fluorescence technique where contrast is based on the lifetime of individual fluorophores
The fluorescence lifetime is defined as the average time that a molecule remains in an excited state before returning to the ground state. Fluorescence lifetimes can be measured in two ways:
- Frequency-domain measurements or phase modulation - where a modulation of the excitation source causes a measurable phase shift in fluorescence and reduction in its amplitude. The fluorescence lifetime can be calculated from these measures.
- Time-domain measurements - where pulsed light is use to excite a fluorophore and fluorescence lifetimes are measured directly from the fluorescence signal or by photon counting
Fluorescence lifetimes have the advantage that they are not affected by fluorophore concentration, excitation intensity fluctuations, absorption by the sample, or photobleaching. They are, however, affected by environment - pH, oxygen concentration, molecular binding, and FRET enabling studies on local molecular environments.
FLIM is able to discriminate between fluorescence emanating from different fluorophores and autoflorescing molecules in a specimen, even if their emission spectra are similar. It is, therefore, ideal for identifying fluorophores in multi-label studies. FLIM can also be used to measure intracellular ion concentrations without extensive calibration procedures (for example, Calcium Green) and to obtain information about the local environment of a fluorophore based on changes in its lifetime. FLIM is often used to study spatial and temporal protein-protein interactions, properties of membranes and interactions with nucleic acids in living cells. FLIM is a key technique in enabling accurate FRET studies.
FLIM can be implemented on Nikon's research-level confocal microscope systems. Confocal is preferred because out-of-focus signals are greatly reduced to provide higher resolution images. Nikon's LIMO module for FLIM rapidly generates optimized lifetime decay curves (within seconds) and generates sufficient lifetime contrast to distinguish fluorescent probes with small lifetime differences (<0.1 ns). Nikon's LIMO module for FLIM applications can be configured with the C2+ and C2si+ confocal microscopes. In addition to fast (LIMO) confocal imaging, Nikon confocal systems can also be combined with commercial photon counting systems.
Widefield FLIM imaging systems can also be used, especially the combination of white TIRF and FLIM, may be used on Nikon microscope systems.
For ultimate flexibility in FLIM and FRET studies, Nikon recommends the LIMO module configured on the C2si+ confocal system.