Single Molecule FRET

Single molecule experiments offer much more information about the sample which are hidden in ensemble measurements. These include the determination of several subpopulations in a heterogeneous sample, the possibility to detect rare events and the observation of the temporal behavior of single molecules.

 

Fluorescence Resonance Energy Transfer (FRET) is a technique to measure distances of a few nanometers between two fluorescent dyes. A donor fluorophore that is excited by a laser transfers a part of its energy non-radiatively to an acceptor fluorophore. The transfer efficiency is dependent on the interfluorophore distance. Detecting the emission of the donor and acceptor fluorophore seperatly allows us to calculate the transfer efficiency and therefore the distance between the fluorophores.

We use highly diluted solutions so that only one FRET-pair is in the laser focus at the same time. This gives us more detailed information about our sample than bulk measurements since we do not average over many FRET-pairs at once. We are able to detect subpopulations, e.g. different conformational states of a protein or nucleic acid.

Two improvements are planned for the future. Adding Alternating laser excitation (ALEX), where the donor and acceptor are excited alternately, gives a far better signal-to-noise-ratio. Extending the evaluation software with Probability distribution analysis (PDA) will increase the ability to distinguish between subpopulations.

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