Lifetime, fluorescence modulation measurement

Prior to describing the possible applications of laser-diode fluorometry, it is important to understand the two methods now used to measure fluorescence lifetimes these being the time-domain (Tl)/4 5 24 and frequency-domain (FD) or phase-modulation methods.(25) In TD fluorometry, the sample is excited by a pulse of light followed by measurement of the time-dependent intensity. In FD fluorometry, the sample is excited with amplitude-modulated light. The lifetime can be found from the phase angle delay and demodulation of the emission relative to the modulated incident light. We do not wish to fuel the debate of TD versus FD methods, but it is clear that phase and modulation measurements can be performed with simple and low cost instrumentation, and can provide excellent accuracy with short data acquisition times. 

Figure 11.9. Phase and modulation measurement of fluorescence lifetime, tan = 2 jr/r = phase difference.

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. 

Immunoassays based on phase-modulation spectroscopy have been implemented by two distinctly different approaches. Phase-resolved immunoassays rely on fluorescence intensity measurements, in which the emission of one fluorescent species in a mixture is suppressed, and the remainder is quantitated. Phase fluorescence immunoassays utilize measurements of the phase angle and modulation, which change in response to fluorescence lifetime changes. Common aspects of the theory and instrumentation are discussed in this section, followed by individual discussions of the different approaches. 

PFIAs and fluorescence lifetime immunoassays (FLIAs) are uniquely based on measurement of probe emission properties other than the intensity. The phase and modulation are measured, and they directly reflect the fluorescence lifetime of the fluorophore. This provides a major advantage, since the intensity can vary over a broad range, with only minor effects on the results. Phase-modulation measurements can be... 

The techniques described thus far are those most commonly used to measure fluorescence profiles following pulsed laser excitation. As such they are well supported by the availability of commercial instruments and complete systems can readily be assembled. They are not the only methods, however, by which fluorescence lifetimes can be measured using laser excitation. A number of researchers have devised different techniques or modifications to those discussed above to measure lifetimes for example, using multiple lasers for excitation and probing [70, 71] or monitoring the decay via modulated gain spectroscopy [72, 73]. However, in most cases, the only applications have been made by the same workers and these methods will not be discussed here. 

Weber. G. Resolution of the fluorescence lifetimes in a heterogeneous system by pha% and modulation measurements. J. Chem. Phys. In press, communicated privately... 

Tvvo vidcl used approaches are used for lifetime measurcnienis. ilie lime-domain approach and the frt i/iu niy-domain approach. In tinte-domain measurements. a pulsed source is employed and the time-depcndcnr decay of fluorescence is measured. In the frequency-domain method, a sinusoidallv modulated source is used to excite the sample. The phase shift and demodulation of the fluorescence emission relative lo the excitation waveform provide the lifetime information. ( onimercial instrumentation is available to implement both techniques. ... 

Chemical Mechanisms for Fluorescence Modulation. While UV/visible signalling almost always results from the ionization of a conjugated substituent, there exists a plethora of mechanisms by which fluorescence signal transduction may be engendered. It is useful to categorize the mechanisms for fluorescence modulation described to date via the type of measurement that is made. These are intensity, intensity-ratio, and lifetime. A pictoral summary of each is found in Figure 1 of the chapter by Szmacinski and Lakowicz. 

Fluorescence decay kinetics also can be measured by exciting the sample with continuous light whose intensity is modulated sinusoidally at a frequency (m) on the order of 1/t, where t again is the fluorescence lifetime. The fluorescence oscillates sinusoidally at the same frequency, but the amplitude and phase of its oscillatirais relative to the oscillations of the excitation light depend on the product of oo and t (Fig. 1.16 and Appendix A4). If mr is much less than 1, the fluorescence amplitude tracks the excitation intensity closely if an is larger, the oscillations are delayed in phase and damped (demodulated) relative to the excitation [28-30]. Fluorescence with multiexponential decay kinetics can be analyzed by measuring the fluorescence modulation amplitude or phase shift with several different frequencies of modulated excitation. 

In general, a sample will contain molecules that interact with their surroundings in a variety ways, for example because some of the fluorescing molecules are buried in the interior of a protein while others are exposed to the solvent. The fluorescence then decays with multiphasic kinetics that can be fit by a sum of exponential terms (Eq. 1.4). Fluorescence lifetimes can be measured by time-correlated photon counting, by fluorescence upconversion, or by modulating the amplitude of the excitation beam and measuring the modulation and phase shift of the fluorescence (Chap. 1). Pump-probe measurements of stimulated emission become the method of choice for sub-picosecond lifetimes (Chap. 11). For further information on these techniques and ways of analyzing the data see [30-34]. 

The phase shift method is not so well suited to the measurement of non-exponential decays (if, for example, the fluorescence from several levels with different lifetimes overlap). Although measurements at different modulation frequencies enable one to fit the measured phase shifts q to a sum of exponential I Cj (exp(-t/T ), the decay curve cannot be viewed directly, and the fit may not be unambiguous. 

Jablonski (48-49) developed a theory in 1935 in which he presented the now standard Jablonski diagram" of singlet and triplet state energy levels that is used to explain excitation and emission processes in luminescence. He also related the fluorescence lifetimes of the perpendicular and parallel polarization components of emission to the fluorophore emission lifetime and rate of rotation. In the same year, Szymanowski (50) measured apparent lifetimes for the perpendicular and parallel polarization components of fluorescein in viscous solutions with a phase fluorometer. It was shown later by Spencer and Weber (51) that phase shift methods do not give correct values for polarized lifetimes because the theory does not include the dependence on modulation frequency. 

The fluorescent lifetime of chlorophyll in vivo was first measured in 1957, independently by Brody and Rabinowitch (62) using pulse methods, and by Dmitrievskyand co-workers (63) using phase modulation methods. Because the measured quantum yield was lower than that predicted from the measured lifetime, it was concluded that much of the chlorophyll molecule was non-fluorescent, suggesting that energy transfer mechanisms were the means of moving absorbed energy to reactive parts of the molecule. 

Pulsed method. Using a pulsed or modulated excitation light source instead of constant illumination allows investigation of the time dependence of emission polarization. In the case of pulsed excitation, the measured quantity is the time decay of fluorescent emission polarized parallel and perpendicular to the excitation plane of polarization. Emitted light polarized parallel to the excitation plane decays faster than the excited state lifetime because the molecule is rotating its emission dipole away from the polarization plane of measurement. Emitted light polarized perpendicular to the excitation plane decays more slowly because the emission dipole moment is rotating towards the plane of measurement. 

It is important to note that if a mixture of fluorophores with different fluorescence lifetimes is analyzed, the lifetime computed from the phase is not equivalent to the lifetime computed from the modulation. As a result, the two lifetimes are often referred to as apparent lifetimes and should not be confused with the true lifetime of any particular species in the sample. These equations predict a set of phenomena inherent to the frequency domain measurement. 

At present, two main streams of techniques exist for the measurement of fluorescence lifetimes, time domain based methods, and frequency domain methods. In the frequency domain, the fluorescence lifetime is derived from the phase shift and demodulation of the fluorescent light with respect to the phase and the modulation depth of a modulated excitation source. Measurements in the time domain are generally performed by recording the fluorescence intensity decay after exciting the specimen with a short excitation pulse. 

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