Imaging Lifetimes

Abstract

This chapter discusses the critical contributions of Gregorio Weber to the development of techniques to measure fluorescence lifetimes. The fluorescence lifetime is the average time required for a population of fluorophores in the excited state to decay to the ground state. Events in a fluorophore's environment that influence the excited state can alter the lifetime, and this is measured using fluorescence lifetime imaging microscopy (FLIM). This chapter describes the application of FLIM to quantify F€ orster resonance energy transfer (FRET) between labeled proteins inside living cells. FRET is a non-radiative pathway through which a donor fluorophore in the excited state transfers energy to nearby acceptor mole-cules. The transfer of energy reduces the donor's fluorescence lifetime, and this can be quantified by FLIM. Since energy transfer occurs through near-field electromag-netic interactions, it can only occur over a distance of 80 angstroms or less. Thus, FRET microscopy has become a valuable tool for investigating biochemical net-works inside living cells. In this regard, Gregorio Weber recognized the importance of measuring the biological and physical properties of proteins as integrated systems. Here, proteins labeled with the genetically encoded fluorescent proteins (FPs) are used to demonstrate how FRET-FLIM enables robust and sensitive measurements of protein interactions inside living cells.