Proximity Measurements

Proximity measurements between two or more fluorophore-tagged molecules using the well-understood characteristics of fluorescence - namely energy transfer (Foerster's resonance energy transfer; FRET) - is another popular method of determining whether molecules are brought into nanometer proximity of each other.

Fig. 3.5 Schematic depiction of the fluorescence resonance energy transfer process and its implementation. (A) Orientation of donor and acceptor transition dipoles. The relative angle between the two transition dipole is responsible for depolarization of fluorescence upon energy transfer. (B) Overlap integral J(l) between the donor emission (Ed) and acceptor absorption spectra (Aa). Ad and Ea are the donor absorption and acceptor emis-

Fig. 3.5 Schematic depiction of the fluorescence resonance energy transfer process and its implementation. (A) Orientation of donor and acceptor transition dipoles. The relative angle between the two transition dipole is responsible for depolarization of fluorescence upon energy transfer. (B) Overlap integral J(l) between the donor emission (Ed) and acceptor absorption spectra (Aa). Ad and Ea are the donor absorption and acceptor emis-

sion spectra, respectively. Arrows depict decrease in donor emission and increase in acceptor emission intensities upon energy transfer. Observation windows show excitation and emission wavelength bandwidths for a typical imaging experiment, indicating the potential for cross-talk between the different imaging channels. D = donor; A = acceptor; exc = excitation; em = emission. (Reprinted with permission from [86].)

FRET may be theoretically described as an interaction between a dipole (excited state of a fluorophore) and an induced dipole (nearby ground-state fluorophore). This interaction decays extremely sharply with distance (1/R6), which provides this technique with the characteristics of a spectroscopic ruler in a typical range of 1 to 10 nm for organic fluorophores [74]. Since there is a transfer of energy between an excited-state fluorophore (donor) and a ground-state fluorophore (acceptor), the detection of this phenomenon may be related to changes in donor characteristics (donor lifetime, bleaching rate, or net fluorescence emission intensity or aniso-tropy) or acceptor characteristics (emission intensity or anisotropy) (for a review, see [75]). In the detection of "rafts" - which are defined by this technique as an anomalous proximity between fluorophore-tagged molecules - two types of FRET experiments have been carried out, with differing results. In hetero-FRET, the donor and acceptor are of different molecular and spectral characteristics, whereas in homo-FRET the donor and acceptor are the same species. In all cases the extent of energy transfer was dependent upon spectral overlap between the donor and acceptor species, the distance between dipoles, and the orientation of dipoles with respect to each other (Fig. 3.5).

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