Frequency-domain lifetime measurement

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. 

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... 

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]. 

Fluorescence is unique among spectroscopic techniques due to its inherently multidimensional nature, the emission process containing a wealth of orthogonal information that is related to the fluorophore and its surroundings. Time- and frequency-domain fluorescence methods are instrumentally sophisticated, but they improve both the sensitivity and selectivity of fluorimetry. Any dye that is used for steady-state fluorescence detection can be used for time-resolved detection as well. Most of these fluor-ophores display lifetimes from 1 to 10 ns, which requires fast electronics for time-domain lifetime measurements or modulation frequencies from 10... 

The second chapter by Peter Verveer and Quentin Hanley describes frequency domain FLIM and global analysis. While the frequency domain technique for fluorescence lifetime measurement is sometimes counterintuitive, the majority of the 10 most cited papers using FLIM have taken advantage of the frequency domain method as stated by these authors. The global analysis of lifetime data in the frequency domain, resolving both E and /d has contributed significantly to this advantage. 

It is important to note that if a mixture of fluorophores with different fluorescence lifetimes is analyzed, the lifetime computed from the phase is not equivalent to the lifetime computed from the modulation. As a result, the two lifetimes are often referred to as apparent lifetimes and should not be confused with the true lifetime of any particular species in the sample. These equations predict a set of phenomena inherent to the frequency domain measurement. 

At present, two main streams of techniques exist for the measurement of fluorescence lifetimes, time domain based methods, and frequency domain methods. In the frequency domain, the fluorescence lifetime is derived from the phase shift and demodulation of the fluorescent light with respect to the phase and the modulation depth of a modulated excitation source. Measurements in the time domain are generally performed by recording the fluorescence intensity decay after exciting the specimen with a short excitation pulse. 

At the end of the 1980s and early 1990s, first experiments were carried out to combine fluorescence lifetime measurements with imaging using both time domain [1-4] and frequency domain [5-7] based approaches. This chapter will deal exclusively with time domain based fluorescence lifetime imaging methods. For the frequency domain based methods, refer Chapter 2. 

Two different approaches for measuring fluorescence lifetimes are commonly employed to study FRET-related phenomenon the frequency-domain FLIM (see Chapter 2) and the time-domain... 

The most sophisticated techniques require time-resolved measurements (lifetime, anisotropy, spectra) either in the time or frequency domain ([6-10] for a focused journal issue on the subject see [11]). Thus, the significance of new, versatile, commercially available light... 

Dual lifetime referencing (DLR) is another powerful technique that enables referenced measurements in case of fluorescent indicators [23]. In this method, the analyte-dependent signal from an indicator is referenced against the signal from an inert luminophore. This can be realized in both the time domain [24] and in the frequency domain [25]. Often, a luminescent reference dye is embedded into gas blocking nanobeads to avoid oxygen quenching. Polymers with very low gas permeability such as poly(acrylonitrile) [24] or poly(vinylidene chloride-co-acry-lonitrile) [26] are the best choice here. 

In frequency-domain FLIM, the optics and detection system (MCP image intensifier and slow scan CCD camera) are similar to that of time-domain FLIM, except for the light source, which consists of a CW laser and an acousto-optical modulator instead of a pulsed laser. The principle of lifetime measurement is the same as that described in Chapter 6 (Section 6.2.3.1). The phase shift and modulation depth are measured relative to a known fluorescence standard or to scattering of the excitation light. There are two possible modes of detection heterodyne and homodyne detection. 

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. 

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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