The dynamic interaction between proteins and other biomolecules in living cells plays a significant role in a wide spectrum of essential processes. Although classically investigated in fluorescence microscopy using co-localization techniques, these interactions can provide additional information when applied to fluorescent proteins in live-cell imaging microscopy using resonance energy transfer techniques. The references listed below point to review articles in the scientific literature that should provide an excellent starting point for investigators seeking information on FRET methodology.
Fluorescent Protein FRET: the good, the bad, and the ugly. Trends in Biochemical Sciences 32: 407-414 (2007). One of the best review articles for investigators new to resonance energy transfer methodology. The authors discuss the various pitfalls associated with the application of FRET techniques to live-cell imaging using fluorescent proteins. Also reviewed are the physical basis of FRET and the various approaches to accurately measure FRET.
Fanciful FRET. Science STKE 331: re2 (2006). An introduction to FRET techniques with emphasis on the use of standards to determine FRET efficiency as a baseline for further investigations. Included is a description of the physical principles surrounding FRET, a discussion of how FRET can be used to decipher intermolecular interactions, and a review of the techniques involved. Also described are the accuracy and interpretation of FRET measurements.
Fluorescence energy transfer as a spectroscopic ruler. Annual Reviews of Biochemistry 47: 819-846 (1978). Originally published over 30 years ago, this article remains one of the cornerstone reviews of FRET theory and experimental application. Topics covered include Förster's original theory, measurement of transfer efficiency, fluorescent reagents for labeling intracellular structures, and example applications.
Resonance energy transfer: methods and applications. Analytical Biochemistry 218: 1-13 (1994). Practical aspects of FRET methodology are reviewed in this seminal paper. These include sample preparation, distance determinations, detection strategies, time-resolved measurements, and data analysis.
FRET imaging. Nature Biotechnology 21: 1387-1395 (2003). Perhaps one of the most comprehensive reviews ever published on FRET technology, this paper contains an exhaustive discussion of theory, methodology, and applications ranging from model systems to single molecules and live-cell imaging. The article also includes a helpful primer on the photophysical phenomena of molecules potentially involved in FRET assays.
FRET or no FRET: a quantitative comparison. Biophysical Journal 84: 3992-4010 (2003). Designed to provide a roadmap for quantitative comparison of various FRET measurement techniques, this review article proposes a surface FRET system with controlled levels of donor and acceptor fluorophores at fixed distances. The system is supported by theoretical calculations and experimental verification under a variety of conditions.
Observing proteins in their natural habitat: the living cell. Trends in Biochemical Sciences 25: 631-637 (2000). An excellent review on the application of FRET and related techniques using fluorescent proteins in living cells. In addition to a discussion of FRET and lifetime imaging (FLIM) methodology, the article also briefly covers recovery after photobleaching (FRAP) and correlation spectroscopy (FCS), as well as multi-color fluorescence imaging.
Protein localization in living cells and tissues using FRET and FLIM. Differentiation 71: 528-541 (2003). Focusing on fluorescent proteins, this paper examines a variety of microscopy illumination techniques applied to FRET measurements, including widefield, confocal, and multiphoton. Also reviewed are the theory of FRET and a variety of data analysis algorithms designed to correct for artifacts such as spectral bleed-through.
Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophysical Journal 74: 2702-2713 (1998). A thorough discussion of the potential artifacts often encountered when undertaking FRET measurements. Among the pitfalls described are direct acceptor excitation and the dependence of FRET on the concentration of the acceptor. The methodology introduced in this paper corrects for cross talk and concentration dependence. Several experimental examples are included.
Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24: 289-296 (2001). This paper reviews techniques to determine whether proteins that are colocalized at the level of light microscopy interact with one another. Rather than fluorescent proteins, the system under investigation employs the acceptor photobleaching technique using synthetic dyes in fixed cells that are targeted at the plasma membrane.