Phase modulated fluorescence

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. 

Szmacinski, H. Lacowicz, J. R. Lifetime-based Sensing Using Phase-Modulation Fluorometry. In Fluorescent Chemosensor for Ion and Molecule Recognition. ACS Symposium Series 538, 1993. 

Lakowicz, J. R. and Balter, A. (1982). Theory of phase-modulation fluorescence spectroscopy for excited-state processes. Biophys. Chem. 16, 99-115. 

In phase-fluorimetric oxygen sensors, active elements are excited with periodically modulated light, and changes in fluorescence phase characteristics are measured. The delay or emission (phase shift, ( ), measured in degrees angle) relates to the lifetime of the dye (x) and oxygen concentration as follows ... 

Knowledge of the dynamics of excited states is of major importance in understanding photophysical, photochemical and photobiological processes. Two time-resolved techniques, pulse fluorometry and phase-modulation fluorometry, are commonly used to recover the lifetimes, or more generally the parameters characterizing the S-pulse response of a fluorescent sample (i.e. the response to an infinitely short pulse of light expressed as the Dirac function S). 

The principles of pulse and phase-modulation fluorometries are illustrated in Figures 6.5 and 6.6. The d-pulse response I(t) of the fluorescent sample is, in the simplest case, a single exponential whose time constant is the excited-state lifetime, but more often it is a sum of discrete exponentials, or a more complicated function sometimes the system is characterized by a distribution of decay times. For any excitation function E(t), the response R(t) of the sample is the convolution product of this function by the d-pulse response ... 

Phase-modulation fluorometry The sample is excited by a sinusoidally modulated light at high frequency. The fluorescence response, which is the convolution product (Eq. 6.9) of the pulse response by the sinusoidal excitation function, is sinusoidally... 

An efficient way of overcoming this difficulty is to use a reference fluorophore (instead of a scattering solution) (i) whose fluorescence decay is a single exponential, (ii) which is excitable at the same wavelength as the sample, and (iii) which emits fluorescence at the observation wavelength of the sample. In pulse fluorometry, the deconvolution of the fluorescence response can be carried out against that of the reference fluorophore. In phase-modulation fluorometry, the phase shift and the relative modulation can be measured directly against the reference fluorophore. 

Fig. 6.13. Data obtained by the phase-modulation technique with a Fluorolog tau-3 instrument (Jobin Yvon-Spex) operating with a xenon lamp and a Pockel s cell. Note that because the fluorescence decay is a single exponential, a single appropriate modulation frequency suffices for the lifetime determination. The broad set of frequencies permits control of the proper tuning of the...

To answer the question as to whether the fluorescence decay consists of a few distinct exponentials or should be interpreted in terms of a continuous distribution, it is advantageous to use an approach without a priori assumption of the shape of the distribution. In particular, the maximum entropy method (MEM) is capable of handling both continuous and discrete lifetime distributions in a single analysis of data obtained from pulse fluorometry or phase-modulation fluorometry (Brochon, 1994) (see Box 6.1). 

O Connor D. V., Ware W. R. and Andre J. C. Pouget J., Mugnier J. and Valeur B. (1989) (1979) Deconvolution of Fluorescence Decay Correction of Timing Errors in Multi-Curves. A Critical Comparison of Tech- frequency Phase/Modulation Fluoro-... 

Hresko R. C., Sugar I. P., Barenhoiz Y. and Thompson T. E. (1986) Lateral Diffusion of a Pyrene-Labeled Phosphatidylcholine in Phosphatidylcholine Bilayers/Fluorescence Phase and Modulation Study, Biochemistry 25, 3813-3823. 

In phase-modulation fluorometry, it is worth noting that the transfer rate constant can be determined from the phase shift between the fluorescence of the acceptor excited directly and via donor excitation. 

Figure 1.1. Schemes for fluorescence sensing intensity, intensity ratio, time-domain, and phase-modulation, from left to right.

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. 

H. Szmacinski and J. R. Lakowicz, Optical measurements of pH using fluorescence lifetimes and phase-modulation fluorometry, Anal. Chem. 65, 1668-1674(1993). 

J. R. Lakowicz and B. P. Maliwal, Fluorescence lifetime energy transfer immunoassay quantified by phase-modulation fluorometry, Sensors andActuators B 12, 65-70 (1993). 

A. J. Ozinskas, H. Malak, J. Joshi, H. Szmacinski, J. Britz, R. B. Thompson, P. A. Koen, and J. R. Lakowicz, Homogeneous model immunoassay of thyroxine by phase-modulation fluorescence spectroscopy, Anal. Biochem. 213, 264-270(1993). 

The use of high-speed modulated excitation (f> kr + knr) combined with coherent detection methods has resulted in the popular techniques of frequency domain fluorometry, also known as phase-modulation fluorometry. These techniques can be used to determine the temporal characteristics of both fluorescence and phosphorescence and will also be addressed later in this chapter. 

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... 

A single modulation frequency is sufficient to measure the fluorescence phase angle and modulation and hence the analyte concentration. These intensity decay... 

Figure 12.2. Pulse and phase modulation fluorescence Lifetime techniques.

It will be seen that, as in the case of the LED, control of the bias voltage gives simple modulation of the laser output intensity. This is particularly useful in phase-modulation fluorometry. However, a measure of the late awareness of the advantages of IR techniques in fluorescence is that only recently has this approach been applied to the study of aromatic fluorophores. Thompson et al.(51) have combined modulated diode laser excitation at 670 and 791 nm with a commercial fluorimeter in order to measure the fluorescence lifetimes of some common carbocyanine dyes. Modulation frequencies up to 300 MHz were used in conjunction with a Hamamatsu R928 photomultipler for detecting the fluorescence. Figure 12.18 shows typical phase-modulation data taken from their work, the form of the frequency response curves is as shown in Figure 12.2 which describes the response to a monoexponential fluorescence decay. 

Figure 12.18. The lower portion of the figure shows diode laser phase-modulation fluorescence lifetime...

The helium-neon (HeNe) laser immediately comes to mind, having a very useful spectral line at 633 nm for steady-state red/near-IR fluorescence studies. Kessler and Wolfbeis have demonstrated the fluorescence assay of the protein human serum albumin using the probe albumin blue excited with a red HeNe laser.(71) Another useful wavelength available from the green HeNe laser is at 543.5 nm and this has been used with phase-modulation fluorometry by Lakowicz etal. to study probes such as carboxy seminaphtorhodafluor-6 (SNARF-6) as a means of measuring pH.(72)... 

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

Figure 13.5. Methods of fluorescence sensing, (a) Single excitation/emission wavelength intensity changes with analyte concentration (b) wavelength-ratiometric A/B changes with analyte concentration (c) liftetime based (time domain) r changes with analyte concentration (d) lifetime-based (phase modulation) Am and A change with analyte concentration.

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... 

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