxic injection, being careful not to have cells up the needle track), bring to the confocal - a user of mine in L.A. on a Leica SP1 confocal, 10x objective lens (probably 0.4 NA), went 800 um. On a City of Hope LSM510/MP, I helped image hemisectioned mouse brains previously implanted with GFP+ neural stem cells (Argon ion laser) plus DAPI (Coherent Chameleon laser, probably 750 nm excitation) several hundred micrometers deep. Again, one of the keys is to flush out the blood cells from the mouse vasculature - they scatter a lot more than mouse brain tissue. I have never been involved with brain slices - hopefully those protocols flush the blood cells after sac'ing the mouse.

George

On 2/5/2012 2:53 PM, Petr Busek wrote:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear all,
I am trying to view fluorescently labeled glioma cells invading into a 400um
thick brain slice on an Olypus FV300. Has anyone experience with this and
how "deep" it is reasonable to expect to see in the slice using a confocal
microscope? How can you maximize this depth? (selection of objectives,
processing of the slice....)
Thanks for any suggestions, Petr.

Petr Busek, MD, PhD
Charles University in Prague
First Faculty of Medicine
Laboratory of Cancer Cell Biology
Institute of Biochemistry and Experimental Oncology
U Nemocnice 5
128 53 Prague 2
Czech Republic
www.lf1.cuni.cz/lbnb
Fax +420 224 965 826

--

George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

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Martin Wessendorf-2

Re: How "deep" can you see in a brain slice?

In reply to this post by Petr Busek

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Dear Dr. Busek--

What do you want to see? The presence or absence of glia, or details on
the glial cells themselves? If all you want to do is to establish the
presence of the glia, you may be able to get reasonably good results by
mounting the tissue between two coverslips and taking images from either
side of the "sandwich". Gel-coat a coverslip, mount the tissue on it,
and then dehydrate, clear, and mount with another coverslip using DPX.
I'd image the tissue using oil-immersion optics, bearing in mind that
you want the longest working distance possible. --Different
manufacturers' objectives have different capabilities in that regard and
you can't necessarily trust the specifications.

By mounting the tissue between two coverslips, you can get good optical
access to either side of the tissue. You simply place the coverslip
"sandwich" on a slide and hold it in place with cellophane tape. If you
don't need to create a single image through the entire thickness of the
tissue, it should work.

Good luck!

Martin Wessendorf

On 2/5/2012 1:53 PM, Petr Busek wrote:

--
Martin Wessendorf, Ph.D. office: (612) 626-0145
Assoc Prof, Dept Neuroscience lab: (612) 624-2991
University of Minnesota Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
Minneapolis, MN 55455 e-mail: [hidden email]

Neeraj Gohad-2

Re: How "deep" can you see in a brain slice?

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Since we are on the topic, this question is for folks who have tried or regularly use the Sacle-A2 method of clearing tissues. In the original Hama et al. paper they have prescribed the pH of the Scale-A2 to be 7.7. Scale-A2 is not a great buffer and it's difficult to adjust the pH to 7.7, has anybody ran into the same problem?

Best,

Neeraj.

Neeraj V. Gohad, Ph.D.
Research Assistant Professor
Department of Biological Sciences
132 Long Hall
Clemson University
Clemson,SC-29634
Phone: 864-656-3597
Fax: 864-656-0435

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Monday, February 06, 2012 11:35 AM
To: [hidden email]
Subject: Re: How "deep" can you see in a brain slice?

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To join, leave or search the confocal microscopy listserv, go to:
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Dear Dr. Busek--

What do you want to see? The presence or absence of glia, or details on the glial cells themselves? If all you want to do is to establish the presence of the glia, you may be able to get reasonably good results by mounting the tissue between two coverslips and taking images from either side of the "sandwich". Gel-coat a coverslip, mount the tissue on it, and then dehydrate, clear, and mount with another coverslip using DPX.
I'd image the tissue using oil-immersion optics, bearing in mind that you want the longest working distance possible. --Different manufacturers' objectives have different capabilities in that regard and you can't necessarily trust the specifications.

By mounting the tissue between two coverslips, you can get good optical access to either side of the tissue. You simply place the coverslip "sandwich" on a slide and hold it in place with cellophane tape. If you don't need to create a single image through the entire thickness of the tissue, it should work.

Good luck!

Martin Wessendorf

On 2/5/2012 1:53 PM, Petr Busek wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
> I am trying to view fluorescently labeled glioma cells invading into a
> 400um thick brain slice on an Olypus FV300. Has anyone experience with
> this and how "deep" it is reasonable to expect to see in the slice
> using a confocal microscope? How can you maximize this depth?
> (selection of objectives, processing of the slice....) Thanks for any
> suggestions, Petr.
>
> Petr Busek, MD, PhD
> Charles University in Prague
> First Faculty of Medicine
> Laboratory of Cancer Cell Biology
> Institute of Biochemistry and Experimental Oncology U Nemocnice 5
> 128 53 Prague 2
> Czech Republic
> www.lf1.cuni.cz/lbnb
> Fax +420 224 965 826

Glen MacDonald-2

Re: How "deep" can you see in a brain slice?

In reply to this post by Petr Busek

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It partly depends on your resolution requirements. George's response was characteristically thorough.

