The NvVref method is subject to the volume-reference trap. 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. 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. 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 you obtain a numerical density, Nv, by estimating where you sampled, it is called the NvVref method. For both methods systematic random sampling should be used. When making an estimate using unbiased stereology there are two methods.
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