Fluorescence time-domain FLIM

Implementation of time domain FLIM methods is comparatively straightforward in laser scanning microscopes (LSMs). Here, pointscanning is used so that single channel lifetime detection suffices. In principle, standard fluorescence lifetime detection equipment developed for spectroscopy can be used in combination with point-scanning systems and a pulsed laser. 

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. 

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. 

Fig. 2 Data acquisition for time-domain FLIM. FI fluorescence intensity, h gated image no 1,12 gated image no 2. Left Excitation pulse of the light source and synchronized timegated detection with a CCD camera. Right Lifetime determination by two subsequent time-gates according to Eq. 2...

Lifetime imaging can be implemented both in wide field and in scanning microscopes such as confocal microscopes and two-photon excitation microscopes. The most common implementations in time-domain fluorescence lifetime imaging microscopy (FLIM) are based on TCSPC [8, 9] and time-gating (TG) [2, 10],... 

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... 

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. 

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. 

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... 

---