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
- Repeated measurement of the time between laser pulse and fluorescence photon at each pixel.
- Calculation of a histogram of photon counts over arrival time after the laser pulse.
- 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.
- Display of lifetime image using a false-color look-up table.