Fluorescence Lifetime Imaging Microscopy (FLIM)

FLIM microscopy measures the fluorescent or phosphorescent decay rate of molecules (typically dyes or fluorescence proteins). In the so-called time-domain implementation, this is achieved by focusing a laser pulses lasting only picoseconds in time on a given point in the sample and measuring the time delay of the first fluorescently/phosphorescently emitted photon relative to the time that the pulse was incident on the sample. This is repeated millions of times a second (with spacing of only 10s of nanoseconds between consecutive pulses) until a statistically acceptable histogram is generated of the number of photons arriving at different time delays after the excitation pulse. This curve can be well described theoretically and fitted with one or a combination of exponential functions. A key fitting parameter is the so-called “lifetime” (denoted by the symbol t), which is the time it takes the exponential function to drop to half its initial value.

The point is then translated through the sample, and the same done again, until one has generated a 2D or 3D map of the “lifetime”. The lifetime is affected by the local environment of the emitter and will decrease if the emitter is placed in a higher density or refractive index environment. It may also be used as an indicator of temperature and pH using certain emitters. Very often it is however used to measure the so-called Foerster Resonant Energy Transfer (FRET) efficiency. FRET is the process by which energy is transferred between an emitting molecule (so-called “donor”) and a molecule that can absorb energy at the emitters emission wavelength (so-called “acceptor”).

In the presence of FRET, the lifetime of the donor molecules will decrease by an amount directly related to the distance between the donor and acceptor molecule as ~1/R6. This allows one to calculate the donor-acceptor separation with almost Angstrom precision! FRET in this measurable form only occurs for separations between 1-10nm. At smaller distances the energy transfer follows a more complicated model limiting the ability to determine the distance, whereas at larger distances the FRET follows a different functional dependence and is too weak to be measurable. There are number of details, such as the relative orientations of the donor and acceptor as well as the quantum yield of the donor and absorption cross section of the acceptor which will affect the FRET rate, that are described in more detail in the references below and references therein.


Examples of use:

  • Measuring the distances (between 1-10nm) or binding dynamics between two (fluorescently labelled) proteins.    

Useful references: