FLIM - The Method

FLIM measurements are carried out with Leica TCS SMD FLIM systems

Fluorescence lifetime definition

The fluorescence lifetime is a measure of how long a fluorophore remains on average in its excited state before returning to the ground state by emitting a fluorescence photon. The emission of a fluorescence photon from a fluorophore does not occur at a fixed time. Instead, a distribution of times is observed, which can be described by an exponential decay function. The characteristic time constant of this decay, the  fluorescence lifetime, is in the range of a few picoseconds (10^-12 s) to several tens of nanoseconds (10^-9 s).

Fluorescence lifetime as probe for nano-environment

This lifetime is a characteristic parameter of each fluorescent dye that may change with its micro-surrounding or its conformational state. Lifetime information probes the molecular environment for its composition, such as ion concentration, pH, lipophilicity or the binding to other molecules.

Combination of lifetime measurement and imaging

FLIM combines lifetime measurements with imaging: lifetimes obtained at the pixel-level are color-coded to produce images. Thus, FLIM delivers information about the spatial distribution of a fluorescent molecule together with information about its nano-environment. This way an additional dimension of information is obtained.

Principle of FLIM data acquisition and analysis

1. Repeated measurement of the time between laser pulse and fluorescence photon at each pixel.
2. Calculation of a histogram of photon counts over arrival time after the laser pulse.
3. Fit of an exponential decay to each  histogram. The amplitude reflects the total number of photons, the time constant τ is called the fluorescence lifetime.
4. Display of lifetime image using a false-color look-up table.