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Concept
Single-molecule FRET
Summary
Single-molecule fluorescence (or Förster) resonance energy transfer (or smFRET) is a biophysical technique used to measure distances at the 1-10 nanometer scale in single molecules, typically biomolecules. It is an application of FRET wherein a pair of donor and acceptor fluorophores are excited and detected at a single molecule level. In contrast to "ensemble FRET" which provides the FRET signal of a high number of molecules, single-molecule FRET is able to resolve the FRET signal of each individual molecule. The variation of the smFRET signal is useful to reveal kinetic information that an ensemble measurement cannot provide, especially when the system is under equilibrium with no ensemble/bulk signal change. Heterogeneity among different molecules can also be observed. This method has been applied in many measurements of intramolecular dynamics such as DNA/RNA/protein folding/unfolding and other conformational changes, and intermolecular dynamics such as reaction, binding, adsorption, and desorption that are particularly useful in chemical sensing, bioassays, and biosensing.
Single-molecule FRET measurements are typically performed on fluorescence microscopes, either using surface-immobilized or freely-diffusing molecules. Single FRET pairs are illuminated using intense light sources, typically lasers, in order to generate sufficient fluorescence signals to enable single-molecule detection. Wide-field multiphoton microscopy is typically combined with total internal reflection fluorescence microscope (TIRF). This selectively excites FRET pairs on the surface of the measurement chamber and rejects noise from the bulk of the sample. Alternatively, confocal microscopy minimizes background by focusing the fluorescence light onto a pinhole to reject out-of-focus light. The confocal volume has a diameter of around 220 nm, and therefore it must be scanned across if an image of the sample is needed. With confocal excitation, it is possible to measure much deeper into the sample than when using TIRF.
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