Phase modulation measurements

Figure 13.6 Principle of phase-modulation measurement and elements of a phase fluorometer (a) sample measurement (b) cross-correlation phase fluorometer,...

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... 

For phase-modulation measurements we have chosen tiis(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) perchlorate ([Ru(Ph2phn)3] ), which is highly sensitive to oxygen. This complex in silicon is also rather specific. The physiologically and medically important gases, CO2, nitrous oxide, cyclopropane,... 

Examination of Hgure 4.2 reveals another effect of the lifetime, tiiis bang a decrease in the peak-to-peak bright of the emission relative to that of the modulated excitation. The modulation decreases because some of the fluoto-phores excited at the peak of the excitation continue to emit when the excitation it at a minimum. The extent to which diis occurs depends on the decay time and light modulation frequency. This effiect is called demodulation and can also be used tocalculate the decay time. At present, pulse and phase-modulation measurements ate both in widespread use. 

A more complete description of the theoiy of emission anisotropy may be fbimd in (19) (which also contaips specific examples of the technique s use as well as the appropriate equations for phase/modulation measurements). 

The type of recording electronics used depends on whether pulsed excitation (either pulse sampling or timesingle photon covmting) or phase/ modulation measurement are being made. Therefore, we discuss the electronics in the context of the method. 

As with alternating electrical currents, phase-sensitive measurements are also possible with microwave radiation. The easiest method consists of measuring phase-shifted microwave signals via a lock-in technique by modulating the electrode potential. Such a technique, which measures the phase shift between the potential and the microwave signal, will give specific (e.g., kinetic) information on the system (see later discussion). However, it should not be taken as the equivalent of impedance measurements with microwaves. As in electrochemical impedance measurements,... 

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. 

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 ... 

Fig. 5 Radio frequency pulse sequences for measurements of Sj and Si in DSQ-REDOR experiments. The MAS period rR is 100 ps. XY represents a train of 15N n pulses with XY-16 phase patterns [98]. TPPM represents two-pulse phase modulation [99]. In these experiments, M = Nt 4, N2+ N3 = 48, and N2 is incremented from 0 to 48 to produce effective dephasing times from 0 to 9.6 ms. Signals arising from intraresidue 15N-13C DSQ coherence (Si) are selected by standard phase cycling. Signal decay due to the pulse imperfection of 15N pulses is estimated by S2. Decay due to the intermolecular 15N-I3C dipole-dipole couplings is calculated as Si(N2)/S2(N2). The phase cycling scheme can be found in the original figure and caption. (Figure and caption adapted from [45])...

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. 

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. 

Noninvasive glucose measurements can potentially be performed with phase-modulation fluorometry. The blood gas application described above requires drawing the blood, i.e., an invasive as well as an unpleasant procedure. Similarly, present measurements of blood glucose also require fresh blood. Insulin-dependent diabetics... 

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

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... 

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