Quantitative FRET Microscopy of Live Cells

Abstract

Quantitative fluorescence resonance energy transfer (FRET) microscopy is a powerful tool for analyzing dynamic protein-protein interaction within living cells. FRET microscopy is increasingly employed to access the molecular mechanisms governing diverse cellular processes such as vesicular transport, signal transduction and the regulation of gene expression. However, evaluation of experi- mental approaches for FRET microscopy and the data they produce requires an appreciation of the techniques at the photophysical, molecular and data-acquisition level. This chapter aims to provide a conceptual framework for comparing FRET technologies and interpreting the data they produce. We begin with a qualitative discussion of FRET physics and the molecular interactions that can be probed by FRET. The discussion then shifts to the aspects of quantitative microscopy neces- sary for FRET-based measurements. With this foundation, we move to an overview of the current techniques in FRET microscopy, including acceptor photobleaching, spectral fingerprinting, FRET stoichiometry, and polarization FRET. Lastly, we discuss interpretation of FRET data and emerging applications to protein network analysis. Altogether, this chapter provides a progressive overview of FRET micro- scopy, beginning with fluorescent excited states, moving to detection methods and ending with interpretation of cell biology data.