Saturation effects on phosphorescence anisotropy measurements at high laser pulse energies

Triplet spectroscopic methods such as time-resolved phosphorescence anisotropy permit successful measurement of slow rotational diffusion of membrane proteins. However, these methods are potentially subject to saturation phenomena. We present theoretical and experimental studies of how high excitation energy densities can complicate measurements of phosphorescence intensity and anisotropy. Increases in excitation laser pulse energy initially increase phosphorescence intensity. Further increases then lead to phosphorescence saturation. As a consequence, the initial phosphorescence anisotropy decreases and approaches zero at very high excitation energies. The relative standard deviation of anisotropies measured in any system reaches a minimum at some particular excitation energy density. These results allow us to define optimum experimental conditions for time-resolved phosphorescence anisotropy measurements. For example, for excitation of erythrosin chromophores at typical wavelengths by the center of a Gaussian laser beam, optimum pulse energies in microjoules are approximately 5.0R ², whereR is the beam 1/e² radius in mm.