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Updated: Mar 10, 2026
Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope
Published on: April 7, 2014
Unbiased estimation of cell number using the automatic optical fractionator
Peter R Mouton1, Hady Ahmady Phoulady2, Dmitry Goldgof2
1Department of Pathology & Cell Biology, University of South Florida Colleges of Medicine and Engineering, 4001 E Fletcher Ave, Tampa, FL, 33613, USA; Byrd Alzheimer's Institute, University of South Florida College of Medicine, 4001 E Fletcher Ave, Tampa, FL, 33613, USA; Department of Computer Sciences and Engineering, College of Engineering, University of South Florida, 4202 E Fowler Ave, Tampa, FL, 33620, USA.
Abstract:
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.
