Over the past decade, remarkable developments have occurred in the application of Förster resonance energy transfer (FRET)-based microscopy and spectroscopy to the biomedical sciences. These advances are being driven by the rapid progress of imaging and data processing technologies combined with the development of novel fluorescent probes that are genetically encoded by transferable DNA sequences. The application of these technologies is shedding new light on protein-protein interactions, protein conformational changes, and the behavior of signaling molecules inside living cells. Given these rapid advances, it may be useful in this preface to offer a brief historical overview of the important early contributions that provide the framework for the modern FRET techniques covered in the following chapters.

As long ago as 1922, Cario and Franck (Z. Physik. 11, 161 (1922)) observed that illumination of a mixture of mercury and thallium vapors at a wavelength absorbed only by mercury resulted in fluorescence emission from both atoms.  In 1927, Jean-Baptiste Perrin recognized that energy could be transferred (transfert d’activation) from an excited donor molecule to its neighbors through direct electrodynamic interactions.  These near-field interactions would allow the donor to transfer of excitation energy without the emission of an intermediate photon. Perrin’s model, however, was based on dye molecules with precisely defined oscillator frequencies, and incorrectly predicted that energy transfer could occur over distances of up to 1000 Å (J. Perrin, C.R. Acad. Sci. (Paris) 184, 1097 (1927)). Several years later, Perrin’s son Francis expanded on his father’s work, supplying a corresponding quantum mechanical theory of excitation energy transfer (F. Perrin, Ann. Chim, Physique 17, 283 (1932)). In his own work, Francis recognized that “spreading of absorption and emission frequency” because of dye molecule collisions with the solvent molecules would decrease the probability of energy transfer. His calculations reduced the intermolecular distance over which efficient energy transfer could occur to 150 - 250 Å, which was still greater, by a factor of 3, than the experimental observations.

In 1948, Theodor Förster extended the work of the Perrins to quantitatively describe the FRET process.  Förster showed that the efficiency of FRET varied as the inverse of the sixth power of the distance between the oscillating dipoles, and defined the critical molecular separation, R0, now called the Förster radius, at which the rate of energy transfer was equal to the rate of fluorescence emission. In contrast to the earlier assumptions made by the Perrins, Förster observed “the absorption and fluorescence spectra of similar molecules are far from completely overlapping” and quantified the spectral overlap integral (T. Forster, Annalen Der Physik, 2,55-75 (1948)). Using this information, Förster determined an R0 value for fluorescein of 50Å, much smaller than the values resulting from the Perrins’ work, and in perfect agreement with the experimental observations. Our colleague, Dr. Robert Clegg, recently suggested that the FRET acronym should refer to Förster resonance energy transfer (FRET) to give credit to these valuable contributions, and we entirely agree (R. Clegg, Biophotonics International, September, 42-45 (2004)). The chapters below illustrate how the techniques pioneered by Förster have been extensively applied to measure protein proximity in biological systems.  

This is the first book to address the technological developments in FRET microscopy from the viewpoint of fundamental concepts, methods, and biological applications. Each chapter includes the theory and basic principles behind the FRET microscopy or spectroscopy technique being discussed. The range of techniques considered is broad: wide-field, confocal, multiphoton, and lifetime FRET, spectral imaging FRET, photobleaching FRET, single- molecule FRET, bioluminescence FRET, time and image correlation spectroscopy. The authors describe the physics of FRET and the basics of the various light microscopic FRET techniques used to provide high spatial and temporal resolution with the goal of localizing and quantitating the protein-protein interactions in live specimens.

Each chapter provides many possible combinations of conventional and green fluorescent protein fluorophores for FRET with appropriate filter combinations and spectral configurations. This will allow readers to address specific biological questions through the choice of probes for FRET experiments, as well as the selection of the most suitable experimental approaches. More importantly, this book covers the extraction of FRET signal from the contaminated FRET images acquired with various microscopy techniques and provides a dedicated step-by-step FRET data analysis algorithm. This should make these methods of molecular imaging easier to understand for all readers, especially graduate students, postdoctoral fellows, and scientists who are new to state-of-the-art FRET microscopy imaging systems.



We would like to extend special thanks to all the contributors for the time spent in preparing the valuable manuscripts included in this book. We also want to thank Mr. Jeffrey House and Ms. Nancy J. Wolitzer, Oxford University Press, for constant feedback and timely answers to all our questions. Our special thanks to Ms. Ye Chen for her valuable help in editing the art-work for this book. Also, we wish to thank Mr. Hal Noakes for the design of the cover for the book.

Importantly, we would also like to thank the University of Virginia and the listed organizations below for their support in bringing out this valuable book

Carl Zeiss, Inc

Chroma Technology Corporation

Nikon, Inc

Olympus America, Inc

Omega Optical, Inc

Universal Imaging Corporation


                    Ammasi Periasamy, Ph.D.

                    Richard N. Day, Ph.D