Fluorophore decay time

In time-resolved fluorescence, rare earths are frequently used as fluorescent labels. The fluorophores have large Stokes shifts, ie, shifts of the emitted light to a higher wavelength relative to the absorption wavelength, and comparatively long decay times, approximately 0.5 ms. This simplifies the optical... 

Lifetime [3,9-11] based sensors rely on the determination of decay time of the fluorescence or phosphorescence. Typically, the fluorescence lifetime is 2-20 ps and phosphorescence lifetime is 1 ps to 10 s. Lifetime-based sensors utilize the fact that analytes influence the lifetime of the fluorophore. Thus all dynamic quenchers of luminescence or suitable quenchers can be assayed this way. The relationship between lifetimes in the absence (t0) and presence (t) of a quencher is given by Stern and Volmer ... 

When the fluorescence decay of a fluorophore is multi-exponential, the natural way of defining an average decay time (or lifetime) is ... 

A solution of a pure fluorophore may reasonably be expected to display a single exponential decay time. The emission from fluorophore-protein conjugates, on the other hand, may be best characterized by two or three exponential decay times (Table 14.2). In labeling proteins with fluorophores, a heterogeneity of labeled sites results in fluorophore populations that have different environments, and hence different lifetimes. The lifetime distribution of a fluorophore-protein conjugate in bulk solution may vary further when immobilized on a solid support (Table 14.2). 

There are several conformational arrangements with different interactions of the fluorophores with surrounding groups of atoms. Such interactions may affect differently nonradiative deexcitation processes in the excited state, and the decay times for these conformational states will differ. If each of these states is characterized by singleexponential decay kinetics, then the number of constants f, will correspond to the number of aromatic groups in the protein that are in conformationally different states. 

The inert reference luminophore and indicator fluorophore need to have highly different decay times... 

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

The refractive index of the medium also has an affect upon the radiative rate constant for decay of a fluorophore, as shown in Eq. (27) (see ), which can thus affect observed decay times and quantum yields. 

A related method has been used to demonstrate that lifetimes as ort as 200 ps can be measured usii the mode noise in a free-running a on-ion laser to produce variations in the excited state population of a fluorophore . Meaairement of the rf power spectmm of the resilting fluctuations then reveals the excited state lifetime. Mode noise contains very high frequency fluctuations which the excited state population cannot follow because of its finite lifetime, and thus these hi frequency components are absent from the rf spectrum of the fluorescence fluctuations. The fluorescence process thus acts like a low pass exponential filter, and comparison of the fluorescence power spectmm with that of the source provides the decay time data, as demonstrated below. 

As an example of excitation energy transfer studied by time-resolved fluorescence, let us take again the case of the inclusion complex of the multichromophoric cyclodextrin CD-St with oxazine 725 described in Section 7.2.4.2 [15]. Figure 7.9 shows the fluorescence decay of CD-St the very first part of the decay is due to energy transfer [13] from the steroidic naphthalene fluorophores to oxazine 725. Data analysis led to an average decay time for transfer of about 25 ps, which is quite fast, as expected from the short average distance between donor and acceptor ( 9-10 A). 

Evidently, fluorescers with decay times much longer than the lamp pulse characteristics can be analyzed in much the same way as radioactive decay curves. A semilogarithmic plot of fluorescence intensity against time is linear, with a slope proportional to the decay time and the ordinate intercept providing a quantitative measure of the amount of fhiorophore. If the lamp pulse time and the decay time of the fhiorophore are comparable, the fluorophore s decay charac-... 

One common approach to fluorescence sensing is to rely on fluorophores which are collisionally quenched by the analyte. There are many known collisional quenchers (andytes) which alter the fluorescence intensity and decay time. These include O2 (27-31), chloride (32-33), chlorinated hydrocarbons (34), iodide (35), bromate (36), xenon (37), acrylamide (38), succinimide (39), sulfur dioxide (40), and halothane (41), to name a few. The quenching process obeys the Stem-Volmer equation ... 

The nucleotides and nucleic acids are generally non-fluorescent. However, some notable exceptions are known. Phenylalanine transfer RNA from yeast (tRNA ) contains a single highly fluorescent base, called the Y-base, which has an emission maximum near 470 nm. The presence of this intrinsic fluorophore has resulted in numerous studies of tRNA by fluorescence spectroscopy. Regarding the non-fluorescent nucleic acids, it should be noted that they do fluoresce, but with very low yields and with short decay times. 

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