http://confocal-microscopy-list.275.s1.nabble.com/How-deep-can-you-see-in-a-brain-slice-tp7256661p7258951.html
glycerol. In our hands, oil immersion optics have never matched the
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> 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]>
>>
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>>>
>>>
>>> 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:
>>>
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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
>>>>
>>>>
>>>>
>>>
>>>
>>> --
>>>
>>>
>>> George McNamara, PhD
>>> Analytical Imaging Core Facility
>>> University of Miami
>>>
>