Fluorescence Lifetime Imaging (FLIM)
FLIM is a microscopy technique that is based on measuring the lifetime of the dyes that are used to stain the sample. After absorbing a photon, the fluorescent molecule stays in an excited state until it emits a photon or returns to the ground state by a non-radiative process (quenching, energy transfer). The fluorescence lifetime describes the mean time that a fluorophore remains in the excited state. This time is characteristic for a certain fluorophore (under constant environmental conditions) and can thus be used to discriminate fluorescence signals of dyes even with strongly overlapping spectra.
The fluorescence lifetime is strongly influenced by the chemical environment in the vicinity of the fluorophore. This way it is possible to get information on parameters such as local pH-value, oxygen or ion concentrations.
FLIM is generally based on a laser scanning microscope, either a confocal or a 2-photon microscope. A pulsed laser beam scans the sample pixel wise and the time between an excitation pulse and the emission of a single fluorescence photon is measured. This is repeated several times. The arrival times of the photons are registered in a histogram, resulting in an exponential decay curve. From this curve, the fluorescence lifetimes for every pixel are calculated. The result is a color-coded image of the sample. FLIM combines the 3D resolution of the confocal/2-photon microscope with a time-resolved measurement, thus giving full 4D information about the sample.
Ideal light sources for FLIM microscopy are tunable visible fiber lasers such as the FemtoFiber pro TVIS or the iChrome TVIS, a fully automated tunable fiber laser which can be easily integrated in a microscope setup. Both lasers are continuously tunable from 488 nm up to 640 nm, delivering picosecond pulses with an intrinsically narrow bandwidth.