Fluorescence resonance energy transfer (FRET) microscopy

Introduction

FRET is an interaction of two fluorochromes depending on the distance of these two fluorochromes. One fluorochrome (the Donor) transfers the excitation energy directly to a second fluorochrome (the Acceptor) without the emission of a photon. Typically two fluorochromes are used that have overlapping emission and excitation wavelengths but FRET can be achieved in several ways. The two interacting fluorochromes need to be close together so that FRET takes place; roughly speaking in the range of a few nanometers. In a FRET experiment this interaction of the two fluorochromes needs to be detected. Therefore it is necessary to detect the emission of the acceptor when the excitation of the donor is applied. It has to be kept in mind that the measurement of the biological effect can also mean to measure the quench of the FRET-signal.

The quantitative or semi-quantitative determination of FRET is quite complex.

Applications

Using the FRET principle, a lot of experimental approaches can be realized, like for example the investigation of conformation changes in proteins or binding assays.