By T.W.J. Gadella (Eds.)
This quantity studies the suggestions F?¶rster Resonance power move (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) supplying researchers with step-by-step protocols and convenient tricks and tips. either became staple ideas in lots of organic and biophysical fields.
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Extra resources for Fret and Flim Techniques
This is usually done by analyzing a Stern-Volmer plot [82, 106]. Fo¨rster used the known properties of dynamic quenching in order to show the dynamic nature of FRET by the competition with quenching [54, 55]. In general, consider two competing processes leading to de-excitation of D*, process A and process B. Process A can be measured by determining the rate or extent of process B in the presence or absence of process A. EVectively, we measure B in order to determine A. These simple considerations cover essentially all the indirect methods of measuring the energy transfer process.
But as we have shown, fluorescence (the emission of a photon) is not the mechanism of the transfer, and one can measure FRET without ever measuring fluorescence. 2. The acronym FRET and extensions beyond Fo¨rster transfer FRET is often referred to as the acronym for ‘‘fluorescence resonance energy transfer’’. This name is a misnomer, but this use has 40 FRET AND FLIM TECHNIQUES become common, and is spread throughout the literature (also by the author). However, it is better to reserve the acronym FRET for ‘‘Fo¨rster resonance energy transfer’’, because then it is clear when one is referring to the conditions for which the Fo¨rster mechanism is valid.
The form, amplitude, and distance dependence of the time-dependent electric field of a dynamic Hertzian dipole changes as the distance from the dipole increases (near-, intermediate- and far-field). Propagating waves (emission of radiation) are only formed in the far field at distances of approximately one-half the wavelength from the dipole center. The nearfield amplitude is immense compared to the fields farther away. Classically, the energy ‘‘leaks’’ out into the far field as radiation (fluorescence) from the oscillating dipole only very slowly compared to 18 FRET AND FLIM TECHNIQUES the oscillation frequency.