Abstract

Fluorescence probes which are sensitive to their environment, to the proximity of other chromophores and other structural or dynamic parameters have many applications in the investigation of biological systems on the molecular level. Their use in complex biological assemblies has been hampered by artifacts resulting from the absorption and scattering of excitation and emitted light within the sample. We have demonstrated that by measuring fluorescence intensity (I) or anisotropy (r) at a series of concentrations (h), the limiting values of these parameters at vanishing concentration (h - o) are given by the intercepts of the straight lines obtained by plotting log (I/h) or log r versus h. The slope of the line is a function of the absorption and scattering characteristics of the particles (e.g., cells) at the excitation and emission wavelengths and of the configuration of the fluorometer.

© 1984 Optical Society of America