Intrinsic fluorescence excitation effect

From a practical point of view the consequences of TOF dispersion are important only for short intrinsic fluorescence decay times of to < 1 nsec. Figure 8.15 shows an example with to = 50 psec and realistic optical constants of the substrate. The intensity maximum in Fb(t) is formed at At 30 psec after (5-excitation. After this maximum, the fluorescence decays with an effective lifetime of r ff = 100 psec that increases after long times to t > > 500 psec. The long-lived tail disappears as soon as there is some fluorescence reabsorption, and for Ke = K there is practically no difference to the intrinsic decay curve (curve 3 in Figure 8.15). 

Titration with chelators of a metalloenzyme preparation from which extraneous metals and chelators have been removed produces a characteristic enhancement of the intrinsic protein fluorescence (excitation at 280 nm, emission at 350 nm) (13). This fluorescence enhancement by nonfluorescent chelators is instantaneous, reversible by excess added divalent metal ions, and can occur without loss of activity. Different chelators give different characteristic amounts of fluorescence enhancement at saturation, demonstrating the specific effect of the chelator on the fluorescence of the apparent metalloenzyme-chelator complex. In contrast, if the effect of chelators were simply to complex with Mg2 after its dissociation from the metalloenzyme, the resulting apoenzyme should have identical fluorescence properties regardless of which chelator was utilized. 

Figure 15.2 Effect of pH on the intrinsic fluorescence of an odorant-binding protein from P diversa, PdivOBP2, with a large conformational transition between pH 6 and pH 5.5. Spectra were obtained on a Shimadzu RF-5301PC spectrofluorophotometer with a 6 pg/ml protein solution in 20 mM buffers, with excitation at 235 nm and emission monitored from 280 to 420 nm. (1) pH 7, (2), pH 6 in sodium phosphate, (3) pH 5.5, (4) pH 5, (5) pH 4.5, and (6) pH 4 in sodium acetate.

Figure 1. The effect of dimethyl sulfoxide on the structure of fi-galactosidase, as monitored by (A) the intrinsic fluorescence (B) the intrinsic uv absorption. Conditions 0°C, pH 7.0, excitation at 285 nm, x = 300 nm, A = 290 nm,...

Here, L(ti) is the hei t and At the width of the excitation pulse s 6-fimction component at time t/. F(t) (the intrinsic fluorescence decay) is given by equation 1 and its argument t - t accounts fijr the fiict that the elapsed time varies between when the different subpopulations are created (at times t, ) and when the fluorescence intensity is measured. The (relative) height of L at t( appropriately scales the magnitude of the resulting fluorescence response. The effect of convolving an exponential decay with an excitation pulse of finite width will be seen in Section 2.6.4, where we describe the analysis of pulsed-excitation data. A fuller discussion of the convolution integral may be found in (9). ... 

Two-photon excitation provides intrinsic 3-D resolution in laser scanning fluorescence microscopy. The 3-D sectioning effect is comparable to that of confocal microscopy, but it offers two advantages with respect to the latter because the illumination is concentrated in both time and space, there is no out-of-focus photo-bleaching, and the excitation beam is not attenuated by out-of-focus absorption, which results in increased penetration depth of the excitation light. 

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