Fluorescence time scale

In Eqs. (4.43)—(4.47), use is made of the well-known rclation(23)P = K/kBT, where k is the bending rigidity. Equation (4.45) differs from the corresponding BZ result by the inclusion of the D L / term. Normally, this is negligible on the fluorescence time scale, but for sufficiently short filaments it could make a significant contribution. It appears with the coefficient 1.0 instead of 2.0 because the correction term from the Euler-McLaurin summation formula actually cancels out one of the two D t terms in Eq. (4.43). Equation (4.43) or (4.45) is then inserted in Eq. (4.26) to obtain the BZ tumbling correlation function for the filament. 

Fluorescence lifetime measurements can give information about collisional deactivation processes, about energy transfer rales, and about excited-slate reactions. Lifetime measurements can also he used analytically to provide additional selectivity in the analysis of mixtures containing luminescent species. The measure-mciil of luminescence lifetimes was initially restricted to phosphorescent systems, where decay limes were long enough to permit the easy measurement of eiiiil-ted intensity as a function of lime. In recent years, however, it has become relatively routine to measure rates of luminescence decay on the fluorescence time scale (10 Mo -r 10 M). 

Finally, tlie ability to optically address single molecules is enabling some beautiful experiments in quantum optics. The non-Poissonian photon arrival time distributions expected tlieoretically for single molecules have been observed directly, botli antibunching at short times [112] and bunching on longer time scales [6, 112 and 113]. The fluorescence excitation spectra of single molecules bound to spherical microcavities have been examined as a probe... 

Figure 4.7 Changes in intraceiiuiar calcium in cultured rat ventricular myocytes exposed to oxidant stress. Calcium was measured using the fluorescent probe Fura>2. The ratio of the Fura-2 fluorescence measured at 340 and 380 nm excitation is shown and this is proportional to the intracellular calcium concentration. The fast-speed traces shown (note the 3.5 s time-scale) were recorded after various durations of oxidant stress. Myocytes under control conditions (before t = 0) show spontaneous calcium transients. These transients decreased in frequency with oxidant stress until cells failed to show spontaneous activity but continued to maintain a low intracellular calcium.

The lifetime detection techniques are self-referenced in a sense that fluorescence decay is one of the characteristics of the emitter and of its environment and does not depend upon its concentration. Moreover, the results are not sensitive to optical parameters of the instrument, so that the attenuation of the signal in the optical path does not distort it. The light scattering produces also much lesser problems, since the scattered light decays on a very fast time scale and does not interfere with fluorescence decay observed at longer times. 

Gadella, T., Jovin, T. and Clegg, R. (1994). Fluorescence lifetime imaging microscopy (FLIM) Spatial resolution of microstructures on the nanosecond time scale. Biophys. Chem. 48, 221-39. 

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