The principal methodology utilized is unbiased stereology. Stereology is a statistical method for estimating the morphological properties of three-dimensional structures from a number of two-dimensional images (e.g. histological material). Estimates include: volume, area, surface area boundary length, population size and numerical density among others. At the core of the technique is the idea that measurements are made using a systematic random sampling paradigm, so that neither human of methodological bias can skew the results.

Estimates are generated using a library of "probes" that are specifically designed to reveal a specific characteristic (length, number etc). A probe is applied to the tissue being studied using a random sampling scheme that helps prevent bias' imposed by shape orientation and so on. In the past theses analyses were performed manually from drawings or photographs using transparent overlays and tedious mathematical calculations. The advent of powerful personal computers has changed that.

Our lab employs a sophisticated software/hardware suite made by MicroBrightField, Inc. (Colchester, VT) called StereoInvestigator. The system consists of a Microscope with a computer controlled stage and focus (Z-axis) encoder. A high-resolution digital camera mounted to the microscope produces the image of the tissue on the computer monitor. The software generates a graphical overlay of the particular probe being used and not only moves the stage as needed, but encodes the positional data for all three axes.

In some analyses, the image produced by the camera and monitor my not be suitable. For example if one were trying to count neurons but did not want to include glia in that count, the native optics of the microscope are preferable. In this case a second option for the analyses is a small (1" diagonal) SVGA monitor, that projects the software interface into the microscope, so that it appears as an overlay on the tissue as it is seen through the microscope.