http://confocal-microscopy-list.275.s1.nabble.com/How-deep-can-you-see-in-a-brain-slice-tp7256661p7258400.html
clearing. There are objectives available specifically for BABB, which
would make it the ideal clearing tool. If index matching the objective
oil. A while back we tested multiple mounting media from Citifluor (no
had the same refractive index as immersion oil. The tissue slices were
> 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
>>