Anisotropy decays instrumentation

Figure 4.7 Fluorescence anisotropy decay curves for the PMMA brush swollen in benzene (filled circles) and the free PMMA chain in benzene solution at concentrations of 0.33 (triangles) and 2.9 X 10 g (open circles). The graft density of the brush is 0.46 chains nm . The solid curve indicates the instrument response function. Reproduced with permission from the American Chemical Society.

Anisotropic rotati onal diffusion has been more Nct sively studied using FD methods. In fact, the earliest reports on the anisotropic rotation of fluorophores concerned experiments performed using fixed-frequency phase-modulation fluorometers. At that time the phase-modulation instruments operated at only one or two fixed hrequendes. Hence, it was not possible to recover the anisotropy decay law. The experiments were performed by measuring the differential polarized phase angles as the temperature was varied. It is relatively simple to predict the maximum value of Ao) for known values of the lifetime and fundamental anisotropy. For an isotropic rotor, the predicted value of Aw is given by... 

In the previous chapter we presented an overview of protein fluorescence. We described the spectral properties of the aromatic amino acids and how these properties depend on protein structure. We now extend this discussion to include time-resolved measurements of intrinsic protein flu( scence. Prior to 1983, most measurements of time-resolved fluorescence were performed using TCSPC. The instruments employed for these measurements typically used a flashlamp etcitation source and a standard dynode-chain-type PMT. Such instruments provided instrument response functions with a half-width near 2 ns, which is comparable to thedecay time of most proteins. The limited repetition rate of the flashlamps, near 20 kHz. resulted in data of modest statistical accuracy, unless the acquisition times were excessively long. Given the complexity of protein intensity and anisotropy decays, and the inherent difficulty of resolving multiexponential processes. ii was difficult to obUun definitive information on the decay kinetics of proteins. 

The data in Figure 17.18 illustrate another problem encountered when one is measuring protein anisotropy decays. Examination of the data reveals that the measured time-zero anisotropy [ 0)1 less than the fundamental anisotropy (ro=0.3) for the 300-nm excitation wavelength. This frequently occurs owing to the limited time resolution of the instrumentation. If the correlation time is too sh< t, the anisotropy decays within the instrument response function, and the apparent time-zero anisotropy is less than die actual value. ... 

TRAMs carried out on dilute solutions of P(ACE) in MTHF glasses are shown in Figure 2a. The anisotropy decays to zero within the time resolution of our current TCSPC instrumentation (<100 ps), indicating extremely rapid and efficient EEM. The additional emission depolarizing... 

Definition and Uses of Standards. In the context of this paper, the term "standard" denotes a well-characterized material for which a physical parameter or concentration of chemical constituent has been determined with a known precision and accuracy. These standards can be used to check or determine (a) instrumental parameters such as wavelength accuracy, detection-system spectral responsivity, and stability (b) the instrument response to specific fluorescent species and (c) the accuracy of measurements made by specific Instruments or measurement procedures (assess whether the analytical measurement process is in statistical control and whether it exhibits bias). Once the luminescence instrumentation has been calibrated, it can be used to measure the luminescence characteristics of chemical systems, including corrected excitation and emission spectra, quantum yields, decay times, emission anisotropies, energy transfer, and, with appropriate standards, the concentrations of chemical constituents in complex S2unples. 

The rotational relaxation of DNA from 1 to 150 ns is due mainly to Brownian torsional (twisting) deformations of the elastic filament. Partial relaxation of the FPA on a 30-ns time scale was observed and qualitatively attributed to torsional deformations already in 1970.(15) However, our quantitative understanding of DNA motions in the 0- to 150-ns time range has come from more accurate time-resolved measurements of the FPA in conjunction with new theory and has developed entirely since 1979. In that year, the first theoretical treatments of FPA relaxation by spontaneous torsional deformations appeared. 16 171 and the first commercial synch-pump dye laser systems were delivered. Experimental confirmation of the predicted FPA decay function and determination of the torsional rigidity of DNA were first reported in 1980.(18) Other labs 19 21" subsequently reported similar results, although their anisotropy formulas were not entirely correct, and they did not so rigorously test the predicted decay function or attempt to fit likely alternatives. The development of new instrumentation, new data analysis techniques, and new theory and their application to different DNAs in various circumstances have continued to advance this field up to the present time. 

At present, there is widespread interest in directly measuring the time-dependent processes. This is because considerably more information is av lilable from the time-dependent data. For example, the time-dependent decays of protein fluorescence can occasionally be used to recover the emission spectra of individual tryptophan residues in a protein. The time-resolved anisotropies can reveal the shape of the protein and/or the extent of residue mobility within the protein. The time-resolved energy transfer can reveal not only the distance between the donor and acceptor, but also the distribution of these distances. The acquisition of such detailed information requires high resolution instrumentation and careful data acquisition and analysis. 

Thus after a period of tf the intensity has dropped to 37% of h, that is 63% of the molecules return to the ground state before tf. In many cases the above expression needs to be modified into more complex expressions. First of all it is assumed that the instrument yields an infinite (or very) short light pulse at time zero. In cases where tf is small Ig must be replaced by a function, which describes the lamp profile of the instrument. Also, more than one lifetime parameter is often needed to describe the decay profile, which is l(t) must be expressed as a sum of exponentials. Finally the concept of anisotropy should be mentioned. Anisotropy is based on selectively exciting molecules with their absorption transition moments aligned parallel to the electric vector of polarized light. By looking at the polarization of the emission the orientation of the fluorophore can be measured. The anisotropy of the system is defined as (Equation 6) (Rendell, 1987 Lakowicz, 2006) ... 

For fluorescence measurements, by far the most versatile and widely used time-resolved emission technique involves time-correlated single-photon counting [8] in conjunction with mode-locked lasers, a typical mo m apparatus being shown in Figure 15.8. The instrument response time of such an apparatus with microchannel plate detectors is of the order of 70 ps, giving an ultimate capability of measurement of decay times in the region of 7 ps. However, it is the phenomenal sensitivity and accuracy which are the main attractive features of the technique, which is widely used for time-resolved fluorescence decay, time-resolved emission spectra, and time-resolved anisotropy measurements. Below ate described three applkations of such time-resolved measurements on synthetic polymers, derived from recent work by the author s group. 

Some of these applications are complementary to the steady state methods discussed in section 8.2. Investigations of fluorescence lifetimes and of anisotropy or fluorescence quenching phenomena in the lifetime mode, that is during the decay after a single flash, require more elaborate instrumentation and theory than steady state investigations. On the whole applications rather than detail of methods are discussed here. The use of the lifetime method for the study of molecular rotation, domain movement and more local dynamic events can often, some experts say always, provide additional information even for those problems which can be investigated with considerable success by steady state measurements. 

By the use of fluorescence lifetime instrumentation, one can further determine the evolution of the fluorescence anisotropy with time during and beyond the lifetime of the excited state. In the simplest case of a fluorophore in solution, with a single fluorescence lifetime and depolarization through rotational relaxation alone, the fluorescence anisotropy will decay according to... 

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