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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:[log in to unmask]] On Behalf Of Craig Brideau
Sent: Monday, 6 February 2012 2:22 PM
To: [log in to unmask]
Subject: Re: How "deep" can you see in a brain slice?
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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 <[log in to unmask]>
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> 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
>
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