Graduation Year


Document Type




Degree Granting Department

Computer Science and Engineering

Major Professor

Dmitry B. Goldgof, Ph.D.

Co-Major Professor

Lawrence O. Hall, Ph.D.

Committee Member

Peter R. Mouton, Ph.D.


Stereology, Thresholded Absolute Gradient, Z-stack, Contrast, Depth From Focus


The study of microstructures in brightfield microscopy using unbiased stereology plays a large and growing role in bioscience research. Stereology enables objective quantitative analysis of biological structures within a tissue sample. A first step in the stereology process is to calculate the thickness of a tissue sample by locating the top and bottom surfaces of the sample. The aim of this project is to fully automate this location process by using the relative optical focus measure as an indicator of tissue surface boundary.

The current method for identification of focus bounding planes requires a trained user to manually select the top and bottom of the tissue at each sample position examined. To automate finding the correct focal planes, i.e. the "just out of focus" planes at the top and bottom surfaces of the tissue sections, a novel approach was developed. Several gray scale focusing functions were analyzed, but while the traditional emphasis of microscopy focus functions is to find global maximums on the focus curve, in this project the aim was to find the sharp "knees" on the focus curve. Starting with a low focus measure value when the focal plane of the objective lens is out of focus above the tissue sample, the objective focal plane is moved downward through the tissue. The ideal focus measure should increase sharply as the upper surface of the tissue passes into the depth of field of the objective lens. As the focal plane is moved through the tissue, the focus measure value rises and falls as objects within the tissue come in and out of focus. As the bottom tissue surface passes into the depth of field the ideal focus measure should reflect some level of focus, dropping precipitously as the surface passes out of the depth of field into the unfocused region below the tissue.