Photonics Dictionary

single-molecule FRET

Single-molecule Förster resonance energy transfer (smFRET) is a specialized technique in biophysics and molecular biology used to study the distances and dynamic interactions between two fluorophores (molecules that fluoresce) at the single-molecule level. FRET is based on the transfer of energy between a donor fluorophore and an acceptor fluorophore when they are in close proximity.

Here is a breakdown of the key components and principles of smFRET:

Förster resonance energy transfer (FRET): FRET is a non-radiative energy transfer process that occurs between a donor fluorophore and an acceptor fluorophore. It relies on the dipole-dipole interaction between the excited state of the donor fluorophore and the ground state of the acceptor fluorophore. FRET is highly sensitive to the distance between the two fluorophores and is commonly used to study molecular interactions and conformational changes.

Single-molecule techniques: Unlike traditional ensemble methods that provide average measurements for a population of molecules, smFRET allows researchers to study individual molecules in real time. This is particularly valuable when dealing with heterogeneity within a sample, as it enables the observation of molecular behaviors that might be masked in ensemble averages.

Experimental setup: In a smFRET experiment, a biomolecule of interest (e.g., a protein, DNA, or RNA) is labeled with donor and acceptor fluorophores. The labeled molecule is then immobilized or tracked as it undergoes dynamic processes. Laser excitation is used to initiate fluorescence, and the emitted photons are detected. Changes in FRET efficiency provide information about the distance and relative orientation between the donor and acceptor fluorophores.

Data analysis: The collected data are analyzed to extract information about the molecular structure, conformational changes, or interactions occurring at the single-molecule level. This analysis often involves calculating FRET efficiency, which is a measure of how efficiently energy is transferred between the donor and acceptor fluorophores.

Single-molecule FRET has been instrumental in studying a variety of biological processes, including protein folding, DNA and RNA dynamics, molecular binding events, and conformational changes in biomolecules. Its ability to provide insights into the heterogeneity and dynamics of individual molecules makes it a powerful tool in the field of molecular and cellular biology.

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