Phase and Modulation Measurement

the fluorescence lifetime r can be derived from a measurement of p- 

This technique is of high accuracy and is meant to be used in precision measurement instrumentation, for it is inherently insensitive to the DC-offset and the AC-noise in the sinusoidal signal which can be substantially reduced by a great variety of electronic devices ranging from various electronic analogue filters, and digital filters to the most effective lock-in amplifiers. 

At the early stages of development, the lack of a convenient and economic excitation modulation scheme has limited the use of such a phase shift technique in fluorescence thermometry. Now with the wide availability of cheap and easily modu- 

However, since the kind of system requires the measurement of fluorescence during the excitation period, it is highly sensitive to the excitation light leakage to the detector and the optics required to prevent such leakage can make the system very costly. 

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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 multiexponential decay law of the emission from a mixture of fluorophores can be recovered from phase and modulation measurements over a range of multiple frequencies by... 

This procedure involves selecting a fluorophore of known lifetime and placing it in the microscope and measuring the phase and modulation depth [11]. Rearranging Eqs. (2.5 and 2.6) allows the expected phase and modulation to be predicted. These may then be used to compute the position of zero phase and the modulation depth of the light source. An advantage of the method is that it may be done under conditions exactly matching those of a sample. 

Jameson D. M., Gratton E. and Hall R. D. (1984) The Measurement and Analysis of Heterogeneous Emissions by Multi-frequency Phase and Modulation Fluorometry, Appl. Spectrosc. Rev. 20, 55—... 

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... 

Membranes and vesicles were labeled at a DPH/lipid ratio of 1/400 and measured using phase-modulation fluorometry at ten frequencies between 5 and 90 MHz at 37°C. y2 values were calculated assuming errors of 0.2 and 0.002 in the phase and modulation, respectively, except where otherwise noted. /, 2, Fraction of Exponential term or Lorentzian t, 2, lifetime (ns) cu, center of Lorentzians (ns) w12, half-width of Lorentzians (ns). 

All fluorescence measurements were performed with an SLM 48000 MHF multifrequency cross-correlation phase and modulation fluorometer. The excitation... 

A glycogen solution placed in the emission compartment will scatter fight and is used as reference (rrf = 0) to determine the phase delay and fluorescence demodulation. For each measurement, the reference intensity is adjusted so that it is equivalent to the intensity of the fluorescence signal of the sample. Phases and modulations of the fluorescence and scattered fight are obtained relative to the reference photomultiplier or instrumental... 

The use of multifrequency cross correlation phase and modulation phosphorometry, based on a technology usually associated with fluorescence lifetime determination, has also now been applied to the measurement and analysis of triplet state decay times . ... 

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. 

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