Fluorescence frequency-domain data

Frequency-domain measurements of fluorescence energy transfer are used to determine the end-to-end distance distribution of donor-acceptor D-A) pairs linked by flexible alkyl chains. The length of the linker is varied from 11 to 2B atoms, and two different D-A pairs are used. In each case the D-A distributions are recovered from global analysis of measurements with different values for the FSrster distance, which are obtained by collislonal quenching of the donors. In all cases essentially the same distance distribution Is recovered from the frequency-domain data for each value of tha Ffirster distance. The experimentally recovered distance distributions are compared with those calculated from the RIS model. The experimentally recovered distance distributions for the largest chain molecules are In agreement with the predictions of the RIS model. However, the experimental and RIS distributions are distinct for the shorter D-A pairs. 

Fig. 17. Frequency-domain data for the intrinsic fluorescence of S] Nuclease and melittin.

A general discussion of the use of least-squares fitting in fluorescence measurements may be found in (28). The global analysis of fluorescence data is discussed in (29). Commercially available time-resolved fluorimeters are typically sold with data analysis software included. Available stand-alone packages include the Globals Unlimited suite, which is capable of analysing both time- and frequency-domain data, stopped-flow kinetics, etc. The Center for Fluorescence Spectroscopy at the University of Maryland (USA) also offers software for frequency- and time-domain fluorescence lifetime analysis. 

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. 

To analyze frequency domain FLIM data, first the phase shift and demodulation of the fluorescence light with respect to the excitation light are estimated. In the case of single frequency data, this reduces the FLIM data to only three parameters phase shift, demodulation, and total intensity. This step can be done in various ways as described in the following sections. From these parameters, the lifetimes can be estimated either by Eqs. (2.6 and 2.7), or by more elaborate approaches as described below. 

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 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. 

In spite of its prevalence in the fluorescence decay literature, we were not universally successful with this fitting method. Most reports of hi- or multiexponential decay analysis that use a time-domain technique (as opposed to a frequency-domain technique) use time-correlated photon counting, not the impulse-response method described in Section 2.1. In time-correlated photon-counting, noise in the data is assumed to have a normal distribution. Noise in data collected with our instrument is probably dominated by the pulse-to-pulse variation of the laser used for excitation this variation can be as large as 10-20%. Perhaps the distribution or the level of noise or the combination of the two accounts for our inconsistent results with Marquardt fitting. 

In phase-modulation fluorometry, the sample is excited by a sinusoidally modulated light at high frequency. The fluorescence response, which is the convolution product (Eq. (7.6)) of the d-pulse response by the sinusoidal excitation function, is sinusoidally modulated at the same frequency but delayed in phase and partially demodulated with respect to the excitation. The phase shift and the modulation ratio M (equal to m/mo), that is the ratio of the modulation depth m (AC/DC ratio) of the fluorescence and the modulation depth of the excitation mg, characterize the harmonic response of the system. These parameters are measured as a function of the modulation frequency. No deconvolution is necessary because the data are directly analyzed in the frequency domain. 

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... 

Fluorescence lifetime data of 1, 4, 5, and 6 in presence of 10 M -CD were collected with frequency-domain fluorometry. These probes gave only 1 1 complexes with P-CD [58] and, given the association constant values, the complex molar fraction was >0.95 for 4 and 5 and 0.1 for 6. The fluorescence decay of all the probes was best described by unimodal Lorentzian lifetime distribution [51,59] rather than by a mono- or biexponential function corresponding to the emission of the complexed and the free probe. This distribution was attributed to the coexistence of molecules included in the cavity to different extents. It was proposed that, in the case of 4, the apolar benzene ring enters the cavity first and penetrates until the whole naphthalene is included. This is the most stable and, hence, the most populated conformation of the complex. The distribution of the lifetimes suggests that at any time there is an ensemble of molecules in different stages of complexation which have slightly different lifetimes. 

Fluorescence anisotropy decay of [Leu ] enkephalin tyrosine was measured using the frequency- domain up to 10 GHz. The data indicate a 44 ps cori elation time for local tyrosine motions and a 219 ps correlation time for overall rotational diffusion of the pentapeptide (Lakowicz et al. 1993). Also a rotational correlation time of 26 ps was measured by H NMR for Ha of tyrosine in position 1 of L-dermorphin (Simenel, 1990). These ps values determined by NMR and by fluorescence spectroscopy are the result of possible significant atomic fluctuations that occur in the picosecond time scale (Karplus and Me Gammon, 1981). Since it was difficult in quenching experiments performed on DREK to measure such short correlation times we do not know whether these atomic fluctuations would depend on the conformation of the peptide or not. However, our results clearly put into evidence the presence of a local rotation within DREK. 

Multi-exponential decays of fluorescence can also be recovered by measurements in the frequency-domain. This has only become practical within the past four years [27,28]. The resolution of multi-exponential decays requires measurements over a wide range of light modulation frequencies. Earher instrumentation could operate at only one to three frequencies, and these hmited data were not adequate to determine the four parameters in equation 10 (two and two t,). The new instruments... 

Brochon JC, Livesey AK (1990) Data analysis in frequency-domain fluorometry by the maximum entropy method - recovery of fluorescence lifetime distributions. Chem Phys Lett 174 517-522... 

The kinetics of excited-state reactions can be characterized from time-resolved measurements of the concomitant fluorescence emission. Current methodologies in time [1,2] and frequency domain [3-8] provide data with sufficient accuracy allowing an elaborate data analysis. 

Fig. 2.4. (A) Sketch of the cryostat insert for single-molecule spectroscopy by fluorescence excitation. The focus of lens L is placed in the sample S by the magnet/coil pair M, C. (B) Scan over the inhomogeneous line (a) with a 2 GHz region expanded (b) to show isolated single-molecule absorption profiles. (C) Three-dimensional pseudo-image of single molecules of pentacene in p-terphenyl. The measured fluorescence signal (z-axis) is shown over a range of 300 MHz in excitation frequency (horizontal axis, center = 592.544 nm) and 40 pm in spatial position (axis into the page). (D) Rotation of the data in (c) to show that in the spatial domain, the single molecule maps out the shape of the laser focal spot. Bar, 5 pm. For details, see [33]...

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