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BOX 43.1 THE OPTICAL FRACTIONATOR STEREOLOGICAL METHOD The figure shows the key features of a stereological technique designed to provide accurate and efficient estimates of total neuron number in a brain region of interest. The hippocampal formation is used as an example. The method consists of counting the number of neurons in a known and representative fraction of a neuroanatomically defined structure in such a way that each cell has an equal probability of being counted. The sum of the neurons counted, multiplied by the reciprocal of the fraction of the structure that was sampled, provides an estimate of total neuron number. Serial histological sections are prepared through the rostrocaudal extent of the hippocampus and are stained by routine methods for visualizing neurons microscopically. An evenly spaced series of the sections is then chosen for analysis (positions represented schematically in top panel). This first level of sampling, the “section fraction,” therefore comprises the fraction of the total number of sections examined. For example, if every tenth section through the hippocampus is analyzed, the section fraction equals 1/10. The appropriate sections are then surveyed according to a systematic sampling scheme, typically carried out using a microscope with a motorized, computer-controlled stage. The lower part of the figure illustrates this design in which the microscope stage is moved in even X and Y intervals, and neurons are counted within the areas defined by the small red squares (“a (frame)” in the inset). The second level of the fractionator sampling scheme is therefore the “area fraction,” or the fraction of the XY step from which the cell counts are derived. The last level of sampling is counting cells only within a known fraction (h) of the total section thickness (t), avoiding a variety of known errors introduced by including the cut surfaces of the histological preparations in the analysis. This is accomplished using a high-magnification microscope objective (usually 100X) with a shallow focal depth. In the illustration provided, the “thickness fraction” is defined as h/t. Neurons are counted as they first come into focus, according to an unbiased counting rule, called the “optical disector,” that eliminates the possibility of counting a given cell more than once. Finally, total neuron number in the region of interest (N) is estimated as the sum of the neurons counted (sumQ−), multiplied by the reciprocal of the three sampling fractions; the “section fraction,” “area fraction,” and the “thickness fraction.” For the present example, the total estimated neuron number is given by N = (sumQ−) × (10/1) × (XY step area/a(frame)) × (t/h) Original illustration design by L. E. Mekiou and P. R. Rapp. Peter R. Rapp