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:
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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