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Single Molecule Fluorescence Resonance Energy Transfer

=Methodology= Single molecule FRET measurements are typically performed on fluorescence microscopes, either using surface-immobilized or freely-diffusing molecules. 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). In freely-diffusing experiments, the same biomolecules are free to diffuse in solution while being excited by a small excitation volume (usually a diffraction-limited spot). In freely-diffusing experiments, bursts of photons due a single-molecule crossing the excitation spot are acquired with SPAD detectors. 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 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. Conversely, 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 the sample in order to generate an image. 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.[1] In freely-diffusing smFRET, the confocal spot is kept at a fixed 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. For surface-immobilized experiments, data collection with cameras will produce movies of the specimen which must be processed to derive the single molecule intensities with time. Conversely, measurements employing SPADs can acquire photon timestamps and therefore more directly yield timetraces of intensity vs. time. 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.[2]

=Applications=