Frequency-domain lifetime determinations

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 ... 

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. 

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...

The value of fEdetermines all other variables in the equations above. In turn, fE is determined by the temporal resolution of interest of the system studied. To resolve an average excited state lifetime t, the required data sampling rate, in frequency domain techniques is at least an order ofmagnitude slower than it is in the time domain as stated by the following relation (when Np > 32 and Nw= 1) ... 

The correction of systematic phase errors in frequency domain spectroscopy can be achieved by use of a fluorophore of known lifetime as standard. It has been pointed out that a simple scattering solution can be used as standard. This ingenious suggestion very conveniently dispenses with the need for a fluorescent standard with a previously-determined lifetime value. Phase noise, another troublesome factor encountered in frequency domain fluorimetry, can be eliminated by use of a differential method. 

There are two methods that have been used to determine fluorescence lifetimes in DNA sequencing. In the first method, known as the frequency-domain or phase-modulation method, the excitation beam is intensity modulated. The a.c. portion of the resulting emission is phase-shifted relative to the laser modulation this phase-shift contains information about the fluorescence lifetime, or lifetimes if more than one fluor is present [140]. McGown and coworkers [144,145] used this method for four-color sequencing. In that work, 488 nm or 514 nm laser light was electronically modulated with a Pockels cell before being focused onto a capillary column. Detection, made normal to the laser direction, was optically filtered to reduce laser scatter and was focused onto the detector of a... 

The methods used for determining the lifetimes and emission intensities can be divided into time-domain and frequency-domain methods. In the time-domain methods, the excitation of the system is performed by a light pulse or pulse... 

The methods based on RET are conunonly used for determining intramolecular donor-acceptor distances in macromolecules. The use of lanthanide chelates as donors and prompt fluorophores as acceptors is especially advantageous because of the large difference in their lifetimes. The present example shows the use of the frequency-domain method as an alternative in determining the relevant lifetimes in connection with a recently published work on RET [16],... 

The idea to use frequency-domain detection for the measurement of fluorescent lifetimes dates back to 1921 [4], although the idea to measure small phase changes to determine short time intervals is much older [5]. The first instruments to measure non-spatially-resolved lifetimes in the 1920s were all based on frequency-domain detection. 

In contrast to the emission lifetime determination techniques reported above, the phase shift does not require the collection of the excited states decay curve. Indeed this frequency-domain technique provides emission lifetimes by measuring unusual parameters. 

There are two ways to collect FLIM data freqnency-domain or time-domain data acqnisition (Alcala et al. 1985 Jameson et al. 1984). Briefly, in freqnency domain FLIM, the fluorescence lifetime is determined by its different phase relative to a freqnency modulated excitation signal nsing a fast Fourier transform algorithm. This method requires a frequency synthesizer phase-locked to the repetition freqnency of the laser to drive an RF power amplifier that modulates the amplification of the detector photomultiplier at the master frequency plus an additional cross-correlation freqnency. In contrast, time-domain FLIM directly measures t using a photon connting PMT and card. 

An electronic or vibrational excited state has a finite global lifetime and its de-excitation, when it is not metastable, is very fast compared to the standard measurement time conditions. Dedicated lifetime measurements are a part of spectroscopy known as time domain spectroscopy. One of the methods is based on the existence of pulsed lasers that can deliver radiation beams of very short duration and adjustable repetition rates. The frequency of the radiation pulse of these lasers, tuned to the frequency of a discrete transition, as in a free-electron laser (FEL), can be used to determine the lifetime of the excited state of the transition in a pump-probe experiment. In this method, a pump energy pulse produces a transient transmission dip of the sample at the transition frequency due to saturation. The evolution of this dip with time is probed by a low-intensity pulse at the same frequency, as a function of the delay between the pump and probe pulses.1 When the decay is exponential, the slope of the decay of the transmission dip as a function of the delay, plotted in a log-linear scale, provides a value of the lifetime of the excited state. 

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