FLIM

Fluorescence-lifetime imaging microscopy or FLIM is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography.

The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample.

A fluorophore which is excited by a photon will drop to the ground state with a certain probability based on the decay rates through a number of different (radiative and/or nonradiative) decay pathways. To observe fluorescence, one of these pathways must be by spontaneous emission of a photon. In the ensemble description, the fluorescence emitted will decay with time according to

where

In the above, ${\displaystyle t}$ is time, ${\displaystyle \tau }$ is the fluorescence lifetime, ${\displaystyle I_{0}}$ is the initial fluorescence at ${\displaystyle t=0}$, and ${\displaystyle k_{i}}$ are the rates for each decay pathway, at least one of which must be the fluorescence decay rate ${\displaystyle k_{f}}$. More importantly, the lifetime, ${\displaystyle \tau }$ is independent of the initial intensity and of the emitted light. This can be utilized for making non-intensity based measurements in chemical sensing.

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