Phase fluorometers

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. 

The use of phase sensitive detection with the phase fluorometer to analyze multicomponent systems was first described in 1970 by Veselova and coworkers (76). 

If the signal decay is a single-exponential curve, equations 16 and 17 result in values for X that are in agreement with each other. Dissimilar values indicate multiexponential decay, which usually means that the sample contains more than one fluorophore. Multiexponential decay can be resolved by using a phase fluorometer with phase sensitive detection. A time-independent, direct-current signal is produced that is proportional to the cosine of the difference between the phase angle of the detector ( D) and the phase angle of the fluorescence ( ) ... 

Spencer, R. D. and Weber, G. (1969). Measurement of subnanosecond fluorescence lifetimes with a cross-correlation phase fluorometer. Ann. N. Y. Acad. Sci. 158, 361-76. 

Gratton, E. and Limkeman, M. (1983). A continuously variable frequency cross-correlation phase fluorometer with picosecond resolution. Biophys. J. 44, 315-24. 

Hedstrom, J., Sedarous, S. and Prendergast, F. G. (1988). Measurements of fluorescence lifetimes by use of a hybrid time-correlated and multifrequency phase fluorometer. Biochemistry 27, 6203-8. 

The ability of fluorescence to provide temporal information is of major importance. Great progress has been made since the first determination of an excited-state lifetime by Gaviola in 1926 using a phase fluorometer. A time resolution of a few tens of picosecond can easily be achieved in both pulse and phase fluorometries by using high repetition rate picosecond lasers and microchannel plate photo-... 

Historically, the first instrument for the determination of lifetime was a phase fluorometer (designed by Gaviola in 1926) operating at a single frequency. Progress in instrumentation enabled variable modulation frequency by employing a cw laser (or a lamp) and an electro-optic modulator (0.1-250 MHz), or by using the harmonic content of a pulsed laser source (up to 2 GHz). These two techniques will now be described. 

Phase fluorometers using a continuous light source and an electro-optic modulator... 

Phase fluorometers using the harmonic content of a pulsed laser... 

The time resolution of a phase fluorometer using the harmonic content of a pulsed laser and a microchannel plate photomultiplier is comparable to that of a single-photon counting instrument using the same kind of laser and detector. 

K. W. Berndt and J. R. Lakowicz, Electroluminscent lamp-based phase fluorometer and oxygen sensor, Anal. Biochem. 201, 319-325 (1992). 

Figure 6-8. Incorporation of directly modulated loser diode into phase fluorometer. Reproduced from Ref. 25 with permission.

J. R. Alcala, E Gratton, and D. M. Jameson, A multifrequency phase fluorometer using the harmonic content of a mode-locked laser, Analytical Instrumentation 14, 225-250 (1985). 

E. Gratton, D. M. Jameson, N. Rosato, and G. Weber, A multi-frequency cross-correlation phase fluorometer using synchrotron radiation, Rev. Sci. Instrum. 55, 486-494 (1984). 

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

Phase fluorometers utilize continuous irradiation by a beam of light that is sinusoidally modulated. If the frequency of the modulation is set correctly, there will be a phase difference in the modulation of the fluorescent emission that will depend upon x. 

Phase Fluorometers using a Continuous Light Source and an Electro-optic Modulator... 

Phase Fluorometers using the Harmonic Content of a Pulsed Laser... 

Phase fluorometers utilize continuous irradiation by a beam of lighf thaf is sinusoidally modulated. If the frequency of fhe modulation is sef correcfly, there will be a phase difference in the modulation of the fluorescent emission that will depend upon x. Phase fluorometry can yield the same information as does pulse fluorometry.327432,133 gy ysing two or more modulation frequencies the decay rates and fluorescence lifetimes for various chromophores in a protein can be observed. For example, the protein colicin A (Box 8-D) contains three tryptophans W86, W130, and W140. Their fluorescence decays with lifetimes Xj, Xy X3 of -0.6-0.9 ns, 2.0-2.2 ns, and 4.2-4.9 ns at pH 7. While X3 originates mainly from W140, both of the other tryptophans contribute to x and X2. Changes in fluorescence intensify with pH reflect a pfC value of... 

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