confocal of whole mount retina

[Cromey, Douglas W - (dcromey)]

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I have a lab that is starting a project that will involve confocal imaging of whole mount retinas (small rodents).  We were able to image to-pro3 labeled nuclei through several layers to a depth of approximately 100um using a 63x water immersion objective.

Is there anyone that does something similar that would be willing to offer up some sample prep tips?

I recognize that spherical aberration becomes a significant issue when imaging that deep.  Any suggestions for possible clearing techniques (hopefully something not too complicated)?

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
voice:  520-626-2824       fax:  [phone]

http://swehsc.pharmacy.arizona.edu/micro
Home of: "Microscopy and Imaging Resources on the WWW"

[JOEL B. SHEFFIELD]

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Hi Doug,

A significant problem in imaging through the layers of the retina is that
some of the structures that you may be looking at are, themselves, in
overlapping layers.  Thus one nucleus in the inl may very well block proper
access to one that is deeper in the tissue.  That problem aside, the other
problem is the contribution of all of the plexiform layers to the optical
path.  Over the years, we have been able to circumvent this by mounting in
various concentrations of glycerol, which might match much of the
refractive index of the fiber layers.  We have also begun to look at the
effect of 2,2 thiodiethanol, which does wonders for cells in culture,
although it seems to extract certain lipophylic dyes.  see:  Staudt T, et
al (2006). "2,2′-Thiodiethanol: A new water soluble mounting medium for
high resolution optical microscopy". *Microscopy Research and Technique* *70
*: 1–9. doi <http://en.wikipedia.org/wiki/Digital_object_identifier>:
10.1002/jemt.20396 <http://dx.doi.org/10.1002%2Fjemt.20396>

Although I'm sure you're aware of it, you should also keep in mind that the
psf can be quite elongated in the z direction, and might yield misleading
results.  Often, if we're not careful, spheres end up appearing like tubes
when viewed from the side.

Joel



On Wed, Jun 19, 2013 at 5:29 PM, Cromey, Douglas W - (dcromey) <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have a lab that is starting a project that will involve confocal imaging
> of whole mount retinas (small rodents).  We were able to image to-pro3
> labeled nuclei through several layers to a depth of approximately 100um
> using a 63x water immersion objective.
>
> Is there anyone that does something similar that would be willing to offer
> up some sample prep tips?
>
> I recognize that spherical aberration becomes a significant issue when
> imaging that deep.  Any suggestions for possible clearing techniques
> (hopefully something not too complicated)?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  AHSC 4212         email: [hidden email]
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/micro
> Home of: "Microscopy and Imaging Resources on the WWW"
>

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs

[Glen MacDonald-2]

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Hi Doug,
Aside from TDE and glycerol for ~aqueous RI range, there are
Sca/e; Hama etal, 2011
Benzyl alcohol-glycerol; Clendenon etal 2011
ClearT; Kuwajima etal 2013

the last 3 can be adjusted for final RI.  I've used Sca/e with only fair results, but not yet tried the BA-Gly or ClearT.  To-Pro-3 can withstand dehydration and solvents, I've used with methyl salicylate-benzyl benzoate clearing.  

The retina is challenging,  you will need to verify and correct for axial distortions.
Glen MacDonald
        Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
        Cellular Morphology Core
Center on Human Development and Disability
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]







On Jun 19, 2013, at 2:29 PM, "Cromey, Douglas W - (dcromey)" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have a lab that is starting a project that will involve confocal imaging of whole mount retinas (small rodents).  We were able to image to-pro3 labeled nuclei through several layers to a depth of approximately 100um using a 63x water immersion objective.
>
> Is there anyone that does something similar that would be willing to offer up some sample prep tips?
>
> I recognize that spherical aberration becomes a significant issue when imaging that deep.  Any suggestions for possible clearing techniques (hopefully something not too complicated)?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  AHSC 4212         email: [hidden email]
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/micro
> Home of: "Microscopy and Imaging Resources on the WWW"

[Glen MacDonald-2]

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To join, leave or search the confocal microscopy listserv, go to:
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Hi Doug,
Since a water pipe burst in the ceiling above the confocal in the Ophthalmology dept,, we are providing confocal service until their system is repaired.  I just spoke with someone imaging a whole mouse retina, he was using a 60X/1.35 oil immersion lens with a retina mounted in Vectashield,and  routinely images to ~150 um depth.  I can't say whether that is the optimal preparation, but its a leading lab in the field.   Vectashield is essentially glycerol, so that may be a good place to start.    
Regards,
Glen
Glen MacDonald
        Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
        Cellular Morphology Core
Center on Human Development and Disability
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]







On Jun 19, 2013, at 2:29 PM, "Cromey, Douglas W - (dcromey)" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have a lab that is starting a project that will involve confocal imaging of whole mount retinas (small rodents).  We were able to image to-pro3 labeled nuclei through several layers to a depth of approximately 100um using a 63x water immersion objective.
>
> Is there anyone that does something similar that would be willing to offer up some sample prep tips?
>
> I recognize that spherical aberration becomes a significant issue when imaging that deep.  Any suggestions for possible clearing techniques (hopefully something not too complicated)?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  AHSC 4212         email: [hidden email]
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/micro
> Home of: "Microscopy and Imaging Resources on the WWW"