Fluorescence intrinsic lifetimes

Here t. is the intrinsic lifetime of tire excitation residing on molecule (i.e. tire fluorescence lifetime one would observe for tire isolated molecule), is tire pairwise energy transfer rate and F. is tire rate of excitation of tire molecule by the external source (tire photon flux multiplied by tire absorjDtion cross section). The master equation system (C3.4.4) allows one to calculate tire complete dynamics of energy migration between all molecules in an ensemble, but tire computation can become quite complicated if tire number of molecules is large. Moreover, it is commonly tire case that tire ensemble contains molecules of two, tliree or more spectral types, and experimentally it is practically impossible to distinguish tire contributions of individual molecules from each spectral pool. 

INTRINSIC AND EXTRINSIC FLUORESCENCE. Intrinsic fluorescence refers to the fluorescence of the macromolecule itself, and in the case of proteins this typically involves emission from tyrosinyl and tryptopha-nyl residues, with the latter dominating if excitation is carried out at 280 nm. The distance for tyrosine-to-tryp-tophan resonance energy transfer is approximately 14 A, suggesting that this mode of tyrosine fluorescence quenching should occur efficiently in most proteins. Moreover, tyrosine fluorescence is quenched whenever nearby bases (such as carboxylate anions) accept the phenolic proton of tyrosine during the excited state lifetime. To examine tryptophan fluorescence only, one typically excites at 295 nm, where tyrosine weakly absorbs. [Note While the phenolate ion of tyrosine absorbs around 293 nm, its high pXa of 10-11 in proteins typically renders its concentration too low to be of practical concern.] The tryptophan emission is maximal at 340-350 nm, depending on the local environment around this intrinsic fluorophore. 

FRET interactions are typically characterized by either steady-state or transient fluorescence emission signals from the donor or acceptor species. Efficient nonradiative energy transfer results in donor PL loss associated with acceptor gain in photoluminescence intensity (if the acceptor is an emitter). The rate of this energy transfer is related to the intrinsic lifetime of the isolated donor and depends strongly on the donor-acceptor separation distance ... 

The longest wave absorption band of anthracene is short axis polarized. The substitution in 9,10 positions leads to a bathochromic shift in this band. The intrinsic lifetimes are proportional to Jandean be obtained from the area under the respective absorption curves. The molar extinction coefficients are 9, 10-dichloro-A > 9-chloro-A > A. The lifetime decreases with increase of absorbance and at the same time the fluorescence efficiency f is observed to increase. The values of f f°r various anthracenes in CC14 and the quantum efficiencies of their reactions with the solvent, both in absence of oxygen, are presented in Table 11.5. 

In the electron-transfer process generalized in Eq. 1, one of the components of the reactant state may be fluorescent. This spin-allowed radiative process will thus be in competition with the nonradiative electron-transfer reaction and the two processes will contribute to the overall decay of the reactant state. The intrinsic lifetimes of fluorescent molecular states range typically from 10 to longer than 10 s. The occurrence of electron transfer involving the fluorescent state will shorten its lifetime and measurement of this quantity will therefore allow computation of the rate constant for electron transfer. 

One-dimensional velocity distribution Specific conductivity, hard-sphere diameter for a collision, length parameter in Lennard-Jones potential, symmetry number of a molecule Intrinsic lifetime of a photoexcited state Azimuthal angular velocity in spherical polar coordinates, azimuthal angle in spherical polar coordinates, angle of deflection Quantum yield at wavelength A Fluorescence (phosphorescence) quantum efficiency... 

The effect is significant for reactions with short half-times. In an ordinary diffusion-limited reaction under conditions where the species B is in great excess, the half-time is l/(/ diff[B]). If / diff 10 ° sec-, a steady-state treatment of the kinetics will suffice as long as [B] < 0.1 If [B] is larger, the effect of transient behavior cannot be ignored. Another instance where transients are important is in the kinetics of fluorescence quenching. Here, the intrinsic lifetimes of the photoexcited species are typically 10 sec. The photostationary state is not established and effects attributable to the time dependence of fcapp can be observed. 

The experimental results for 6 were very encouraging. Excitation at 590 nm populates the two porphyrin excited singlet states, but steady state and time resolved fluorescence measurements revealed that singlet energy transfer from the Pzn to the free base P occurs with a time constant of ca. 40 ps and an efficiency of 90% (step 1 in Fig. 7), resulting in the excitation being localized on P. This result confinned the expectation that the added porphyrin would act as an antenna for the system. In a model dyad consisting of just the PZn-P species, the intrinsic lifetime of the firee base... 

Because of the underlying photophysics, fluorescence lifetimes are intrinsically short, usually on the order of a few nanoseconds. Detection systems with a high timing resolution are thus required to resolve and quantify the fluorescence decays. Developments in electronics and detector technology have resulted in sophisticated and easy to use equipment with a high time resolution. Fluorescence lifetime spectroscopy has become a popular tool in the past decades, and reliable commercial instrumentation is readily available. 

Temporal characteristics at early stages were elucidated by measuring fluorescence intensity with the gate time of 1.74 ns as a function of the delay time. Compared to the laser pulse, the time where the maximum intensity is attained shifts to the early stage as the laser fluence becomes high. Of course, we could not find out any decay component with intrinsic fluorescence lifetime of 17 and 35 ns. It is concluded that an Si - Si annihilation occurs quite efficiently during the pulse width. 

In the passive mode, the optical device measures the variation in fluorescence characteristics (intensity, lifetime, polarization) of an intrinsically fluorescent analyte. The optical device can have different optical configurations involving in most cases an optical fiber (passive optode) (Figure 10.44). 

The various possible schemes for fluorescence sensing are summarized in Figure 1.1. At present, most fluorescence assays are based on the standard intensity-based methods, in which the intensity of the probe molecule changes in response to the analyte of interest. However, there has been the realization that lifetime-based methods possess intrinsic advantages for chemical sensing. (A more detailed description of... 

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). 

From the practical point of view, the radiative decay rate kr may be assumed to be independent of the external parameters surrounding the excited sensor molecule. Its value is determined by the intrinsic inability of the molecule to remain in the excited state. The radiative decay rate kr is a function of the unperturbed electronic configuration of the molecule. In summary, for a given luminescent molecule, its unperturbed fluorescent or phosphorescent decay rate (or lifetime) may be regarded to be only a function of the nature of the molecule. 

In summary, the use of fluorescence lifetime monitoring for temperature sensing at high temperatures is based on the phenomenon of thermal quenching of fluorescence, while this phenomenon is j u st the very obstacle that blocks the extending of the measurement further into higher temperatures. Therefore, fluorescence thermometry is intrinsically more effective for measurement within moderate temperature regions, due to this fundamental nature of the fluorescence emission itself. 

Problems still remain in overcoming the intrinsic optical cross-talk in arrays of avalanche photodiodes, which at present precludes equivalent applications to multianode MCP-PMs in such as multiplexed lifetime measurements at different fluorescence wavelengths. 

The lifetime of monomer fluorescence in the absence of routes to excimer formation is tm = (kFM + koM + kTM) 1 = ky1, and the intrinsic quantum yield is Qm = kpM/kM. The lifetime and intrinsic quantum yield of excimer fluorescence, td and Qd, will be considered in a later section. 

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