Single molecule fluorescence techniques FRET

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. 

More recently the FRET technique has been widely applied to a broad range of biochemical problems. Sensitivity has been improved to the extent that fluorescence of single molecules can be detected. 

The distance between two different fiuorophore molecules can be probed by fluorescence resonance energy transfer (FRET) [308]. The energy transfer rate from the donor to the aeeeptor depends on the sixth power of the distance. FRET becomes noticeable at distanees on the order of a few mn and therefore occurs only if the donor and aeeeptor are physically linked. With FLIM techniques, FRET results are obtained from a single lifetime image of the donor [15, 32, 38, 61, 62, 63, 73, 80, 93, 147, 209, 405, 508]. 

As we saw in Chapters 2 and 3, the fluorescence emitted from single molecules can be collected by two distinct methods. In diffusion experiments the fluorescence emitted by a single molecule is collected as it passes through a small ( 0.1 fl) detection volume. This is repeated many thousands of times and burst analyses (such as FRET) and correlation techniques (FCS) can be used to extract both equilibrium and kinetic parameters of interest. The advantage of this technique lies in its relative simplicity, however the time over which a single molecule can be observed is limited to the time... 

A sophisticated experimental setup capable of simultaneous measurement of intensity, lifetime and anisotropy of both the donor and acceptor dyes has been reported by Margittai et al. [11]. The advantage of this demanding approach which they have termed single molecule multiparameter fluorescence detection (smMFD) is that it overcomes the problems associated with simpler FRET schemes such as the bleaching of donor or acceptor (see also the ALEX technique Chapter 2 and [12,13]) and assumptions concerning the quantum yields and... 

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