Are variable tissue thicknesses problematic?

Question

I have found that the amount of tissue shrinkage between experiments really varies, leaving me with a final thickness between 10 and 18um, depending on the experiment. I am unsure about whether or not to maintain the height of my counting brick, even if it means using a 5um counting brick for an 18um thick section. Is it acceptable to change the counting brick height between experiments, as long as it is maintained between different samples within an experiment? Any advice would be greatly appreciated. Thanks!

Discussion

Just as a point of clarification I assume you mean the height of the optical disector when you say the counting brick height. The optical disector is usually bounded by guard zones on top and bottom so that only the middle portion of the tissue is used in counting.

The original thickness of the tissue is not given here, but for the purposes of discussion I can assume that the material was cut at 30 microns. If that is the case, then a final section thickness of 10 microns means that the tissue has shrunk to 33% of its original thickness and 18 microns means that the tissue shrunk to 60% of it’s original thickness. An optical disector height of 5 microns in the 10 micron tissue is really a 15 micron high optical disector in the original tissue. In the 18 micron tissue the 5 micron optical disector is an 8 micron high optical disector.

I mention this because I want to point out that in a sense the optical disector height is changing between animals. This is reflected in the varying height sampling fraction values.

It is perfectly fine to change the sampling parameters between specimens. What does not work out is changing them in the middle of an organ. Not only is it okay to change the sampling parameters, it is also the smart thing to do. Beginning with the very first specimen knowledge is learned about how many counts are made. If only 50 counts are made then it is likely that the estimate is not that good. If the counts total to 1000 then it is likely that the estimate is must better than it actually needs to be. In other words, too much work was done. Going on to the next animal, sampling parameters are adjusted to bring the number of counts to a usable level – say 150 to 250.

In the case given here, a check of section thickness up front can be used to select sampling parameters appropriate for specimens. A short disector may require the use of more sampling sites to get enough counts. Smaller grid step sizes produces more sampling sites. A taller disector may be able to use larger gird stepping sizes to reduce the number of sampling sites and still count enough cells.

You may want to take a look at the CE formulas. Understanding all of the nitty gritty is not necessary. Notice that the equations are based on the counts and not on the sampling parameters themselves. The results from different specimens can be compared even though the sampling parameters differ.

There are other considerations if the tissue has varying thickness within the sections. Please ask again if this is an issue.

If you have a significant variation of tissue section thickness, then this will effect the accuracy of the estimation of total cell number when using the standard Optical Fractionator.

If you are using Stereo Investigator, I would suggest that you utilize the number-weighted variation of the Optical Fractionator. This technique requires that you measure the section thickness at each sampling site. Then the measured section thickness is used in the results calculation to make a more accurate estimate of total cell number.

A good paper that covers this topic is: 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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