Methods

The basic fundamentals of FRET have been known for many years (16-18). FRET consists of a quantum mechanical process resulting in radiationless energy transfer from the first excited state of a fluorescent molecule, called the donor (D), to the first excited state of another fluorescent molecule, called the acceptor (A). As a consequence, under conditions that are given subsequently, excitation of the donor may induce the fluorescence of the acceptor (Fig. 1).

Fig. 2. FRET. (A) Example of overlap spectral region (in gray) required for FRET: a, donor absorption spectrum; b, acceptor absorption spectrum; c, donor emission spectrum; d, acceptor emission spectrum. (B) Minimal filter set required for FRET monitoring: 1, recording of donor fluorescence intensity; 2, recording of acceptor fluorescence intensity without FRET; 3, recording of FRET intensity.

Fig. 2. FRET. (A) Example of overlap spectral region (in gray) required for FRET: a, donor absorption spectrum; b, acceptor absorption spectrum; c, donor emission spectrum; d, acceptor emission spectrum. (B) Minimal filter set required for FRET monitoring: 1, recording of donor fluorescence intensity; 2, recording of acceptor fluorescence intensity without FRET; 3, recording of FRET intensity.

For FRET to occur, the two fluorophores must be conveniently selected: the donor emission spectrum must significantly overlap the absorption spectrum of the acceptor, and the reabsorption of the fluorescence emitted by the donor must be minimal to avoid tedious data corrections (Figs. 1,2). Furthermore, FRET intensity is sensitive to the relative orientation of the transition dipoles of the fluorophores. Then, because FRET has to compete with other deactivation processes of the donor excited state, the donor should have a high fluorescence quantum yield. Finally, the distance between D and A must be <10 nm, for FRET efficiency decreases with the sixth power of the distance between them. It is just this last property that makes FRET an invaluable tool for probing protein conformation and protein conformational changes. Moreover, both D and A must be insensitive to photobleaching when FRET is used for probing protein conformational changes, for that goal requires successive periods of irradiation.

Application of FRET techniques to biomedical research started quite recently (19,20). As a matter of fact, interpretation of FRET data on biological events is a difficult task for the following reasons. Observation is generally carried out on a population of biological molecules that are known to experience different 3D conformations even when sharing the same free-energy level. Thus, even if the time necessary for data collection is "ideally short," data will reflect the distribution of individual molecules in the same energy level, i.e., the relative weight of their different potential 3D conformations. Furthermore, technical limitations do not allow the collection of significant amounts of data within periods of time short enough compared to intracellular movements. Such a situation is common to every optical method of collecting information, making it difficult to assign an experimental value to a precise conformation. For these reasons, FRET is often used as an "all or nothing" method, separating the studied molecules into two subpopulations, that in which FRET occurs (D-A < 10 nm) and that in which FRET does not (D-A > 10 nm).

As indicated before, FRET can be used for experiments in solution with a conventional spectrofluorometer or under the microscope for studying biomolecules in living cells. In the latter case, owing to the concentration of light at the objective, the risk of photobleaching is highly increased. Thus, besides the use of direct single-photon irradiation, other methods have been developed. Multiphoton microscopy takes advantage of the existence of the "optical window," the wavelength domain between 700 and 1100 nm where the biological material is practically transparent to radiation (21,22). The simultaneous absorption of two photons at 800 nm may allow reaching the first excited state, since it is usually done with a single 400-nm photon. This occurs when two coherent photons "reach" the fluorescent target at the same time and requires the use of a high-energy, high-frequency (<100 fs) pulsed laser. The efficiency of two-photon excitation follows a power-squared relation and can be achieved only inside a tiny volume of the sample at a time. The whole cell is then not damaged by irradiation, and the risk of photobleaching is restricted to the tiny volume where the two photons collide. Nevertheless, some risk of photobleaching may remain for some fluorescent probes (23).

B Teflon ri"g Adherent cell

Beam

Laser

Laser

Evanescent field

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Getting Started With Solar

Getting Started With Solar

Do we really want the one thing that gives us its resources unconditionally to suffer even more than it is suffering now? Nature, is a part of our being from the earliest human days. We respect Nature and it gives us its bounty, but in the recent past greedy money hungry corporations have made us all so destructive, so wasteful.

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