Reconstituting the three-dimensional structure of biofilms represents a significant concern in microbiological studies. Previous studies have attempted to compute or at least approximate of the biovolume. Since the scale of microscopic images is important to ultimately assessing spatial (and temporal) variability in samples, calibration was performed using fluorescent microspheres of known sizes.  Refractive index matching between the microscope lens, lens media and mounting media is very critical. The beads were mounted in the same oil used by the immersion objective of the microscope. Even though some of the problems have been partially corrected, the optical properties of the microspheres produced a diffraction effect resulting into several distortions. The most noticeable effects were: (1) a hallo effect in the lower half of the microsphere, (2) fuzziness occurring to a lesser extent in the upper half and more so in the lower half, and (3) an apparent elongation of the lower half. To overcome these optical problems, imaging was limited to the upper half of microspheres. Nevertheless, the estimates of volume, based on a sample of 30 microspheres, were not significantly different from the hypothesized value. We concluded that, even though a measure of the ground reality should be used for calibration purposes in quantitative analyses of microbial images, fluorescent microspheres might not provide an ideal solution. Alternatively, the novel GIS approach provides a good tool to reconstitute and compute volumes based on parallel sections through microbiological samples.