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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) <
[log in to unmask]> 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: [log in to unmask]
> voice:  520-626-2824       fax:  520-626-2097
>
> 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: [log in to unmask]
URL:  http://astro.temple.edu/~jbs