True emission and absorption anisotropies for the study of protein rotation obtained from fluorescence depletion measurements in various experimental geometries

The utility of fluorescence depletion methods for the measurement of slow protein rotational diffusion has been limited by the lack of a rigorous mathematical model to obtain, from depletion data, anisotropies directly comparable to those obtained from phosphorescence emission or triplet absorption measurements. A generalized theory to meet this need is described. The experimental method requires the acquisition of, at most, three separate measurements to calculate absorption or emission anisotropies. Each measurement is made with a different orientation of either the probe beam polarization, pump beam polarization, or emission polarizer. The results of the theory are applied to two experimental configurations. The first of these involves collecting emission at 90 degree(s) to colinear pump and probe beams. From such data we are able to calculate the absorption anisotropy, the emission anisotropy, and the interdipole angle. The second configuration represents a system where all polarization axes lie in a single plane as would occur in a microscope-based system. For this configuration we are able to calculate, given the interdipole angle derived from the 90 degree(s) case, the true absorption anisotropy, the true emission anisotropy, or both.