We've fixed whole mouse brain and cleared in 100% glycerol to record infiltration by GFP-expressing G6 glioma cell with 2 mm thick optical volume on a FV-1000 confocal, 4X and 10X/.4 objectives, as well as 400 um thick cultured slices. Same instrument with 20X.75 objective could get >400 mm into 1 mm fixed mouse brain slices cleared with methyl salicylate-benzyl benzoate to create montages tracing axons (It was left over from imaging intact mouse cochlea at 800 um depth with 10X/.4, down to working distance of any high NA lens). If using immersion lenses, don't overlook the benefits of adjusting the RI of your immersion medium.

Regards,
Glen
Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923 USA
(206) 616-4156
[hidden email]

On Feb 5, 2012, at 11:53 AM, Petr Busek wrote:

George McNamara

Re: How "deep" can you see in a brain slice?

In reply to this post by Barbara Foster

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Hi Barbara,

Olympus now has 2 mm, 4 mm and 8 mm working distance "Scale" objective
lenses. Pricey.

Recent Nature product focus - oppostie page 535 of the 26 January 2012
issue is a Zeiss plan-apochromatic 20x/1.0 VIS-IR lens with working
distance of 5.6 mm.

The Erturk et al paper used both a multiphoton/confocal and the
Ultramicroscope from LaVision Biotec -
http://www.lavisionbiotec.com/en/microscopy-products/ultramicroscope/
... My next door neighbors (at work) are getting their Ultramicroscope
delivered in March. I don't know which model. Don't know when/if I'll
get to see it head to head with the Leica MP/SP5 I manage (my neighbors
have their own confocal scope so don't come over much).

Enjoy,

George

On 2/5/2012 11:12 PM, Barbara Foster wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> I was shocked when I visited Neuroscience this past Fall and saw what
> they were doing with special clearing agents and special objectives
> with multiphoton. The new limit is.... (ready?) at least 4 mm (no,
> that is not microns) and I think that there may be some new
> combinations which go as far as 8mm. This will be one of the topics
> for the Editor's Page for the April issue of American Lab. I will be
> writing that article this next week and can get you the current stats
> then.
>
> Good hunting!
> Barbara Foster, President and Sr. Consultant
>
> Microscopy/Microscopy Education
> P: (972)924-5310
> W: www.MicroscopyEducation.com
>
> We are now scheduling courses through June 2012
>
> At 12:08 PM 2/5/2012, you wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>>
>> I'm no medical guy but I work for Japanese microscope company that
>> will not be named.
>> Brain isn't very translucent so I wouldn't hold out much hope. I
>> would say 100um tops, maybe even closer to 50um. After this your
>> image will start to degrade quite a bit. Any deeper than that and you
>> are going to want a 2P-Confocal. I've imaged colloidal suspensions
>> that are ~100um thick, and they are very close to being index
>> matched. At the maximum depth the image wasn't that pretty.
>>
>> Maybe some more Neuro people will have a better answer for you.
>>
>> Good luck
>>
>> H
>>
>> Hugh Newman
>>
>> Graduate Researcher
>>
>> Dept. Physics and Physical Oceanography
>>
>> Memorial University
>>
>> St. Johns, Newfoundland, Canada
>>
>>
>>
>> > Date: Sun, 5 Feb 2012 13:53:33 -0600
>> > From: [hidden email]
>> > Subject: How "deep" can you see in a brain slice?
>> > To: [hidden email]
>> >
>> > *****
>> > To join, leave or search the confocal microscopy listserv, go to:
>> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> > *****
>> >
>> > Dear all,
>> > I am trying to view fluorescently labeled glioma cells invading
>> into a 400um
>> > thick brain slice on an Olypus FV300. Has anyone experience with
>> this and
>> > how "deep" it is reasonable to expect to see in the slice using a
>> confocal
>> > microscope? How can you maximize this depth? (selection of objectives,
>> > processing of the slice....)
>> > Thanks for any suggestions, Petr.
>> >
>> > Petr Busek, MD, PhD
>> > Charles University in Prague
>> > First Faculty of Medicine
>> > Laboratory of Cancer Cell Biology
>> > Institute of Biochemistry and Experimental Oncology
>> > U Nemocnice 5
>> > 128 53 Prague 2
>> > Czech Republic
>> > www.lf1.cuni.cz/lbnb
>> > Fax +420 224 965 826
>>
>

--

George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

Jeremy Adler-4

efficient detection of fluorescence - widefield

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In fluorescence emitted photons are often the limiting factor so in
widefield fluorescence filter blocks need to be optimised.

A possible problem is that for a molecule with a Stokes shift of say
20nm the detector will only detect the shortest emitted photons it
includes a range that starts at less than the Stokes shift, say
excitation + 15nm. But looking at the width of excitation filters used
in widefield fluoresence the excitation range, perhaps 460/40, then
many of the emitted photons will not even reach a detector with an
emission filter set to exclude excitation light, perhaps a 490nm Long
pass.
This argues that if photon detection efficiency is the primary concern
then excitation ranges need to be narrow. In the real world the power
output of widefield light sources is limited and an excitation range
is needed to effective produce fluorescence is useful amounts and the
emission spectrum of most fluorophores has a very long tail - but
should consideration be given to the the excitation range with respect
to the Stokes shift ?

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607