Time resolved fluorescence measurement method

M. G. Badea and L. Brand, Time-resolved fluorescence measurements, Methods Enzymol. 61, 378-425 (1979). 

The quantum yields of fluorescence and phosphorescence, 4>f and d>p, may be determined experimentally by means of a fluorescent standard such as a rhodamine B solution whose independent of the exciting wavelength within a wide range. Lifetimes rp and rp are also experimentally accessible through time-resolved fluorescence measurements (phase method or single-photon counting) or by measuring the time dependence of phosphorescence. (Cf. Rabek, 1982.) In Table 5.2 the observable quantities and their relationship to rate constants are collected. 

A streak camera which has 12 ps shot-to-shot jitter has been developed. With this degree of precision, a simple method of signal averaging of picosecond fluorescence events may be employed direct summation. This results in a substantial increase in the signal-to-noise ratio of time-resolved fluorescence measurements over single-shot records. 

There are several ways to perform time-resolved fluorescence measurements. Since the time dependence of fluorescence emission is typically on a picosecond to nanosecond time scale it is very difficult to achieve. To overcome this difficulty either a frequency domain method or the single photon counting approach is used. 

The second method for measuring FRET involves time-resolved fluorescence measurements. This method provides a way of obtaining average lifetimes without a precise knowledge of donor concentrations. The technique enables the quantitative determination of donor-acceptor separation distances, and is based on the measurements of the donor lifetime in the presence and absence of the acceptor. Measuring fluorescence intensity decay as a function of time elucidates the emission dynamics of... 

The measurement of the time-dependent depolarization of the fluorescence from molecules rotating on a time-scale comparable to the fluorescence decay time, enables information to be derived concerning the molecular reorientation motion. A review of these techniques has been published. A method involving an optical delay line has been used to record time-resolved fluorescence depolarization methods using only 1 photodetector, and thus some of the possible instrumental distortions are removed. ... 

Time resolved anisotropies can simply be measured by adding polarization sensitivity to time resolved fluorescence measurements described in Section 2.7.4. The qualitative interpretation of the data follows intuitively from the discussion of steady state anisotropy in Section 2.7.5, so we do not describe the method further. For more information we refer the reader elsewhere [1,127]. 

The time behaviour of transient spectra is very essential for the assignment of these spectra to energy levels or species. If possible, independent experimental methods should be used to gain data of the time behaviour. Therefore, we performed time resolved fluorescence measurements on the oligothiophenes and obtained the fluorescence lifetimes of T [3]. Today other references are also available [11]. 

A new era of research in fluorescence spectroscopy has emerged with the advent of powerful lasers capable of generating short-lived pulses and with the simultaneous development of sophisticated detection methods. While research groups were previously limited to the study of processes on the microsecond and nanosecond time scale, these developments have expanded the accessible time scale to the pico- and femtosecond. Time-resolved fluorescent measurements are being used, for example, to unravel the dynamics of excited states (excitons) generated in conjugated polymer films (such as stimulated emission) and the processes that... 

Another dynamic method is to use a time-resolved fluorescence measurement in which the kinetics of intramicellar excimer formation and dissociation are taken into consideration. The excimer is a dimer made of activated and nonactivated molecules. The excimer of pyrene in micelles has been investigated in detail, and pyrene has been employed in most cases. 

In the past ten years, numerous applications of fluorescence methods for monitoring homogeneous and heterogeneous immunoassays have been reported. Advances in the design of fluorescent labels have prompted the development of various fluorescent immunoassay schemes such as the substrate-labeled fluorescent immunoassay and the fluorescence excitation transfer immunoassay. As sophisticated fluorescence instrumentation for lifetime measurement became available, the phase-resolved and time-resolved fluorescent immunoassays have also developed. With the current emphasis on satellite and physician s office testing, future innovations in fluorescence immunoassay development will be expected to center on the simplification of assay protocol and the development of solid-state miniaturized fluorescence readers for on-site testing. 

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