Schematic representation of the FRET spectral overlap integral (shown in gray shadow).
Fluorescence Lifetime Imaging Microscopy (FLIM) is a fluorescent imaging
technique that utilizes a microscope equipped with a detector capable of high-frequency modulation and/or fast gating. FLIM is widely used for biomedical applications, and can distinguish the molecular environment of labeled macromolecules inside cells. FLIM works by using the decay kinetics of the excited state of chromophores in a sample to spatially map various components of a specimen. Because of this, factors like dye concentration, photobleaching, light scattering, and excitation light intensity do not impact imaging. FLIM is versatile, in that it can utilize a single decay time or entire decay profile, in 2 or 3D.
There are many variations in FLIM instrumentation but they all fall into three categories based on how measurements are made, including; the time-domain method (using a pulsed light source), frequency-domain method (using a megahertz modulated sources), or the continuous wave (using a steady state light source) method.
FLIM is made possible by fluorescence resonance energy transfer (FRET)
, which relies on the distance-dependent interaction that occurs between the excited states of two dye molecules. In other words, FRET relies only on the physical interaction of two fluorophores, termed the donor and acceptor, significantly improving the spatial resolution of produced images. In principle, excitation is transferred from a donor to an acceptor molecule without the emission of photons. Dependence of the energy transfer efficiency on the donor-acceptor separation enables this phenomenon to be used for the study of cell component interactions.
Optical microscopy combined with FRET allows temporal as well as spatial information of proteins, lipids, enzymes
, DNA, and RNA in vivo
interactions to be acquired and quantified. In imaging the colocalization of these molecules, the spatial resolution of FRET techniques is significantly better than that of other microscopy methods. It is important to keep in mind that quantitative measurements may be affected by a few limitations. Distortions in the image must be corrected and accounted for, and the number of donor and acceptor pairs can produce significant cross talk
, which may misreport true quantitative results.
Table 1. Common FRET donor and acceptor pairs and their R0 values.
FLIM comes with a number of advantages, including that image acquisition is fast enough, down to the hundredths of a millisecond, to allow for imaging live cells for use in vivo
as well as in situ
. FLIM does not rely on chromophore concentration, which can be challenging to control within a cellular population. As the lifetime of the fluorophore signal is used to create an image, photon scattering in thicker samples is minimized. FLIM has also been used to image fixed and archived diseased tissue samples to determine the functional state of proteins involved in the pathology of disease.
There do exist a number of factors that will, however, affect the fluorescence lifetime, including ion intensity, hydrophobic properties, oxygen concentration, molecular binding, and the energy transferred in protein-protein interactions.