Basics of smFRET microscopy
smFRET is the acronym for single-molecule Förster Resonance Energy Transfer. smFRET exploits a natural phenomenon that can occur between 2 fluorophores when they come within 1-10nm distance. The emission energy of one fluorophore, the donor, can then be transferred to the acceptor which subsequently fluoresces.
The technique involves labeling the sample with donor and acceptor fluorophores, which can be on the same or on different molecules. By measuring the FRET fluorescence intra- or inter-molecular interactions can be characterized.
For smFRET to work it is required that the emission spectrum of the donor fluorophore partially overlaps with the absorption spectrum of the acceptor.
Applications of single-molecule FRET
smFRET offers insight into molecular interactions by quantifying and characterising:
frequency and duration of binding events
intramolecular transitions and dynamics (protein folding)
specific interactions and complex assembly
kinetics and dwell times for single molecules
Using the Nanoimager for smFRET
The Nanoimager is the only commercially available microscope capable of carrying out single-molecule FRET microscopy. It comes with software that can interpret the smFRET imaging data and enables reactions to be followed in real-time.
With the large field of view, thousands of FRET fluorescence events can be traced in a single acquisition, speeding up the process and improving the accuracy of results. Individual FRET traces can be plotted and population averages as a whole can calculated.