Unbiased estimation of cell number using the automatic optical fractionator.

A novel stereology approach, the automatic optical fractionator, is presented for obtaining unbiased and efficient estimates of the number of cells in tissue sections. Used in combination with existing segmentation algorithms and ordinary immunostaining methods, automatic estimates of cell number are obtainable from extended depth of field images built from three-dimensional volumes of tissue (disector stacks). The automatic optical fractionator is more accurate, 100% objective and 8-10 times faster than the manual optical fractionator. An example of the automatic fractionator is provided for counts of immunostained neurons in neocortex of a genetically modified mouse model of neurodegeneration. Evidence is presented for the often overlooked prerequisite that accurate counting by the optical fractionator requires a thin focal plane generated by a high optical resolution lens.