Lifetimes, frequency domain

Examination of Eqs. (2.9-2.11) suggests that having frequency domain lifetimes measured at a variety of frequencies is desirable, as it will allow a mixture of fluorophores to be determined. With this in mind, two approaches may be taken to obtain multifrequency results. The first of these is simply to make a series of FLIM measurements while stepping through a predetermined set of frequencies. In practice, this is of limited utility for biological systems because of photo-induced damage to the specimen. 

Division of the observed fluorescence fluctuation spectrum (Fig. 8b) by such a pattern yields the frequency domain lifetime directly through (50). This is shown for Rhodamine B in Fig. 9 to yield a decay time of 3.2 ns. 

Phase shift fluorimetry, the other important method for measuring fluorescent lifetimes, also continues to be developed and improved. The effects of Inaccurate reference lifetimes on the interpretation of frequency domain fluorescence data can be removed or minimized by a least squares analysis method.The direct collection of multi-frequency data for obtaining fluorescence lifetimes can be achieved by the use of digital parallel acquisition in frequency domain fluorimetry. Frequency domain lifetime measurement has been used for on-line fluorescence lifetime detection of eluents in chromatography. An unusual use of frequency domain measurement which has been reported is for the examination of photon migration in living tissue. Photons in the... 

Lifetimes. The theory of frequency-domain lifetime determinations has been described in detail elsewhere (15-19). Briefly, a high-frequency (MHz - GHz) sinusoidally-modulated light source is used to excite the fluorescent sample. The time-dependent mathematical representation of the excitation waveform (Ex(t)) is given by ... 

A particular advantage of frequency domain lifetime measurements is thaL by measuring a number of frequencies simultaneously, it is possible to make lifetime measurements very quickly, in as short a time as a few ms. Thus changes in excited-state lifetimes during chemical reactions can be studied in real time across the ms time domain. This matches that of the most commonly used chemical and biochemical fast reaction technique, stopped-flow, and stopped-flow accessories are available for commercial frequency domain fluorimeters. Because the measurements are quick, frequency domain measurements over a wide spectral range also provide an attractive method for obtaining time-resolved fluorescence spectra. 

Fig. 1 Principle of frequency-domain lifetime measurement (see text). By using sinusoidally modulated excitation light and measuring the phase-shift and demodulation of the emitted fluorescence the lifetime of the fluorochrome is determined...

Conceptually, time-domain lifetime measurement is easier to understand than frequency-domain lifetime measurement. In time-domain lifetime measurement, a short (relative to the fluorescence lifetime) pulse of excitation light is given, after which the emitted fluorescence is measured time resolved [15], resulting directly in decay curves like that described in Eq. (1) (see also Fig. 2). Due to the requirement of short light pulses and fast detection, time-domain measurements became possible only about 40 years later than frequency-do-main measurements using a flashlamp as excitation source [16]. 

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