Intrinsic fluorescence time-resolved emission

Polymers of + ) catechin and (-)-epicatechin have an intrinsic fluorescence because chromophores are an integral part of each monomer unit. The time-resolved emission from well-characterized dimers can be used to determine the relative populations of two rotational isomers at the interflavan bond between monomer units. When combined with the solid-state conformations, molecular mechanics calculations, and rotational isomeric state analysis, the interpretation of the time-resolved fluorescence leads to the unperturbed dimensions of the polymers. Significant population of both rotational isomers causes the chains to have unperturbed dimensions comparable with those in atactic polystyrene molecules of the same molecular weight. 

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. 

For chromophores that are part of small molecules, or that are located flexibly on large molecules, the depolarization is complete—i.e., P = 0. A protein of Mr = 25 kDa, however, has a rotational diffusion coefficient such that only limited rotation occurs before emission of fluorescence and only partial depolarization occurs, measured as 1 > P > 0. The depolarization can therefore provide access to the rotational diffusion coefficient and hence the asymmetry and/or degree of expansion of the protein molecule, its state of association, and its major conformational changes. This holds provided that the chromo-phore is firmly bound within the protein and not able to rotate independently. Chromophores can be either intrinsic—e.g., tryptophan—or extrinsic covalently bound fluorophores—e.g., the dansyl (5-dimethylamino-1-naphthalenesulfonyl) group. More detailed information can be obtained from time-resolved measurements of depolarization, in which the kinetics of rotation, rather than the average degree of rotation, are measured. For further details, see Lakowicz (1983) and Campbell and Dwek (1984). 

To further characterize the mobility of the IRE loop, time-resolved isotropic fluorescence emission decay components of the IRE RNAs were determined as a function of temperature. Some details of the measurements and data assessment will be necessary here to appreciate both the utility of the information and caveats about its literal interpretation. Considering first the TCSPC instrument itself, some uncertainty in the measurements arise from its intrinsic parameters. With 300 nm incident light, the IRF of the photomultiplier tube ranged from 190 to 276 ps full-width at half-height (FWHH). The width of the IRF and the time resolution (32.5 ps/channel) limit the short components that can be reliably extracted from the fit, and certainly those <200 ps will have large errors on their amplitudes and lifetimes. Fluorescence emission decay components as short as 9—20 ps (Larsen et al., 2001) and 30—70 ps (Guest el al., 1991) (and much shorter by Wan et al., 2000) have been measured for 2AP in a stacked conformation, but in our instrument, a fit to such a short lifetime would be inaccurate. 

The latter point to a conformational transition of the protein at Tin. The time-resolved fluorescence studies indicated that the intrinsic Trp fluorescence emission of the protein was represented by a bimodal distribution with Lorential shape and was strongly affected by the protein conformational dynamics (Bismuto et al., 1999 D Auria et al.,1999). Parameters of the temperature dependence of the bimodal lifetime distribution, such as fraction relative intensity, the position of centres, and the distribution line widths,... 

It should be noted that the decay curve measured in the absence of an emission lar-izer will be In(t) + Ix(t) whereas inspectfon of the above demonstrates that the true fluorescence decay curve will be proportional to I (t) + 2Ix(t). Thus, for molecules rotating on the same timescale as the fluorescence decay, the fluorescence decay curve measured in the absence of a polarizer, will be distorted, and so time-resolved fluorescence depolarization must be considered even when the only desired measurement is the intrinsic fluorescence decay of the chromophore. To overcome this problem the fluorescence ould be monitored through a polarizer set at 54.7° to the excitation polarization vector ... 

In previous work W, we also saw a weak emission near 400-450 nm, which increases intensity upon photo-oxidation ( ). Interestingly, in the present work, involving rigorously degassed samples, little or no trace of this emission could be detected at any temperature, suggesting that it was entirely due to photo-oxidation in the previous work. For one sample, an extremely weak (<10"2 times as intense as intrinsic fluorescence) emission was seen in the 400-450 nm region, which at 17K was resolved in-... 

Among nonisotopic techniques, fluorescence (both intrinsic and extrinsic) offers a convenient mode of detection, and the sensitivity of some fluorescent labels is comparable to that of radiolabeled iodine. Recent innovations include the use of polarized light for excitation, such that the degree of polarization of the emission as well as its intensity can provide information about the concentration and size-related behavior (e.g., rotational diffusion) of the fluorescent-labeled molecule. One disadvantage of steady-state fluorescence techniques is that many analytical samples either autofluoresce or quench the fluorescence of the substance of interest. A recent development that circumvents this problem utilizes long-lived fluorophores such as the lanthanide metal ions as labels. Detection is time resolved and data are collected after the decay of spurious or otherwise unwanted fluorescence, i.e., after 100-200 psec. 

Another aspect, also considered in Subsection 1.8.3.3.2, concerns fundamental time-resolved fluorescence studies. Here, the emphasis is placed on fiuores-cence depolarization measurements, which are very helpful in following rotational and segmental motions and for studying the flexibility of macromolecules. If the polymer under investigation does not contain intrinsically fluorescent probes (e.g., certain amino acid moieties in proteins), then the macromolecules have to be labeled with fluorescent markers. Information concerning the rate of rotation or segmental motion then becomes available, provided that the emission rate is on a similar time scale. Only when this condition is met can the rate of depolarization be measured. If the emission rate is much faster, there is no depolarization, whereas if it is much slower, the depolarization will be total. 

A quantitative analysis and microscopic assignments of the time resolved fluorescence emission components from polymers in the solid or solution phase to specific physical entities is generally exceedingly difficult. This stems partly from (1) the intrinsic heterogeneity of the emission which in some cases arises from the preponderance of shallow traps, (2) the formation of intramolecular and in solid films or powders intramolecular excimers and (3) the effects of dimensionality and size. 

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