Theory and Application of Differential Polarized Phase Fluorometry

Abstract

As discussed in the preceding contributions, polarized pulse fluorometry is widely used to investigate the rotational motions of fluorophores. Differential polarized phase fluorometry (DPF) provides comparable information. For differential phase measurements, a sinusoidally modulated exciting light is employed, and the difference in phase angle between the parallel and perpendicular components of the fluorescence emissions measured. These phase angles depend on the rates of fluorophore rotation and the isotropy and freedom of these rotations. Although polarized phase measurements date back to 1935, the theory and practice of DPF have only recently become available and been applied in biological research. These advances are primarily a result of the efforts of Weber.¹,² In the following sections, the theory of differential phase fluorometry, and the measurement of phase angles will be described. In addition, examples from the literature will be reviewed, and DPF compared with pulse methods.