A Comparison of Image Histograms and Biovolume by Threshold Curves on an Oral Biofilm Taken with Three Different Gains. Three CLSM image stacks were acquired with identical image acquisition parameters except for gain. The first column shows grayscale histograms as well as the maximum intensity projection of the biofilm image shown in gray. The second column shows biovolume as a function of threshold, the fitted power law curve, and the maximum intensity projection of saturated voxels shown in blue. (a) Scenario where signal sensitivity is too low, yielding no saturated voxels. In this scenario, the BEM, Otsu, and IS are comparable in threshold detection. (b) Scenario where signal sensitivity is optimized by a confocal operator for the stain mixture, producing saturated voxels. BEM selects for a lower threshold compared to Otsu and IS methods. (c) Scenario where signal sensitivity is too high. BEM threshold selection is no longer applicable whereas Otsu and IS methods show robustness to operator error or inexperience. Correlation coefficients are high in all three scenarios, with the highest belonging to the image optimized by an operator.