Fluorescence resonance energy transfer single molecule

Although much of the book focuses on physical theory, 1 have emphasized aspects of optical spectroscopy that are especially pertinent to molecular biophysics, and 1 have drawn most of the examples from this area. The book therefore covers topics that receive little attention in most general books on molecular spectroscopy, including exciton interactions, resonance energy transfer, single molecule spectroscopy, high-resolution fluorescence microscopy, femtosecond pump-probe spectroscopy, and photon echoes. It says less than is customary about atomic spectroscopy and about rotational and vibrational spectroscopy of... 

Deniz A A, Dahan M, Grunwell J R, Ha T, Faulhaber A E, Chemla D S, Weiss S and Schultz P G 1999 Single-pair fluorescence resonance energy transfer on freely diffusing molecules observation of Forster distance dependence and subpopulations Proc. Natl Acad. Sc/. USA 96 3670-5... 

Oswald B, Gruber M, Bohmer M, Lehmann F, Probst M, Wolfbeis OS (2001) Novel diode laser-compatible fluorophores and their application to single molecule detection, protein labeling and fluorescence resonance energy transfer immunoassay. Photochem Photobiol 74 237-245... 

Ha, T. (2001). Single-molecule fluorescence resonance energy transfer. Methods 25, 78-86. 

Selvin, P. R., Ha, T., Enderle, T., Ogletree, D. F., Chemla, D. S. and Weiss, S. (1996). Fluorescence resonance energy transfer between a single donor and a single acceptor molecule. Biophys. J. 70, Wp302-Wp302. 

Scheme 17. Bis-chromophore 54 serves as an example for the detection of fluorescence resonance energy transfer at the single molecule level.

The intersubunit rotation is required for translocation as ribosomes trapped in the nonrotated state by an engineered intersubunit disulfide bridge fail in tRNA-mRNA movement. Real-time observation of intersubunit movement by fluorescence resonance energy transfer (FRET) showed that intersubunit movement occurs concomitantly with hybrid state formation, and that the rotated state can be trapped by the antibiotic viomycin. Similarly to the fluctuation of tRNAs between classical and hybrid states, single-molecule studies have detected spontaneous intersubunit movement where the 3 OS subunit fluctuates between a rotated... 

Fluorescence resonance energy transfer (FRET) is a spectroscopic means of obtaining distance information over a range up to 80 A in solution. It is based on the dipolar coupling between the electronic transition moments of a donor and acceptor fluorophore attached at known positions on the RNA species of interest. It can be applied in ensembles of molecules, either by steady-state fluorescence or by lifetime measurements, but it is also very appropriate for single-molecule studies. In addition to the provision of distance information, recent studies have emphasized the orientation dependence of energy transfer. 

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

Ha T. Single-molecule fluorescence resonance energy transfer. 

Jahn R, Seidel AM. Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and 107. open conformations of syntaxin. Proc. Natl. Acad. Sci. U.S.A. 2003 100 15516-15521. 

T. Ha, Single-Molecule Fluorescence Resonance Energy Transfer, Methods 25, 78 (2001). 

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