Single-molecule FRET

Single molecule fluorescence 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 on 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. Single molecule fluorescence 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 on 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. 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 signal 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. Fluorescence signal is detected either using ultra sensitive CCD or scientific CMOS cameras for wide field microscopy or SPADs for confocal microscopy. Once the single molecule intensities vs. time are available the FRET efficiency can be computed for each FRET pair as a function of time and thereby it is possible to follow kinetic events on the single molecule scale and to build FRET histograms showing the distribution of states in each molecule. However, data from many FRET pairs must be recorded and combined in order to obtain general information about a sample or a dynamic structure. In surface-immobilized experiments, biomolecules labeled with fluorescent tags are bound to the surface of the coverglass and images of fluorescence are acquired (typically by a CCD or scientific CMOS cameras). Data collection with cameras will produce movies of the specimen which must be processed to derive the single molecule intensities with time. An advantage of surface-immobilized experiments is that many molecules can be observed in parallel for an extended period of time until photobleaching(typically 1-30 s). This allows to conveniently study transitions taking place on slow time scales. A disadvantage is represented by the additional biochemical modificationsneeded to link molecules to the surface and the perturbations that the surface can potentially exert on the molecular activity. In addition, the maximum time resolution of single-molecule intensities is limited by the camera acquisition time (> 1 ms). SmFRET can also be used to study the conformations of molecules freely diffusing in a liquid sample. In freely-diffusing smFRET experiments (or diffusion-based smFRET), the same biomolecules are free to diffuse in solution while being excited by a small excitation volume (usually a diffraction-limited spot). Bursts of photons due a single-molecule crossing the excitation spot are acquired with SPAD detectors. The confocal spot is usually fixed in a given position (no scanning happens, and no image is acquired). Instead, the fluorescence photons emitted by individual molecules crossing the excitation volume are recorded and accumulated in order to build a distribution of different populations present in the sample. Depending on the complexity of this distribution, acquisition times varies from ~5 min to several hours.