Single-molecule Foerster Resonance Energy Transfer (smFRET)
Förster Resonance Energy Transfer (FRET), energy transfer from an excited donor chromophore to an acceptor chromophore via distance-dependent dipole-dipole coupling, has led to numerous fascinating proofs of principle and applications in natural sciences. Using a total internal reflection fluorescence microscope, FRET of surface-immobilised molecules can be monitored over time at the single-molecule level. This dynamic approach has two advantages over ensemble techniques: (i) the properties of individual molecules that would normally be masked by ensemble averaging are uncovered, e.g. revealing conformational subpopulations, (ii) single molecule detection allows to follow a specific molecule for an extended period of time, yielding kinetic parameters of conformational changes. We perform smFRET experiments to study the influence of various biologically relevant metal ions on nucleic acids coformation, following folding reactions of nucleic acids over a time-span of several minutes.
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| Schematic diagram of a two-color emission separation smFRET setup. The laser excitation beam travels through a system of filters, mirrors and lenses and hits the quartz slide at the critical angle. Fluorescence emission of both the donor and the acceptor are spectrally separated and projected side-by-side on CCD camera. | Emission of single donor-acceptor pairs (corresponding green and red spots) are used to calculate FRET efficiency over time (bottom), from which conformational changes and the corresponding kinetic parameters are extracted. |
Group members involved: Richard Börner, Erica Fiorini, Helena Guiset Miserachs, Melodie Hadzic, Mokrane Khier, Sebastian Koenig, Danny Kowerko, Susann Zelger-Paulus, Michelle Schaffer, Anita Schmitz.
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