Single‐Molecule FRET Imaging with Oxford Nanoimaging Nanoimager

The Nanoimager is the world’s first commercial solution for wide‐field single‐molecule Förster resonance energy transfer (smFRET) studies. FRET is a non‐radiative energy transfer between two fluorophores that reports on their intermolecular distance. It operates on the 2–10 nm range. In smFRET studies a ‘donor’ fluorophore is excited by a laser, and depending on their proximity, transfers energy to a second ‘acceptor’ fluorophore. The excited acceptor then emits this energy as fluorescence. The donor and acceptor can be attached to the same or to separate molecules, and the energy transfer signature reports on the separation of the dyes. Any changes to this separation are detected as they happen, with millisecond resolution in time.

smFRET can measure intramolecular distances in a single protein or nucleic acid, or the specific interaction between subunits in a protein complex, in real time. Applications might include the effect of drugs on the conformational dynamics of enzyme binding sites, or the study of protein aggregation in neurodegenerative diseases. smFRET can be used to infer binding constants, reaction pathways and dwell time distributions at the stochastic single‐molecule level, not obscured by ensemble averaging as in techniques such as fluorimetry or calorimetry.

Raw Single‐Molecule Data from DNA Holliday Junctions Experiment
Raw Single‐Molecule Data from DNA Holliday Junctions Experiment

The image above shows raw single‐molecule data from a DNA Holliday junctions experiment. Holliday junctions are involved in homologous recombination and lead to genetic variation. In this experiment, individual Holliday junctions were labeled at two sites, and in the presence of magnesium ions they stochastically and dynamically changed conformation, leading to a change in the separation of the two fluorophores. This dynamic transition was detected by smFRET, and the FRET traces and associated intensity traces were plotted in real time using the Nanoimager software suite.

FRET Traces and Intensity Traces
FRET Traces and Intensity Traces from DNA Holliday Junctions Experiment

Key features of the Nanoimager that highlight its suitability for smFRET include:

  • Simultaneous dual‐color imaging
  • Real‐time analysis of single-molecule intensity traces and population averages
  • High‐throughput imaging of thousands of single molecules per field of view
  • The ability to perform alternating laser excitation (ALEX) smFRET to determine stoichiometry as well as FRET efficiency in the molecules of interest

ALEX extends the potential of smFRET for understanding single molecules. The impact of photophysics and photobleaching on the fluorescence signal from a single molecule becomes fully characterized and decoupled from the FRET measurement. The stoichiometry and labeling properties of individual complexes or molecules are reported, allowing reaction kinetics to be determined even the absence of a FRET signal.