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 usable 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. Fractionator 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 sampling parameters among groups or within groups?
Usually, within a group, there is no need to change sampling parameters; in other words, the biological variability between the subjects of the same group is not enough to warrant changing section interval or area sub-fraction among the members of that group.
It may be that the experimental manipulation causes a change that does indicate different parameters between groups. In order to help stay blind, you can opt to use the more stringent parameters on both groups.
The parameters for a region in a single subject must not change; all sections for a region within a subject should be analyzed with the same parameters.
7) Is it acceptable to change disector height among groups or within groups? I am unsure about whether or not to maintain the height of my disector across tissue specimens that vary in thickness.
As explained in question (6), the parameters must be constant within a subject, and almost always are kept constant within a group. The disector height is one of these parameters. The one parameter that will be allowed to change within an animal/region is section height (section thickness). This should be measured with a frequency to account for variance in the section thickness.
8) How do I know how tall to make my disector?
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.
9) Is it OK if my sections thicknesses do not come out exactly the same?
Yes, you will be measuring the thicknesses during the probe. 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. 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:
10) A counting frame falls on the edge of my region of interest, and the contour is “cutting off” part of the counting frame – is that okay?
The important part of the Fractionator approach to Systematic Random Sampling (SRS) is that the Area Sampling Fraction (ASF) remains constant throughout all of the sections that are counted. The ASF is the ratio between the area of one counting frame and the area of one space of the SRS grid, and therefore is completely independent of the size and shape of your region of interest. As long as you only count cells which are within the portion of the counting frame that is inside the region of interest your results will remain unbiased.
“Variation of fractionator estimates and its prediction” (1998 Schmitz, C. in Anat Embryol (Berl), 198 371-97, see Fig. 9) examines this question in greater detail by performing two separate calculations of the Volume Fraction (VF) and comparing them. First, they calculate the VF by taking the ratio of the area of one counting frame to the area of one SRS grid space. Then, they calculate the VF by breaking each counting frame into 1000 pieces and directly summing only those pieces that fall over the area of interest, and they divide this area by the volume of the region, measured directly. They find that the two separate VF calculations agree, and therefore the final estimate of the total number of cells within the region of interest will not differ significantly based on which method for VF calculation is chosen. Since the first method is much faster it is used for calculating the VF.
11) Is it a problem if the number of counting frames changes from section to section?
Because the unbiased nature of the Fractionator approach depends on whether we sample the same percentage of the tissue for each section (not the total amount of tissue sampled), you’ll still achieve an unbiased estimate as long as you set up the Optical Fractionator using the same parameters for each section of the same subject. Therefore, it’s actually expected that, in most cases, the number of counting frames will vary from section to section; because there is a (systematic) random placement of the disectors and the size and shape of the cross-section of any given region will change as the microtome proceeds through the tissue, the number of counting frames should change accordingly in order to ensure that the same percentage of tissue is sampled for each section.
12) Can I sample several different populations simultaneously?
It depends greatly on how these different cell populations are distributed within the region of interest. If they share a similar distribution, then it’s likely that they can be counted simultaneously by simply using different markings to count the different cell types. However, if there is a difference in how they are distributed, then they may need to be counted separately using different sampling parameters. These differences may include (but are not limited to):
-The approximate number of cells found in the region of interest
-How uniformly they are distributed throughout the region of interest (i.e. are they found in clusters, or are they evenly scattered?)
-Whether the distribution appears to change as a function of an anatomical axis (i.e. do more appear in the caudal portion of the region of interest, or do they appear in similar numbers in all sections?)
There have been recent developments that expand the capabilities of an Optical Fractionator probe, and the “Double Disector” method may prove useful if your cell populations are too different to be counted using the same sampling parameters, but are similar enough that the parameters differ by only 100-200%. The “Double Disector” is a modification of the disector that is used in the Optical Fractionator whereby a small grid of counting frames appears on the screen, but the cell populations are counted differently. One cell population will be counted using only one of the counting frames (typically the more homogeneously distributed population), while another cell population will be counted using all counting frames.
13) If my cell population is very sparsely distributed, do I need to do anything differently?
As with any unbiased stereological procedure, the sampling parameters should be dictated by the distribution of the cell population. For very sparsely distributed populations, it is expected that a higher percentage of the tissue will need to be sampled in order to achieve an acceptable precision than would be necessary for a cell type which is more numerous. Depending on how sparse the population is, it’s possible that 100% of the cross-sectional area will need to be sampled, and each counting frame will always be adjacent to other counting frames. It is OK to scan for the rare cell type at a lower magnification for efficiency, but when you spot one, go back up to the oil lens to check if the leading edge of the particle is in the disector.
14) Is there anything I can do to account for damage that occurs within the region of interest?
Sometimes while conducting one of the probes you discover that due to random circumstances one of the sections that had been randomly and systematically picked that you were planning on sampling is missing or damaged. There are several options listed below.
-You could randomly choose another starting section among the other possible starting points such that the missing section won’t be needed.
-If you are using an interval greater than one and know something about the system you are sampling and you don’t think there are significant changes between sections, you could simply substitute a neighboring section for the missing section. You don’t want to do this to the extent that your section sub fraction is wrong, but a little jitter in the increment of the sections may not hurt.
-If you think the mean of all the section counts or perhaps of the two adjacent sections is the appropriate number to use for the missing section then you can use one of those means. Don’t do this for too many sections though, probably not more than one, as this is not real data for the missing section.
15) How can I convert results of my stereological probe to a density of cells/fibers/vessels?
If the estimate obtained is number, or length, or surface, you would have to obtain the volume and report the volume along with the number, length, or surface. An unbiased method to estimate volume is Cavalieri point-counting.
To avoid confusion, report the number, length, or surface estimate and the volume, do not divide and give the percentage only. This way the reader know what has potentially changed, the number, length, or surface vs. the volume.
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