Time-resolved fluorescence emission spectroscopy of a photoexcited sample is a powerful tool for the study of intricate living cells in both space and time of their internal biochemistry. The experimental challenge of actually visualizing the complex reaction kinetics is feasible by using the state-of-the-art imaging system and the design and synthesis of new fluorescent probes. Fluorescence measurements in the time-domain possess much greater information content about the rates and kinetics of intra- and intermolecular processes than is afforded by wavelength spectroscopy alone.
WHAT IS FLUORESCENCE LIFETIME?
The fluorescence lifetime is defined as the average time that a molecule remains in an excited state prior to returning to the ground state. For a single exponential decay, the fluorescence intensity as a function of time after a brief pulse of excitation light is described as
where I0 is the initial intensity immediately after the excitation pulse.
In practice, the fluorescence lifetime (tau) is defined as the time in which the fluorescence intensity decays to 1/e of the intensity immediately following excitation. Fluorescence decay is often multiexponential, leading to complex decay curves.
Instrumental methods for measuring fluorescence lifetimes are divided into two major categories, frequency-domain and time-domain. Frequency-domain fluorometers excite the fluorescence with light, which is sinusoidal and modulated at radio frequencies (for nanosecond decays), and then measure the phase shift and amplitude attenuation of the fluorescence emission relative to the phase and amplitude of the exciting light. Thus, each lifetime value will cause a specific phase shift and attenuation at a given frequency.
In time-domain methods, pulsed light is used as the excitation source, and fluorescence lifetimes are measured from the fluorescence signal directly or by photon counting.
WHAT IS FLIM?
Many currently available fluorescence microscopic techniques, such as confocal or multi-photon excitation, cannot provide detailed information about the organization and dynamics of complex cellular structures. In contrast, time-resolved fluorescence microscopy allows the measurement of dynamic events at very high temporal resolution and can monitor interactions between cellular components with very high spatial resolution as well. To date, most measurements of fluorescence lifetimes have been performed in solution or cell suspensions. Fluorescence lifetime imaging was developed to overcome this drawback and still provide the ability to use the power of fluorescence lifetime measurements in a single living cell.
FLIM-FRET
The combination of lifetime and FRET (FLIM-FRET) provides high spatial (nanometer) and temporal (nanoseconds) resolution (Bacskai et al., 2003; Elnagovan et al., 2002; Krishnan et al., 2003). The presence of acceptor molecules within the local environment of the donor that permit energy transfer will influence the fluorescence lifetime of the donor. By measuring the donor lifetime in the presence and the absence of acceptor one can accurately calculate the distance between the donor- and acceptor-labeled proteins. While 1p-FRET produces 'apparent' E%, i.e. efficiency calculated on the basis of all donors (FRET and non-FRET), the double-label lifetime data in 2p-FLIM-FRET usually exhibits 2 peaks of donor lifetimes (FRET and non-FRET), allowing a more precise estimate of distance based on FRET donors only. The former may be sufficiently accurate for many situations; the latter may be vital for establishing comparative distances of several proteins from a protein of interest.
