Chapter 18: Visualizing Protein Interactions in Living Cells Using Digitized GFP Imaging and FRET Microscopy

Abstract

Publisher Summary This chapter reviews the theoretical basis of fluorescence resonance energy transfer (FRET) microscopy, with emphasis on its application to the living cell. It describes the characteristics of the green fluorescent proteins (GFPs) that make them useful for FRET imaging studies and outlines the important characteristics of an imaging system suitable for detecting FRET signals. The factors that limit the detection of the weak fluorescent signals associated with FRET include the contribution of autofluorescence, photobleaching, light scattering, and fluctuation in excitation light intensity relative to the image background. These factors can be especially significant for the acquisition of images using ultraviolet light excitation of BFP. The contribution of these factors could be considerably reduced by using two-photon excitation fluorescence imaging microscopy. The chapter uses the expression of genetic vectors encoding protein fusions to GFP and BFP to demonstrate the practical implementation of FRET imaging. It also describes the future direction of FRET imaging technology in biological applications.