Dynamic and static fluorescence anisotropy in biological microscopy (rFLIM and emFRET)

Fluorescence anisotropy, a measure of the polarization state of fluorescence emission, is a sensitive measure of molecular rotational motion and of resonance energy transfer (RET). We report here the formalism and application of dynamic and static fluorescence anisotropy measurements primarily intended for implementation in imaging systems. These include confocal lasre scanning microscopes (CLSM) as well as wide-field instruments, in the latter case adapted for anisotropy-based dynamic frequency domain fluorescence lifetime imaging microscopy (FLIM), a method we denote as rFLIM. Anisotropy RET is one of the modalities used for fluorescence RET (FRET) determinations of the association, and proximity of cellular proteins in vivo. A requirement is the existence of intrinsic or extrinsic probes exhibiting homotransfer FRET (in our nomenclature, energy migration or emFRET) between like fluorophores. This phenomenon is particularly useful in studies of the activation and processing of transmembrane receptor tyrosine kinases involved in signal transduction and expressed as fusions with Visible Fluorescence Proteins (VFPs).