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How to Measure FRET

In my previous article on FRET, I gave you some background on FRET – its mechanism and its applications. Here, I will expand, including what to measure when doing FRET.

There are a number of approaches to FRET quantification:

  1. Sensitized Emission – This two-channel imaging technique uses an algorithm that corrects for excitation and emission crosstalk.
  2. Acceptor Photobleaching – Sometimes called donor dequenching, this technique measures increased donor emission when the acceptor is photobleached.
  3. Fluorescence Lifetime Imaging Microscopy FRET (FLIM FRET) – This technique detects fluorescence lifetime changes of donor.
  4. Fluorophore donor Spectral Imaging – This technique involves exciting at one or two wavelengths and measuring the spectral profiles of both donor and acceptor.
  5. Homo-FRET and polarization Anisotropy Imaging – This technique uses identical donor and acceptor fluorophores and detects FRET through measurements of polarization.

Photo by Goele/wikimedia

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How to Measure FRET

Bitesize Bio – Microscopy & Imaging, March 4, 2015