The Role of the Photon Counting Loss Effect in Time-Resolved Measurements of Fluorescence Anisotropy

Determining the rate of rotation of molecules from their fluorescence anisotropy decay curves is a powerful method for studying molecular systems in biological applications. The single photon count detection systems used for this have a nonlinear dependence of the photon counting rate on the fluorescence intensity flux (photon counting loss effect), which can lead to a number of artifacts. Using metal complexes of phthalocyanines as a test sample, we have shown that such a nonlinearity can cause distortions in the determination of the fluorescence anisotropy lifetime and the asymptotic fluorescence anisotropy. We also assessed the dependence of the described phenomena on temperature and estimated the manifestations of the photon counting loss effect in the case of photobleaching of the fluorophores.