Fluorescence Anisotropy: Probing Rotational Dynamics of Biomolecules

Abstract

Molecules present in the lowest energy state (ground state) absorb light to be raised to the higher energy state (excited state). Fluorescence is an optical phenomenon through which the excess energy is being dissipated in the form of light (emission of photons). Fluorescence can be discriminated from the other emissive deactivation pathway, phosphorescence, by the timescale of the process. The fluorescence is a phenomenon that occurs within nanosecond timescale, while phosphorescence is observed at higher time window (millisecond to second). The fluorescence is highly sensitive to the molecular as well as environmental properties, which makes fluorescence spectroscopy one of the most unique and versatile techniques utilized for chemical and biological research. It is widely used to understand the molecular structure and dynamics in complex systems. The emission properties of fluorophores and its dependence on its immediate environment are being explored by the steady-state and time-resolved fluorescence spectroscopy. Time-resolved fluorescence measurements are preferred over steady-state fluorescence measurements to obtain comprehensive information on the microenvironment of the fluorophore as it captures the competing kinetic processes that are occurring in the timescale of fluorescence lifetime and influence fluorescence, such as collisional quenching, solvent relaxation, energy transfer, and rotational reorientation.