FLIM, Fluorescence Lifetime Imaging Microscopy
FLIM is the abbreviation for "Fluorescence Lifetime Imaging Microscopy" and is the technique that maps the spatial distribution of lifetimes within microscopic images of fixed as well as of living cells. The fluorescence lifetime is the signature of a fluorescent material; it is the exponential decay in emission after the excitation of a fluorescent material. In other words, the lifetime is the average time that fluorescent molecules spend in the excited state.
The fluorescence lifetime does not change upon intensity variations and therefore lifetime measurements are not dependent on the local concentration of fluorophores, the optical path of the microscope, the local excitation light intensity, or on the local fluorescence detection efficiency.
In specific cases, fluorophores change their quantum yield upon the interaction with other fluorescent molecules, or with other small soluble molecules like ions or oxygen. The fluorophores lose their (excited state) energy much faster in those cases and so their fluorescence lifetime decreases. FLIM can measure such lifetime dynamics, and therefore FLIM can be used to measure indirectly biomolecular concentrations, and interactions that are closely related to the fluorescence lifetime of the fluorophores.
technologies
Measurement methods FLIM
FLIM can be measured in two ways: by the time domain method that is mainly applied on confocal laser scanning microscopes and by the frequency domain method that is used on widefield fluorescence microscopes.
The Lambert Instruments FLIM system (LIFA) operates in the frequency domain and can be attached to any wide field fluorescence microscope.
Notes for FLIM users
Confocal FLIM
Confocal microscopy is an imaging technique used to increase micrograph contrast and/or to reconstruct three-dimensional images by using a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. The thickness of the focal plane is defined mostly by the objective lens, and also by the optical properties of the specimen and the ambient conditions. Only the light within the focal plane can be detected, so the image quality is much better than that of widefield images. Typical applications include life sciences, e.g. cell biology.
These images are generated with the LIFA from the same sample (courtesy of P. Bastiaens), either by confocal imaging (CSU10 spinning disk and diode laser coupled to the LIFA system), or by the typical widefield imaging (LED light).
TIRF-FLIM
FLIM-FRET experiments
Förster / Fluorescence Resonance Energy Transfer (FRET) is the non-radiative transfer of energy from a molecule in the excited state (donor) to a molecule in the ground state (acceptor). A fluorescent donor molecule can return to the ground state by losing its energy through emission of a photon (fluorescence), or by transferring its energy to a nearby (1 - 9nm) acceptor molecule (FRET). Compared to a molecule that exhibits no FRET, the donor has more options to lose its energy. Therefore, it returns faster to the ground state, which decreases its lifetime.
Non-FRET experiments
Instead of using FLIM to report FRET, FLIM can also be used for other experiments. Whereas fluorescence lifetimes are mostly independent of the local probe concentration and photobleaching, they can depend on several other factors inside cells.
In the picture a microscopic image of the skin of head is shown. The lifetime image as well as the intensity image is shown that are merged in the bottom part. Lifetime differences are the result of changes in the environment surrounding the (auto)fluorescent proteins.
Papers / Reviews FLIM
A selection of papers and books that describe the technology of FLIM in more detail: