Steady-state fluorescence anisotropy and lifetime measurements of fluorophores and fluorescent-dye-loaded microspheres

We perform steady-state fluorescence anisotropy and lifetime measurements via illumination of fluorophores with a continuous intensity beam of light. In steady-state fluorescence anisotropy, fluorophore molecules are excited when the polarization of the incoming excitation light is parallel to the excitation axis of the fluorophore. Following a delay known as the fluorophore lifetime $\tau $ a molecule will return to its rest state by emitting photons polarized along the instantaneous orientation of the molecule. The steady-state anisotropy r is defined as the average change in orientation of the sample weighted by the average intensity of each polarization axis. Experimental results are used to determine the anisotropy r and the lifetime $\tau $ of the samples: freely diffusing rhodamine as well as yellow-green fluorescent-dye-loaded microspheres with sizes ranging from 0.51 $\mu$m to 6.2 $\mu$m. Experimental outcomes confirm that the custom-made steady-state fluorescence anisotropy optical system and analysis software are properly engineered and optimized. These outcomes include the fluorescence lifetime $\tau $ for each fluorophore type, the fluorescence lifetime $\tau $ for each fluorescent bead size, and the anisotropy r at various temperatures.