Fluorophore decay characteristics

In contrast, time domain instruments attempt to directly measure the decay characteristics of a fluorophore of interest by excitation with ultrashort light pulses and monitoring the decay using either TCSPC [5] or a time gated image intensifier [8],... 

Figure 4.9 illustrates time-gated imaging of rotational correlation time. Briefly, excitation by linearly polarized radiation will excite fluorophores with dipole components parallel to the excitation polarization axis and so the fluorescence emission will be anisotropically polarized immediately after excitation, with more emission polarized parallel than perpendicular to the polarization axis (r0). Subsequently, however, collisions with solvent molecules will tend to randomize the fluorophore orientations and the emission anistropy will decrease with time (r(t)). The characteristic timescale over which the fluorescence anisotropy decreases can be described (in the simplest case of a spherical molecule) by an exponential decay with a time constant, 6, which is the rotational correlation time and is approximately proportional to the local solvent viscosity and to the size of the fluorophore. Provided that... 

The lifetime, therefore, depends not only on the intrinsic properties of the fluorophore but also the characteristics of the environment. For example, any agent that removes energy from the excited state (i.e., dynamic quenching by oxygen) shortens the lifetime of the fluorophore. This general process of increasing the nonradiative decay rates is referred to as quenching. 

Fast librational motions of the fluorophore within the solvation shell should also be consideredd). The estimated characteristic time for perylene in paraffin is about 1 ps, which is not detectable by time-resolved anisotropy decay measurement. An apparent value of the emission anisotropy is thus measured, which is smaller than in the absence of libration. Such an explanation is consistent with the fact that fluorescein bound to a large molecule (e.g. polyacrylamide or monoglucoronide) exhibits a larger limiting anisotropy than free fluorescein in aqueous glycerolic solutions. However, the absorption and fluorescence spectra are different for free and bound fluorescein the question then arises as to whether r0 could be an intrinsic property of the fluorophore. 

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-... 

In the time window between the absorption and emission of a photon, a number of molecular processes can occur. They concern either (a) the fluorophore itself (its rotational and translational diffusion, conformational changes, transition between electronic states differing in dipole moment) or (b) molecules in its immediate vicinity (reorganization of the solvent shell, diffusion of quenchers, etc.). All these processes influence the fluorescence properties (position and shape of the emission band, quantum yield, decay time, etc.). In most cases, both the fluorophore and the surrounding molecules participate in the process and fluorescence characteristics are in fact influenced by their mutual interactions. Figure 3 shows a survey of important... 

Another important characteristic of RET is that the transfer rate is proportional to the decay rate of the fluoro-phore (Eq. [13.1]). This means that for a D-A pair spaced by the value, the rate of transfer will be kjsx ) whether the decay time is 10 ns or 10 ms. Hence, long-lived lanthanides are expected to display RET over distances comparable to those for the nanosecond-decay-time fluorophores, as demonstrated by transfer from Tb " to Co " in thermolysin. This fortunate result occurs because the transfer rate is proportional to the emission rate of the donor. The proportionality to the emissive rate is due to the term Qq/ d in Eq. [13.2]. It is interesting to speculate what would happen if the transfer rate were independent of the decay rate. In this case, a longer-lived donor would allow more time for energy transfer. Then energy transfer would occur over longer distances where the smaller rate of transfer would still be comparable to the donor decay rate. 

Principles and Characteristics The analytical capabilities of the conventional fluorescence (CF) technique (c/r. Chp. 1.4.2) are enhanced by the use of lasers as excitation sources. These allow precise activation of fluorophores with finely tuned laser-induced emission. The laser provides a very selective means of populating excited states and the study of the spectra of radiation emitted as these states decay is generally known as laser-induced fluorescence (LIF, either atomic or molecular fluorescence) [105] or laser-excited atomic fluorescence spectrometry (LEAFS). In LIF an absorption spectrum is obtained by measuring the excitation spectrum for creating fluorescing excited state... 

As in other forms of microscopy, there are three basic kinds of fluorescence microscopy qualitative, quantitative and analytical. Qualitative fluorescence microscopy is concerned with morphology, or with whether something (e.g. an immunological reaction) is present. Quantitative fluorescence microscopy is concerned with finding out how much of a specific substance is present in a specified region of the specimen. Analytical fluorescence microscopy is the characterization of a fluorophore by measurement of excitation and emission spectra or other characteristics such as polarization or decay time. Kinetic studies essentially involve studying the fluorescence parameters described above over a period of time examples include the study of fading rates, enzyme kinetics, time-resolved fluorescence and phosphorescence. 

The emission characteristics of fluorophores (the quantum yield and the wavelength of the emission maximum) are sensitive to their immediate environment and this leads to pronounced changes of fluorescence during protein denaturation (16). The high sensitivity of fluorescence to environmental effects was demonstrated in a particularly effective way in a study in which the decay of tryptophan fluorescence was followed in a number of proteins containing a single tryptophan residue (17). Deviations from simple exponential decay were interpreted as demonstrating that these proteins have a variable conformations, with the rate of their interconversion slow compared with the lifetime of the excited tryptophan. 

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