Frequently Asked Questions (FAQs) about stereology grade tissue
1) How thick should I cut my tissue sections for the optical fractionator probe?
Post-processing tissue thickness is a critical consideration. Probes such as the Optical Fractionator require a tissue thickness that can be broken up optically by the focal point of a high numerical aperture objective. The sections must be thick enough to allow many focal planes through the disector and to allow for guard zones. Typically, tissue between 15-30 microns thick post-processing allows for the loss of upper and lower guard zones while still resulting in several optical Z-planes when using a 1.4 NA objective. Tissue sectioned between 30-60 microns usually gives useable results after staining and dehydration. You should do some experimentation towards developing a staining protocol that does not result in excessive shrinkage in the Z axis.
2) Is my tissue too thin?
That depends on which stereological probe you are using. Optical fractionating probes, such as the Optical Fractionator or Space Balls require relatively thick tissue. For example, If the tissue under study has only one or two focal planes—even with a high numerical aperture—it may be too thin. However, other probes, such as the Cavalieri Estimator for area and volume do not require you to focus up or down through the tissue. With probes such as that, you only need know the sectioning thickness. Decide on which probe or probes to use before preparing the tissue. For details please start with the probes page.
3) How do I identify the ‘top of my tissue’?
Sectioned tissue is not perfectly flat. Think of the surface as a landscape covered with hills and valleys. To identify the top of your tissue, use a high power objective (with a high numerical aperture) to focus up until you are out of focus above the tissue. Slowly focus down until you can see the top of the ‘hills’ come into focus, that is, until only the highest parts of the tissue surface are in focus. Note this Z position. This is the very top of the tissue.
4) Can I use paraffin embedded tissue sections for stereology?
Of course. But remember, paraffin embedded tissue sections are generally very thin (between 2-10 microns) and are not suited for probes that are isotropic because isotropic probes require thick sections. These thin-sectioned tissues may be perfect for probes such as the Cavalieri Estimator.
5) My tissue shrank too much even though I cut it very thick, can I still use it for my Optical Fractionator study?
Remember that the Optical Fractionator probe requires tissue thick enough so you can focus on several distinct focal planes. This is dependent on the post-processing tissue thickness. Your thickly cut sections may shrink so much during post-processing that distinguishing several focal planes is difficult—if not impossible.
6) Is it acceptable to change disector height between experiments or across subjects? I am unsure about whether or not to maintain the height of my disector across tissue specimens that vary in thickness.
It is perfectly fine to change the sampling parameters between specimens. What should be avoided is changing sampling parameters in the middle of a single subject analysis. In other words, all sections of “Control Animal 1” should be analyzed with the same sampling parameters but it is not required that Control Animal 1 and 2 be sampled with the same parameters. In the case of the Optical Fractionator, each subject is evaluated independently for precision and many situations can arise where sampling parameters are not suitable across subjects.
Checking section thickness up front can be helpful in selecting sampling parameters appropriate for each specimen. Thin tissue will limit you to a short disector that may require the use of more sampling sites to obtain your target cell counts—smaller SRS Grid step sizes produce more sampling sites.
Within each subject, significant variation of tissue section thickness will affect the accuracy of the estimation of total cell number when using the standard optical fractionator, if there is a correlation between the number of cells and the thickness of the section at a given counting site. If you are using Stereo Investigator, it is suggested that you consider utilizing the number-weighted variation of the optical fractionator. Obtaining this estimate requires that you measure the section thickness at each sampling site and the measured section thickness is used in the results calculation to make a more accurate estimate of total cell number. When calculating the volume fraction, the average mounted section thickness will be derived by weighting section thicknesses more that have more cells associated with them.
A useful resource on this topic can be found here:
K.-A. DORPH-PETERSEN, J. R. NYENGAARD AND H. J. G. GUNDERSEN, Tissue shrinkage and unbiased stereological estimation of particle number and size, Journal of Microscopy, Vol. 204, Pt 3, December 2001, pp. 232±246.
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developers of Stereo Investigator, the world’s most cited stereology system