Digital Imaging Fluorescence Microscopy: Statistical Analysis of Photobleaching and Passive Cellular Uptake Processes

Abstract

This chapter discusses the fluorescence photobleaching phenomenon and the errors that arise when it is not controlled. Objective criteria are developed to eliminate photobleaching as a variable in kinetic experiments. The photobleaching of a single fluorescent monomolecular film is used as an experimental tool to address a major assumption in fluorescence microscopy: that the specimen in the focal plane, when considered on a pixel-by-pixel basis, is uniformly illuminated. Because of their sensitivity, fluorescent techniques are potentially quite useful, particularly for active and passive mass transport processes. The photobleaching kinetics of only one kind of fluorophore in a uniform microenvironment is easily and accurately described by a simple mathematical model for a first-order reaction: a monoexponential decay and a background term. In poorly defined systems, such as the complex milieu of the cell, the evaluation of kinetic processes is much more complicated, particularly with living cells where both active and passive transport processes occur simultaneously and the cell is continually changing shape and position. The chapter emphasizes the utilization of statistical criteria to evaluate the performance of image processing algorithms.