FLIM: Fluorescence Lifetime Imaging Microscopy is a microscope technique for measuring the rate of fluorescence decay, and therefore the lifetime of fluorescence emission of a population of fluorophores. Fluorescence lifetime is independent of emission intensity and fluorophore concentration, but dependent on enviromental factors such as calcium concentration or FRET-pairing. With FLIM analysis, precise kinetics and localization of fluorophores can be determined.

Background

As previously shown, excitation of a molecule can be graphically represented as a "Jablonski diagram". The salient component for fluorescence lifetime analysis is the timing of the fluorophore states:

• Excitation: 10e-15s between photon absorption and the electron excited state.
• Relaxation: 10e-14 to 10e-11s. This is the time for some energy to be emitted (as heat)
• Fluorescence emission: 10e-9 to 10e-7. Length of time for the Em photon to be emitted, returning the electron to the ground state. This is fluorescence lifetime.

Fluorescence Lifetime (FL) = average time a fluorescent molecule stays in the excited state. It is a probability phenomenon, with the lifetime of a population of molecules being measurable, not that of individual molecules.

Fluorescence Lifetime is characteristic for each fluorophore (under specific environmental conditions). It may be used to distinguish fluorophores with identical fluorescence emission spectra.

Fluorescence Lifetime is independent of the Ex intensity.

FRET decreases the fluorescence lifetime; therefore FLIM can be used as a measure of FRET.

Here is an example showing how the fluorescence lifetime of a molecule (naproxen) changes with environment.

Note the time (in nanoseconds, ns), and the effect of (in this example) the addition of acrylamide. "IRF" is the instrument response function.

Here is a page listing fluorescence lifetime standards, and FL of selected fluorophores.

If fluorescence lifetime is used in an imaging system, it is called: Fluorescence Lifetime Imaging Microscopy.

Here is an image showing the change in fluorescence lifetime of chlorophyll in a damaged leaf. Areas with changed FL are color coded red.