Resonance energy transfer measurement techniques

Fluorescence resonance energy transfer (FRET) is a technique that has been used to measure distances between pairs of proximal fluorochromes. A suitable pair consists of a donor fluorochrome, which has an emission spectrum... 

The possibility to carry out conformational studies of peptides at low concentrations and in the presence of complex biological systems represents a major advantage of fluorescence spectroscopy over other techniques. Fluorescence quantum yield or lifetime determinations, anisotropy measurements and singlet-singlet resonance energy transfer experiments can be used to study the interaction of peptides with lipid micelles, membranes, proteins, or receptors. These fluorescence techniques can be used to determine binding parameters and to elucidate conformational aspects of the interaction of the peptide with a particular macro-molecular system. The limited scope of this chapter does not permit a comprehensive review of the numerous studies of this kind that have been carried and only a few general aspects are briefly discussed here. Fluorescence studies of peptide interactions with macromolecular systems published prior to 1984 have been reviewed. 

The elucidation of the structure, dynamics and self assembly of biopolymers has been the subject of many experimental, theoretical and computational studies over the last several decades. [1, 2] More recently, powerful singlemolecule (SM) techniques have emerged which make it possible to explore those questions with an unprecedented level of detail. [3-55] SM fluorescence resonance energy transfer (FRET), [56-60] in particular, has been established as a unique probe of conformational structure and dynamics. [26-55] In those SM-FRET experiments, one measures the efficiency of energy transfer between a donor dye molecule and an acceptor dye molecule, which label specific sites of a macromolecule. The rate constant for FRET from donor to acceptor is assumed to be given by the Forster theory, namely [59,61-64]... 

Fluorescence Lifetime and Anisotropy Measurements Proteins Fluorescence Resonance Energy Transfer Proteins Fluorescence Techniques Lipids... 

Fluorescence resonance energy transfer (FRET) is a technique that has been used to measure distances between pairs of proximal fluorochromes. A suitable pair consists of a donor fluorochrome, which has an emission spectrum that significantly overlaps with the absorption spectrum of an acceptor fluorochrome (2). With the availability of monoclonal antibodies to many cell-surface determinants, intramolecular distances between nearby epitopes and intermolecular distances between adjacent cell-surface macromolecules can be investigated to analyze molecular interactions influencing important cellular events. Such monoclonal antibodies can be conjugated to fluorescein-isothiocyanate (FITC) as the donor, and either tetramethyl-rhodamine-isothiocyanate (TRITC) or phycoerythrin (PE) as the acceptor. 

This chapter reviews several techniques which combine the use of laser microbeams with antibodies to study molecular and cellular biology. An overview of the basic properties of lasers and their integration with microscopes and computers is provided. Biophysical applications, such as fluorescence recovery after photobleaching to measure molecular mobility and fluorescence resonance energy transfer to measure molecular distances, as well as ablative applications for the selective inactivation of proteins or the selective killing of cells are described. Other techniques, such as optical trapping, that do not rely on the interaction of the laser with the targeting antibody, are also discussed. 

---