Polarized light striking a fluorescent molecule results in polarized fluorescence. This polarized emission gradually returns to unpolarized fluorescence depending on rotational diffusion and other factors. Anisotropy is directly related to the polarization, and is the ratio of the polarizedlight component to the total light intensity. With optional polarizers installed in a spectrofluorometer, we define light intensities: IVV is with excitation and emission polarizers mounted vertically; IHH is for excitation and emission polarizers mounted horizontally. IHV uses an excitation polarizer horizontal and the emission polarizer vertical; IHV requires the excitation polarizer vertical and emission polarizer horizontal. The basic setup, called “Lformat,” is shown in Fig. 1.