Workflow for biosensor processing of LLSM data. The software performs initial image processing steps including flat-field correction, registration, background subtraction, photobleach correction, and bleedthrough correction. Because of great variation in the intensity values at each slice, thresholding (defining the boundary of the cell) can be done manually or with a function fit to changing values specified over a series of slices and/or time points. The surface of the cell is determined through tessellation, a process in which the edge is interpolated based on the threshold values in a rolling local region. The measure of activity (e.g., the FRET ratio or total corrected FRET [Corr. FRET]) is determined for the entire field of view, and then the surface is rendered based on edge segmentation or other surfaces of interest. The advantage of this scheme is that, without additional computation, changes in segmentation can be updated immediately and multiple surfaces such as the outer edge of the cell and regions of high activity can be shown simultaneously. After the activity measures are calculated, the images are deskewed and interpolated on a user-specified grid to generate a solid 3D-cell volume. Finally, ImageTank includes scripts to analyze biosensor activity in any region of interest or in any plane through the cell and to measure these regions or objects in physically meaningful units.