Chapter 4 - Fluorescence Lifetime Spectroscopy and Imaging of Visible Fluorescent Proteins

Abstract

The excited state lifetime of a fluorophore is a fundamental parameter that reports sensitively on the fluorophore nanoenvironment and on interactions between the fluorophore and other molecules. When coupled with the spatial resolution inherent to imaging techniques, fluorescence lifetime spectroscopy provides information about the spatial distributions of the molecules and the dynamics of related processes. In combination with genetically encodable visible fluorescent protein fluorophores, fluorescence lifetime imaging enables the real-time visualization of dynamic biological interactions in the complex context of the living cell. This chapter provides an introduction to fluorescence spectroscopy with an emphasis on fluorescence lifetimes and to the use of visible fluorescent proteins and fluorescence lifetime microspectroscopy for biophotonics applications. The discovery and development of fluorescent proteins as molecular tags in the recent years has led to a revolution by allowing complex biochemical processes to be correlated with the functioning of proteins in living cells. Recent advances like the development and use of new fluorescent tools such as semiconductor nanocrystals (quantum dots), multi-photon excitation, single molecule detection and development of better fluorescent proteins have opened up newer dimensions for the application of fluorescence lifetime for studying biological systems.