Fluorescence frequency-domain FLIM

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. 

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... 

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. 

A.H.A. Clayton, Q.A. Hanley, D.J. Arndt-Jovin, V. Subramaniam, T.M. Jovin, Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (FLIM), Biophys. J. 83, 1631-1649 (2002)... 

With frequency domain FLIM the light source is a continuous wave laser as opposed to a pulsed laser. The continuous wave laser is modulated via an acousto-optical modulator and the sample is excited by a sinusoidally modulated light. The fluorescence response is also sinusoidally modulated at the same frequency but it is delayed in phase and is partially demodulated. For a single exponential decay the lifetime of the donor chromophore can be quickly calculated by either the phase shift (j) (rp) or the modulation ratio M (r ,) using the following equations ... 

In frequency-domain FLIM, the intensity of the excitation light is continuously modulated. Due to the (non-instant) fluorescence decay, the fluorescence emission will display a phase shift and a decrease in modulation. This can be understood if we look at the fluorescence intensity of a mono-exponentially decaying fluorochrome after an infinitesimal short pulse of excitation light at t=0 as described by Eq. (1) ... 

Comparing the frequency-domain and the time-domain approach, it was found that one is not fundamentally better than the other in terms of signal to noise ratio, when measuring the fluorescence lifetime of a mono-exponentially decaying fluorochrome [33]. In both systems, it is crucial, however, to use the optimal settings for each particular measurement. In frequency-domain FLIM, the... 

While publications on fluorescence lifetime imaging microscopy (FLIM) have been relatively evenly divided between time and frequency domain methods, a majority of the 10 most highly cited papers using FLIM have taken advantage of the frequency domain method [1, 2-9]. Both techniques have confronted similar challenges as they have developed and, as such, common themes may be found in both approaches to FLIM. One of the most important criteria is to retrieve the maximum information out of a FLIM... 

Van Munster, E. B. and Gadella, T. W. J. (2004). phi FLIM A new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy. J. Microsc. 213, 29-38. 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

Since TIRF produces an evanescent wave of typically 80 nm depth and several tens of microns width, detection of TIRF-induced fluorescence requires a camera-based (imaging) detector. Hence, implementing TIRF on scanning FLIM systems or multiphoton FLIM systems is generally not possible. To combine it with FLIM, a nanosecond-gated or high-frequency-modulated imaging detector is required in addition to a pulsed or modulated laser source. In this chapter, the implementation with of TIRF into a frequency-domain wide-field FLIM system is described. 

As shown in Section 11.2.1.1, more details can be obtained by confocal fluorescence microscopy than by conventional fluorescence microscopy. In principle, the extension of conventional FLIM to confocal FLIM using either time- or frequency-domain methods is possible. However, the time-domain method based on singlephoton timing requires expensive lasers with high repetition rates to acquire an image in a reasonable time, because each pixel requires many photon events to generate a decay curve. In contrast, the frequency-domain method using an inexpensive CW laser coupled with an acoustooptic modulator is well suited to confocal FLIM. 

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. 

FLIM has its roots in two fields of research 1) microscopy and 2) fluorescence spectroscopy. In the latter field of research, non-spatially-resolved fluorescence hfetime measurements were performed since 1926 [1], i.e. long before FLIM was developed. Typically, bulk measurements were carried out using cuvettes. Not surprisingly, most of the methodology and nomenclature used in FLIM today, e.g. frequency-domain , and time-domain , have their origins in instruments that were used for cuvette-based lifetime measurements. 

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