Three dimensional image volumes collected using optical microscopy exhibit many characteristics that cause difficulty in segmentation. These include decreasing image contrast with increasing tissue depth, poor edge detail with regard to cellular structures, and limited spatial resolution. This paper describes a three dimensional segmentation method utilizing active surfaces to segment microscopy image volumes. We demonstrate this method on a three dimensional sequence of images acquired from stationary/stabilized kidney tissue of a rat. Results from this method are compared against a prior pseudo-three dimensional segmentation method that analyzes slices on a single image-by-image basis, as well as against another native three dimensional segmentation method.