Time-domain lifetime measurement

FIGURE 8. Time-domain lifetime measurement. (Modified from Ref. 11.)... 

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... 

Figure 25.2 Pulse- or time-domain lifetime measurements. Reproduced with permission from Ref. [8] 2006, Springer.

This relationship provides an alternative method to determination of the concentration of the analyte of interest. Specifically, lifetime or decay time measurements can be used in fluorescence based sensors to determine the analyte concentration. These measurements provide better results than steady-state measurements. Time-domain lifetime measurements are typically performed by exciting the sensing element with a short optical pulse which is much shorter than the average fluorophor lifetime. For a single population of fluorophors, the rate at which the intensity decays over time can be expressed as ... 

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]. 

Fig. 2 Principle of time-domain lifetime measurement (see text). Fluorochromes are excited using a short pulse of light, after which the emitted fluorescence is measured time-resolved. Usually, fluorescence is recorded in two or more discreet time intervals...

Figure 11. Experimental He 12(8,v ) and Ne 12(8,v ) excited-state lifetimes, circles and squares, plotted as a function of vibrational quanta, v. The values taken from line width [72] and time-domain [73] measurements are shown as solid and open symbols, respectively.

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. 

The lifetime of an excited state can be calculated by measuring its concentration as a function of time. Most lifetime-measuring techniques are indeed based on the reeording of the excited state concentration as a function of time, and are eoUeetively referred to as time-domain measurements. The techniques described... 

Figure 3.3c shows an example of time-domain fluorescence measurements. The emission decay measured at two different wavelengths, corresponding to and Fj., are plotted as a logarithmic function of intensity (counts) versus time. In the simplest case of irreversibly decaying fluorescent species, fluorescence decay is characterized by a single exponential function whose time constant is the excited-state lifetime. However, in the case of the exdted-state reaction such as tautomerization, it may be a sum of discrete exponentials, or an even more complicated function. In a heterogeneous environment, the system is often better characterized by a distribution of decay times. 

Spectral FLIM involves measuring the apparent lifetimes in a preparation at many wavelengths with the assistance of a spectrograph or a series of filters (see also Chapter 4, Figs. 4.7 and 4.8 depicting hyperspectral FLIM in the time domain). The goal of the measurement is similar to that of the multifrequency approach ... 

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... 

However, FLIM measurements by both frequency and time domain using commercially available software suffers from variation in the measured lifetime from region to region in cells [36], which can be confusing. In the light of the potential pitfalls associated with each of the above-mentioned FRET techniques, potential protein associations should ideally be tested independently by a combination of two or more FRET-based methods in addition to biochemical techniques such as co-immunoprecipitations. 

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... 

Other near-IR applications which use similar pulse and phase instrumentation as used in lifetime measurements include optical time-domain reflectometry(25) and photon migration in tissue. 26 ... 

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