Fluorescence-lifetime imaging microscopy

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

The fluorescence lifetime (FLT) of the fluorophore, rather than its intensity, is used to create the image in FLIM. Fluorescence lifetime depends on the local micro-environment of the fluorophore, thus precluding any erroneous measurements in fluorescence intensity due to change in brightness of the light source, background light intensity or limited photo-bleaching. This technique also has the advantage of minimizing the effect of photon scattering in thick layers of sample. Being dependent on the micro-environment, lifetime measurements have been used as an indicator for pH, viscosity and chemical species concentration.

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.

Fluorescence-lifetime imaging yields images with the intensity of each pixel determined by ${\displaystyle \tau }$, which allows one to view contrast between materials with different fluorescence decay rates (even if those materials fluoresce at exactly the same wavelength), and also produces images which show changes in other decay pathways, such as in FRET imaging.

Fluorescence lifetimes can be determined in the time domain by using a pulsed source. When a population of fluorophores is excited by an ultrashort or delta pulse of light, the time-resolved fluorescence will decay exponentially as described above. However, if the excitation pulse or detection response is wide, the measured fluorescence, d(t), will not be purely exponential. The instrumental response function, IRF(t) will be convolved or blended with the decay function, F(t).

${\displaystyle {d}(t)={IRF}(t)\otimes {F}(t)}$