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Key Words: confocal, Fluorescence, GFP, live cell imaging, spectral imaging, FLIM, fluorophores

Definition:FRET is a fluorescence imaging technique that enables investigation of molecular interactions. It depends on the transfer of energy from one fluorophore to another fluorophore when the two molecules are in close proximity


FRET permits the investigation of molecular interactions beyond the theoretical resolution limit of optical microscopy. It is based on the direct transfer of energy from one molecule to another, which can only occur over very small distances (1-10nm). The following conditions must be satisfied for FRET:

  1. The donor probe should have a sufficiently long lifetime for energy transfer to occur
  2. Donor and acceptor molecules must be approximately 1-10 nm apart.
  3. The absorption spectrum of the acceptor fluorophore must overlap the fluorescence emission spectrum of the donor fluorophore (by approximately 30%).
  4. For energy transfer, the donor and acceptor dipole orientations must be approximately parallel.

Energy transfer is demonstrated by quenching of donor fluorescence together with a reduction in the fluorescence lifetime, and an increase in acceptor fluorescence emission. FRET is very sensitive to the distance between the fluorophores and can be used to estimate intermolecular distances. FLIM imaging can be used in association with FRET studies to identify and characterize energy transfer.

In confocal imaging, there are several methods for FRET studies. These include; filter-based, acceptor bleaching, spectral and FLIM-based methods. The filter-based method requires band-pass filters and specialized software to correct for cross-talk and bleed-through. Acceptor bleaching methods require laser power to bleach fluorophores. Spectral analysis allows spectral changes to be monitored, and FLIM enables quantitative FRET analysis.


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By targeting cellular constituents with fluorescent dyes, fluorescent antibodies and/or fluorescent proteins (GFP and related probes), almost any molecule can be labeled for FRET studies. FRET is a valuable tool for investigating and quantifying interactions in signal transduction, the formation of protein complexes, protein-protein interactions receptor function, membrane dynamics and molecular spatial relationships.


Almost any Nikon upright or inverted epi-fluorescence microscope can be configured to carry out FRET studies. Widefield techniques can be compromized by fluorescence emissions originating from above and below the plane of focus. These can be eliminated using confocal imaging (eC1, C1, and C1si). In confocal FRET applications, fluorophore pairs must be tailored to the available lasers. Spectral confocal imaging has particular advantages for FRET studies in that closely related spectra can be easily and cleanly separated, and quantitative FRET analysis is possible with spectral unmixing and ratio calculation.


For best possible FRET imaging, the Ti-E can be configured with the C1si confocal system and LIMO (This accommodates a range of lasers and offers continuous, variable laser control through AOM technology).


FRET/FLIM on C1si application note:  [download] 

Interactive tutorial on compatible FRET probes: [microscopyu] 

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