Wide-Field, Confocal, Two-Photon, and Lifetime Resonance Energy Transfer Imaging Microscopy

Abstract

The light microscope has been used for almost a century to produce images of cells, and this approach has contributed enormously to our understanding of cellular structure and function (Bright and Taylor, 1986; Herman, 1998; Inoué and Spring, 1997; Pawley, 1995; Periasamy and Herman, 1994). In turn, molecular biological studies over the past few decades have shown that cellular events, such as signal transduction and gene transcription, require the assembly of proteins into specific macromolecular complexes. What we require now are methods to visualize these protein—protein associations as they occur in the living cell. Recent advances in digital imaging coupled with the development of new fluorescent fluorophores now provide the tools to begin the study of protein-protein interactions in the intact cell. In this chapter we describe four different imaging techniques that apply the method of fluorescence resonance energy transfer (FRET) to surpass the optical limitations of the light microscope, allowing detection of the physical interactions of proteins in the living cell.