Phase-modulation method fluorescence lifetime measurement

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. 

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. 

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... 

The principal requirements for photomultipliers in both pulse and phase-modulation methods of measuring fluorescence lifetimes are as follows. 

Fluorescence lifetime measurements are an important aspect of photophysical research. In the past few months the phase-shift measurement technique has become more widely used. This is largely due to the successful achievement of a multifrequency modulation apparatus. An apparatus made from commercially available components has been described and shown to have an accuracy of 10 ps. The performance was checked using mixtures of acridine and quinine sulphate and least-squares-ht procedures. A series of papers from the Illinois group give very detailed account of the state of the art and show the power of the method. The colour delay error arising from the wavelength error in photodetectors can be determined and fluorescence decay times can be obtained with an accuracy of a few picoseconds. ... 

The fluorescence of quinine and the possibility of its quenching or modulation in the presence of external molecules could provide a method for sensing and assaying these molecules. For example, diastereomeric complexes of quinine and quinidine with (+ )-10-camphorsulfonic acid can be discriminated on the basis of their phase modulation-resolved fluorescence (different fluorescence lifetimes for QN and QD). Thus fluorescence lifetimes of 21.79 and 22.89 ns for QN and QD complex, respectively, have been measured, allowing a quantitative determination of QN and QD with a detection limit of 1.8 and 0.97 pM, respectively [142]. Similarly, room-temperature phosphorescence lifetimes were also shown to differ for diaste-reoisomeric complexes of QN and QD [143]. 

What is the advantage of the cross-correlation method in fluorescence lifetime measurements Fluorescence lifetimes are in the order of the ns or / and ps. Thus, high frequencies from the MHz to the GHz are needed to perform fluorescence lifetimes measurements. Still, the accuracy of the measured values is not reached at high frequencies. Therefore, one can translate the high frequency and phase modulation information to lower frequency carrier signal, Aw. In this case, the measured fluorescence lifetimes are highly accurate. 

Because of advances in the technology for measuring lifetimes, particularly by the phase-modulation method (i2-i7), lifetime-based sensing (Figure 1) offers new opportunities for chemical sensing. This is because the fluorescence lifetimes of probes can be sensitive to a variety of factors or chemicals. Moreover, lifetime measurements are insensitive to probe... 

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. 

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. 

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. 

Fig. 6. Experimental arrangement for lifetime measurements by the phase-shift method, using laser excitation. The laser beam is amplitude-modulated by a Pockel cell with analysing Nicol prism and a small part of the beam is reflected by a beam splitter B into a water cell, causing Rayleigh scattering. This Rayleigh-scattered light and the fluorescence light from the absorption cell are both focused onto the multiplier cathode PMl, where the difference in their modulation phases is detected. (From Baumgartner, G., Demtroder, W., Stock, M., ref. 122)).

There are two ways to collect FLIM data freqnency-domain or time-domain data acqnisition (Alcala et al. 1985 Jameson et al. 1984). Briefly, in freqnency domain FLIM, the fluorescence lifetime is determined by its different phase relative to a freqnency modulated excitation signal nsing a fast Fourier transform algorithm. This method requires a frequency synthesizer phase-locked to the repetition freqnency of the laser to drive an RF power amplifier that modulates the amplification of the detector photomultiplier at the master frequency plus an additional cross-correlation freqnency. In contrast, time-domain FLIM directly measures t using a photon connting PMT and card. 

Over a substantial number of years the phase-shift or frequency-domain method has been employed for the measurement of fluorescence lifetimes. The technique requires the continuous excitation of a fluorescent sample with a source of varying intensity. The fluorescence response would normally be expected to increase and decrease to reflect the changes in excitation intensity. However, in a frequency-domain experiment the excitation beam is modulated at a high frequency, (o = 2nf, to produce a sinusoidally changing intensity given by ... 

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. ... 

The phenomena discussed above can be studied using various techniques, and not all methods are equally suitable in a particular case. Two principally different kinds of methods for measuring fluorescence lifetimes exist, namely, pulse methods and modulation or phase-shift methods. Phase-shift methods, despite the fact that they have been known for a longer time, have not found widespread use during the last decade. However, important technical advances have been made in phase-shift methods which in fact have inspired many researchers to apply them more frequently. Nevertheless, pulse methods are still the most widely used today, in particular for high time resolution. If carried out properly both types of methods must and will give the same result. Details of the measuring problem will determine which method is more appropriate in a particular case. 

The mean fluorescence lifetime may also be determined by continuous intensity measurements, if the exciting light intensity is modulated at a high frequency. Fluorescence is excited by light modulated sinusoidally at a known frequency (ajln Hz). The emission is a forced response to the excitation, and is therefore modulated at the same frequency, but with a phase shift, due to the time-lag between absorption and emission. The intensities of the two beams are monitored by photomultipliers. The difference in phase (0) between the two intensities is determined electronically. The lifetime r is given by cox = tan<. The modulation frequency must be made comparable to the decay rate, e.g., around 30 MHz for a mean lifetime of 30 ns. Such frequencies can be achieved by using a hydrogen lamp actuated by a suitably modulated current source. Commercial equipment is available. The method has been applied to quinine sulphate, fluorescein, and acridine, for example, with a precision of 1-2%. It is especially useful for very short (sub-nanosecond) lifetimes. 

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]. 

Two methods of measuring fluorescence lifetime are in widespread use the time-domain (or pulse-fluorometry) and the frequency-domain (or phase-modulation fluorometry) methods. Here, reference will be made only to the first method. 

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