While I had previously been quite familiar with a variety of fluorescence-based molecular biology techniques (immunofluorescence, FACS, FISH), Fluorescence (Forster) Resonance Energy Transfer (FRET) was completely new to me. Learning about this technology during the Techniques Cafe made me realize the diversity of techniques that have been and will continue to be developed for a better understanding of molecular biology moving forward.

The most confusing aspect of this technique is understanding the energy transfer between the two chromophores. Many biologists do not have a strong background in quantum mechanics, which forms the basis of this technology. A strong understanding of the underlying mechanism is not necessary, however. One just needs to know that FRET involves the excitation of a donor fluorophore that is bound to a biomolecule. This donor then transfers its energy to an acceptor fluorophore in a non-radiative fashion – meaning, the transfer of energy does not actually occur through fluorescence. Instead, the energy is transferred through long-range dipole-dipole interactions and resonance energy transfer (RET). The excited fluorophore acts as an oscillating dipole, and transfers its energy to an acceptor with a similar resonance frequency. The measurement that is obtained is E, the FRET efficiency. This is the fraction of energy transfer that is occurring each time a donor chromophore is excited, and can give a significant amount of structural information regarding the donor and acceptor.

In order to assess someone’s understanding of a technique, I think they should be asked what sort of biological questions the technique could be used to investigate. This can show that they understand not only the underlying concept of the technology, but also have a practical understanding of how the technique can be used to investigate scientific hypotheses.

Some examples of questions that could be investigated using FRET:

• Detect in vivo interactions between two proteins
• Examine the distances between two different domains on a protein of interest
• Identify changes in conformation of a single protein in response to a particular physiological event
• Visualize the temporal and spatial localization of molecules within the cell, see how they change over time
• Determine the rate of biochemical reactions (e.g. enzymes)