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We describe a novel algorithm that allows for automatic section thickness determination based on just out-of-focus planes, a prerequisite for fully automatic computerized stereology.Ĭurrent computerized stereology systems require trained users to carry out several manual steps in order to generate design-based estimates of first- and second-order stereological parameters of biological interest. The “modified absolute gradient count” function outperformed all others with an average error of 0.56 μm on a test set of images similar to the training set and, an average error of 0.39 μm on a test set comprised of images captured from a different case, i.e., different staining methods on a different brain region from a different subject rat. Modifications to this function generated four novel functions that outperformed the original. Analysis of fourteen gray-scale focus functions showed, the thresholded absolute gradient function, was best for finding detectable bends that closely correspond to the bounding optical planes at the upper and lower tissue surfaces. In contrast to conventional autofocus functions that locate the optimally focused optical plane using the global maximum on a focus curve, the present study identified by two sharp “knees” on the focus curve as the transition from unfocused to focused optical planes. Section thickness determination in current non-automated approaches requires manual location of upper and lower surfaces of tissue sections. Quantitative analysis of microstructures using computerized stereology systems is an essential tool in many disciplines of bioscience research.