Automatic section thickness determination using an absolute gradient focus function.

Quantitative analysis of microstructures using computerized stereology systems is an essential tool in many disciplines of bioscience research. Section thickness determination in current nonautomated approaches requires manual location of upper and lower surfaces of tissue sections. In contrast to conventional autofocus functions that locate the optimally focused optical plane using the global maximum on a focus curve, this study identified by two sharp 'knees' on the focus curve as the transition from unfocused to focused optical planes. Analysis of 14 grey-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. Modifications to this function generated four novel functions that outperformed the original. 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, that is, different staining methods on a different brain region from a different subject rat. 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.