Time resolved fluorescence measurement phase-modulation

The fluorescence lifetime can be measured by time-resolved methods after excitation of the fluorophore with a light pulse of brief duration. The lifetime is then measured as the elapsed time for the fluorescence emission intensity to decay to 1/e of the initial intensity. Commonly used fluorophores have lifetimes of a few nanoseconds, whereas the longer-lived chelates of europium(III) and terbium(III) have lifetimes of about 10-1000 /tsec (Table 14.1). Chapter 10 (this volume) describes the advantages of phase-modulation fluorometers for sensing applications, as a method to measure the fluorescence lifetime. Phase-modulation immunoassays have been reported (see Section 14.5.4.3.), and they are in fact based on lifetime changes. 

Two time-resolved fluorescence techniques, pulse Jluorimetry and phase-modulation fluorimetjy, are commonly employed to recover the lifetimes. The former uses a short exciting pulse (from femtoseconds to nanoseconds) of light, which leads to the pulsed response of the sample, which should then be deconvolved from the instrument response. In phase-modulation fluorimetry, the intensity of light used for excitation is modulated at a frequency whose reciprocal is similar to the fluorescence decay time. The sample response is also modulated, but with a time delay, measured as phase shift, from which the emission decay time can be calculated. Thus, the first technique works in the time domain, while the second one in the frequency domain. The most widely used technique in the time domain is the time-correlated single-photon counting [10, 11]. The merits of both techniques have been extensively discussed [12]. 

There are two widely used methods for measuring fluorescence lifetimes, the time-domain and frequency-domain or phase-modulation methods. The basic principles of time-domain fluorometry are described in Chapter 1, Vol.l of this series(34) and those of frequency-domain in Chapter 5, Vol. 1 of this series.<35) Good accounts of time-resolved measurements using these methods are also given elsewhere/36,37) It is common to represent intensity decays of varying complexity in terms of the multiexponential model... 

Phase-resolved, phase-modulation, or phase-sensitive lifetime measurements are based on the use of a continuous, sinusoidally modulated excitation source and phase-sensitive detection (Figure 7). The experimentally measured parameters are the modulation (m) and the frequency-dependent phase shift (4 ). The modulation of the excitation is given by bla, where a is the average intensity and b is the modulated amplitude of the incident light. For emission, the modulation is similarly defined, except using the intensities of the emission, BM, relative to the modulation of the excitation, m = B/A)/ b/a). The phase delay or phase angle ( P) is usually measured from the zero-crossing time of the modulated components. For an exponential decay, the fluorescence lifetime Tf can be calculated from the phase shift or... 

Other excitation energies Other than the ones at S, + 1380 and S, + 1420 cm-, there are three prominent bands in the intermediate region of jet-cooled anthracene s excitation spectrum. Time- and frequency-resolved measurements subsequent to excitation of these bands have also been made. Without going into any detail concerning the results of these measurements, we do note that all three excitations give rise to quantum beat-modulated decays whose beat patterns (phases and modulation depths) depend on the fluorescence band detected.42 Figure 16 shows an example of this behavior for excitation to S, + 1514 cm-1. The two decays in the figure correspond to the detection of two different fluorescence bands in the S, + 1514 cm-1 fluorescence spectrum. 

Another technique recently applied for immunoassays is phase-resolved fluorometry (frequency-domain fluorometry). This technique is also based on different fluorescence decay times. The decay time can change upon antigen-antibody binding. Instead of pulsed excitation, in this technique the sample is excited with sinusoidally modulated light. With phase-resolved fluorometry, decay times and decay time differences within the range of subnanoseconds can be measured. The phase-resolved technique can be used also for elimination of background noise. This technique, however, has found only a few applications to immunoassays yet. 

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