When making an estimate using unbiased stereology there are two methods. For both methods systematic random sampling should be used. If you obtain a numerical density, Nv, by estimating where you sampled, it is called the NvVref method. For instance if you count the number of cells in the appropriate virtual spaces while using the optical disector, you can divide that by the total volume of all the virtual spaces to come up with number of cells per volume. If however, you keep track of the volume fraction while sampling, you don’t need to know the volume where you sampled, just the fraction. Take the reciprocal of the volume fraction and multiply it by all the cells you counted where you did sample in order to extrapolate and come up with an estimate of number per region sampled. This is called the fractionator method.
The NvVref method is subject to the volume-reference trap. Because you are reporting on a per volume basis, you don’t know if the volume changed or if the characteristic you are probing, for instance number, has changed. To obtain the absolute estimate you have to multiply the numerical density, Nv, by the reference volume, Vref. Obtaining the reference volume is a step that is not needed for the fractionator method.
Use the fractionator method if you can keep track of the volume fraction. This way you won’t have to obtain Vref and it is powerful to report the estimated characteristic per region instead of per volume. If the volume is also important, instead of reporting densities, give what makes up the density. For instance graph number vs. volume. If you can’t keep track of the volume fraction then use the NvVref method.
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developers of Stereo Investigator, the world’s most cited stereology system