FRET Sensitized Emission Wizard Widefield

FRET SE with the Leica Advanced Widefield Systems AF7000, AF6500 and AF6000

April 15, 2009

Fluorescence Resonance Energy Transfer (FRET) is a technique, which allows insight into the interactions between proteins or molecules in proximities beyond light microscopic resolution.

An excited fluorophore, called the donor, transfers its excited state energy to a light absorbing molecule which is called the acceptor. This transfer of energy is non-radiative. Sensitized Emission is one established method for the evaluation of FRET efficiencies. It can be applied to live cells as well as to fixed samples.

FRET SE is a method mainly used to analyze protein – protein interactions or conformational changes of proteins. There are several prerequisites for FRET to occur:

  • The Donor and Acceptor distance must be <10 nm
  • Sufficient separation of excitation and emission spectra are necessary
  • The Donor emission spectrum must overlap with the Acceptor absorption spectrum
Excitation and emission spectra of CFP/YFP FRET pair. Courtesy: T. Zimmermann, EMBL.

Due to the necessary overlap between Donor emission spectrum and Acceptor absorption spectrum each FRET measurement is done by sequential acquisition of three channels:

  • Donor channel: Donor excitation and Donor emission
  • FRET channel: Donor excitation, Acceptor emission
  • Acceptor channel: Acceptor excitation and Acceptor emission

Donor and Acceptor channel are used to eliminate crosstalk into the FRET channel. FRET sample preparations must therefore include references of donor in the absence of the acceptor (donor only control) and acceptor in the absence of the donor (acceptor only control). Ideally, all references are included in the same preparation. The donor and acceptor references are used to obtain calibration coefficients to correct for excitation and emission cross talk.

As this method is non-invasive, it is most frequently used for live cell experiments.

It is important that throughout the entire experiment and calibration routine, all measuring parameters such as camera exposure time, gain, excitation intensities must remain constant.

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