Phase and modulation fluorometry

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—... 

J. R. Lakowicz, H. Cherek, and A. Balter, Correction of timing errors in photomultiplier tubes used in phase and modulation fluorometry, J. Biochem. Biophys. Methods 15, 131-146 (1981). 

Gratton E, Jameson DM, Hall RD. Multifrequency phase and modulation fluorometry. Ann. Rev. Biophys. Bioeng. 1984 13 105-124. 

Robbins et al. on tryptophan and 3-methylindole since powerful solid-state laser excitation was used. Jameson and Weber have resolved the fluorescence of tryptophan by phase and modulation fluorometry in terms of emission from the zwitterion and anion present in amounts determined by the pH of the solution. The forms interconvert more slowly than fluorescence processes and have similar absorption and emission spectra. Measurements were made with excitation frequencies of 6, 18, and 30 MHz in the pH range 8—10, in which the relative zwitterion concentration varies from 0.82—0.09. Resolved lifetimes were 3.1 0.4 ns for the zwitterion and 8.7 0.1 ns for the anion. The agreement with Gudgin et al. seems satisfactory. 

Gratton, E., Jameson, D. M. Hall, R. D. (1984). Multifrequency phase and modulation fluorometry. Annual Review of Biophysics and Bioengineering,... 

In phase fluorometry, the phase (and modulation) data are recorded at a given wavelength and analyzed in terms of a multi-exponential decay (without a priori assumption of the shape of the decay). The fitting parameters are then used to calculate the fluorescence intensities at various times, 2 > 3 > The procedure is repeated for each observation wavelength X, X2, A3,... It is then easy to reconstruct the spectra at various times. 

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. 

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). 

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. 

Beechem, J. M., Knutson, J. R., Ross, B. A., Turner, B. W. and Brand, L. (1983). Global resolution of heterogeneous decay by phase/modulation fluorometry Mixtures and proteins. Biochemistry 22, 6054-8. 

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). 

Pulse fluorometry uses a short exciting pulse of light and gives the d-pulse response of the sample, convoluted by the instrument response. Phase-modulation fluorometry uses modulated light at variable frequency and gives the harmonic response of the sample, which is the Fourier transform of the d-pulse response. The first technique works in the time domain, and the second in the frequency domain. Pulse fluorometry and phase-modulation fluorometry are theoretically equivalent, but the principles of the instruments are different. Each technique will now be presented and then compared. 

General principles of pulse and phase-modulation fluorometries... 

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 ... 

Fig. 6.6. Principles of pulse fluorometry and multi-frequency phase-modulation fluorometry.

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. 

The least-squares method is also widely applied to curve fitting in phase-modulation fluorometry the main difference with data analysis in pulse fluorometry is that no deconvolution is required curve fitting is indeed performed in the frequency domain, i.e. directly using the variations of the phase shift and the modulation ratio M as functions of the modulation frequency. Phase data and modulation data can be analyzed separately or simultaneously. In the latter case the reduced chi squared is given by... 

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). 

In phase-modulation fluorometry, it is convenient to use the fractional contributions f instead of the fractional amplitudes, as shown in Eqs (6.30) and (6.31). The above equation is thus rewritten as... 

The well-defined statistics in single-photon counting is an advantage for data analysis. In phase fluorometry, the evaluation of the standard deviation of phase shift and modulation ratio may not be easy. 

The time of data collection depends on the complexity of the (5-pulse response. For a single exponential decay phase fluorometry is more rapid. For complex 5-pulse responses, the time of data collection is about the same for the two techniques in pulse fluorometry, a large number of photon events is necessary, and in phase fluorometry, a large number of frequencies has to be selected. It should be emphasized that the short acquisition time for phase shift and modulation ratio measurements at a given frequency is a distinct advantage in several situations, especially for lifetime-imaging spectroscopy. 

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. 

How can phase-modulation fluorometry contribute to this health-care need It now seems possible to construct a lifetime-based blood gas catheter (Figure 1.3), or alternatively, an apparatus to read the blood gas in the freshly drawn blood at the patient s bedside. To be specific, fluorophores are presently known to accomplish the task using a 543-nm Green Helium-Neon laser,(18 19) and it seems likely that the chemistries will be identified for a laser diode source. The use of longer wavelengths should minimize the problems of light absorption and autofluorescence of the samples, and the use of phase or modulation sensing should provide the robustness needed in a clinical environment. For the more technically oriented researcher, we note that the... 

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, Optical sensing of glucose using phase-modulation fluorometry, Anal. Chim. Acta 271, 155-164(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. 

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