Theoretical principles and practical considerations for fluorescence resonance energy transfer microscopy

Abstract

Typically, light microscopic methodologies using conventional optics are limited by the diffraction limit yielding resolutions that cannot be reached lower than approximately 200nm. However, using appropriate donor-acceptor pairs, nonradiative fluorescence resonance energy transfer (FRET) allows the microscopist to detect, and in some cases quantify, molecular interactions on the order of Angstroms. In this chapter, the basic principles of FRET are introduced using both steady state and lifetime modes to detect the close association of fluorescent donor and acceptor molecules. The basic design of experiments and optical and imaging components is discussed to create a microscope that is capable of monitoring dynamic molecular associations in living cells.

Keywords: Donor-acceptor pairs; Fluorescence lifetime imaging; Green fluorescent protein; Nonradiative fluorescence resonance energy transfer; Steady state fluorescence.