Info

'Quartz plate

Laser B

B Teflon ri"g Adherent cell

Laser B

Thin quartz plate

Fluorescence Recoven' Device

Fig. 3. Total internal reflection fluorescence microscopy. (A) Irradiation through the quartz plate used to support cells generates an evanescent field in thin volumes located on both sides of the plate. (B) Illustration of a device allowing grabbing of data from many cells at a time.

Because the source of potential photobleaching is the concentration of the excitation light through the objective, it has been suggested that cells be irradiated in another way. When cells are grown on a thin quartz plate, this plate can be used as a laser light guide inducing an evanescent electromagnetic field in the 0- to 100-nm range above the plate surface, a distance long enough to excite preferentially molecules inside the living cells (24). Moreover, this elegant technique allows the simultaneous collection of data on many individual cells (Fig. 3).

As indicated in Fig. 1, FRET affects the fluorescence intensity of both D and A and changes the lifetime of D. Methods that allow monitoring either fluorescence intensities or lifetime measurements can be used in quantification. When protein conformational changes are studied in solution, fluorescence spectroscopy can be used with conventional equipment (Fig. 4). The fluorescence spectrum resulting from a partial energy transfer will be a combination of the respective fluorescence spectra of the donor and the acceptor. The relative weight of these spectra will depend on the respective quantum yields of D and A for the wavelength used for excitation. The relative contribution of these spectra can be easily obtained even in the presence of other fluorophores or relatively noisy signals, using previously published methods of spectra resolution (25). By contrast, experiments performed on living cells require the use of excitation and emission filter sets (Fig. 5) (26). The use of three sets of filters is generally necessary, which is time-consuming (27). A simplification can be obtained by using fluorophores with minimal overlap of their respective excitation spectra and selecting excitation wavelengths out of this overlap

Fig. 4. Schematic drawing of a conventional spectrofluorometer (PM) photomulti-plier. Respective fluorescence spectra of protein-bound (LP), cation-bound (LM), and free (L) Mag-indo 1 are shown.
Fig. 5. Schematic drawing (not to scale) of a FRET multiwavelength fluorescence imaging microscope. The electronic shutters are necessary to minimize the cell irradiation resulting from illumination and data recording at different wavelengths. CCD, charge-coupled device.

A

Time domain (ns)

\ Excitation nuise

«V

s

y^^^^Fluorescence decay

B Frequency domain

Excitation Sample Fluorescence

Excitation Sample Fluorescence

Fluorescence

Fig. 6. Fluorescence lifetime imaging. (A) In the time domain method, a fast-gated camera is used to repeatedly record during very short periods of time (in gray) data from the multiexponential decay of the fluorescence (bold curve) excited by a very short excitation pulse (light curve). (B) In the frequency domain approach, measurable parameters are the phase shift AO and the relative reduction of amplitude of the fluorescence signal (IF) compared with that of the excitation pulse (IE).

Fluorescence

Fig. 6. Fluorescence lifetime imaging. (A) In the time domain method, a fast-gated camera is used to repeatedly record during very short periods of time (in gray) data from the multiexponential decay of the fluorescence (bold curve) excited by a very short excitation pulse (light curve). (B) In the frequency domain approach, measurable parameters are the phase shift AO and the relative reduction of amplitude of the fluorescence signal (IF) compared with that of the excitation pulse (IE).

range. As shown in Fig. 2B, the first set of filters will allow the monitoring of whole changes of the donor and acceptor fluorescence, respectively, and the second set will measure changes in the acceptor fluorescence resulting only from changes in the chemical microenvironment (without FRET). A combination of these data will allow monitoring of changes in FRET specifically.

Time life measurements can also be used to quantify FRET either in protein solution or in living cells. The fluorescence lifetime is independent of the concentration of the fluorophore, which is an important advantage because the probe concentration is expected to vary inside a living cell. On the contrary, the lifetime strongly depends on molecular interactions between the probe and its chemical environment. Energy transfer itself is expected to influence the lifetime of the donor, for it competes with other deactivation processes of the donor excited state. Thus, a 3D conformational change inducing a change in energy transfer will affect in a complex way the donor lifetime. Nevertheless, fluorescence lifetime imaging has recently been extensively used for FRET monitoring (28-30). Two fundamentally different approaches have been used. In the time domain approach, the fluorescence lifetime (xf) is measured by excitation of the sample with a short pulse of light, much shorter than the fluorescence lifetime (Fig. 6). Such a method requires a camera capable of fast gating, allowing the collection of the fluorescence signal during precise short periods of time (31-33). The use of the time domain approach is thus limited by the difficulty of gating a camera fast enough to collect information during the lifetime of a fluorescence decay (Fig. 6A).

In the frequency domain approach, the sample is excited with sinusoidally modulated light. The optical angular frequency of the light is reciprocal to the fluorescence lifetime to be measured. As a result, the emitted fluorescence is sinusoidally modulated at the same frequency but with a reduction in the modulation depth, M, and shifted in phase, AO (Fig. 6B). This technique requires a detector capable of high-frequency modulation. Parameters related to the respective intensities of the excitation pulse and the fluorescence signal, and to the phase shift AQ, are used to calculate two other parameters, the phase lifetime, Xf, and the modulation lifetime, tm. These last lifetimes are equal only when the sample contains only one fluorescent species, which is never the case inside a cell, owing to molecular interactions. When more than one fluorescent species is present, the phase and modulation lifetimes differ. In such a situation, the true lifetime composition can be obtained by measuring the phase and modulation lifetimes over a range of frequencies and fitting the results to a set of dispersion relationships. Fluorescence lifetime imaging measurements (FLIMs) may become more usual in the near future owing to several advantages associated with spectral discrimination. Simultaneous readout of data and the requirement of only one dichroic and long-pass emission filter result in the use of a lower light dose and reduce the risk of photochemical damage to the cells. Recent articles have reported on progress in numerical analysis of data and on the feasibility of multiwavelength frequency domain FLIM (34-38).

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