Polarization Effects on Fluorescence Measurements.

Abstract

Polarization effects on fluorescence measurements are a function of four independent variables. The first is F, the polarization ratio of the exciting light which reaches the sample. The second is r, the emission anisotropy of the sample, which is the polarization "response" of the sample to plane polarized exciting light. The third is G, the polarization ratio of the emission detection system, which is the ratio of the sensitivities of the detection system to vertically and horizontally polarized light. The fourth is α the viewing angle, which is the angle bet ween the direction of the propagation of the exciting light and the direction from which the emission is being detected. The intensity and the degree of polarization of the fluorescence emission that the sample exhibits are functions of F, r, and a, while the actual readings obtained with a typical spectrofluorimeter are functions of all four variables, F, r, α, and G. A theoretical analysis is made taking all these factors into account, and proper mathematical models are developed for the different modes of operation in which a fluorimeter can be used. These are verified experimentally with data obtained for a sample which has a high degree of emission anisotropy (Nile Blue A Perchlorate in glycerol). A recently designed goniospectrofluorimeter was used. Calibration procedures are developed and recommendations are made for modes of operation and fluorescence standards.