Steady-state fluorimetry

The spectroscopy described thus far is based on the measurement of the intensity of fluorescence produced under steady-state conditions of excitation. Steady-state fluorimetry is derived from the excitation of the sample with a continuous beam of exciting radiation. The lamps and the power supplies used in conventional fluorimeters are sources of continuous radiation. After a short period of initial excitation of the sample, a steady state is established in which the rate of excitation of the analyte is equal to the sum of the rates of all processed, deactivating the lowest excited singlet state including fluorescence. When fhe sfeady state... 

The application of semiconductor lasers to a broad range of areas in spectrometry has recently been reviewed by Imasaka. 67, 68) Topics covered include photoacoustic, absorption, and thermal lens, as well as steady-state and time-resolved fluorescence. Patonay et al. have reviewed the application of diode lasers to analytical chemistry.(69) The performance of several commercially available laser diodes for fluorimetry has recently been compared. 70 ... 

Measurement of the donor lifetime, which typically is 2-25 nsec, requires adequate time resolution. Two techniques, time-correlated singlephoton counting and frequency-domain fluorimetry modulation, can be used (see A. R. Holzwarth, this volume [14]). Excellent books have been written which include discussion of each technique, and Lakowicz and co-workers have discussed advances infrequency-domain instrumentation and applications to FRET. Donor lifetime measurements, unlike steady-state measurements, are capable of detecting multiple donor-acceptor transfer efficiencies in the sample. These lead to multiexponential decays. Donor lifetime measurements are also not affected by an inner-filter effect... 

Fluorimetry has experienced an explosive growth since the early 1980s, much of which has been driven by the use of fluorescence as a noninvasive technique for biology and biochemistry. Fluorescence techniques are widely used to quantify molecular parameters of different chemical, biochemical, and biological processes because of their inherent sensitivity, specificity and temporal resolution. In fact, the luminescence lifetime is an important characteristic of a fluorescent molecule and its environment. Many intra- and intermolecular processes are able to modulate the molecule emission which cannot be investigated by steady-state fluorescence measurements. For example, rotational diffusion,... 

Fluorescence is unique among spectroscopic techniques due to its inherently multidimensional nature, the emission process containing a wealth of orthogonal information that is related to the fluorophore and its surroundings. Time- and frequency-domain fluorescence methods are instrumentally sophisticated, but they improve both the sensitivity and selectivity of fluorimetry. Any dye that is used for steady-state fluorescence detection can be used for time-resolved detection as well. Most of these fluor-ophores display lifetimes from 1 to 10 ns, which requires fast electronics for time-domain lifetime measurements or modulation frequencies from 10... 

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