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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" <[log in to unmask]>:
> *****
> 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 <[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
>>>
>
--
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
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