Single molecule techniques comprise a set of experimental methods that detect, manipulate, and analyze individual biomolecules or molecular complexes, circumventing ensemble averaging and revealing heterogeneity in structure, dynamics, and function. Core modalities include single-molecule fluorescence (e.g., smFRET, TIRF microscopy), optical and magnetic tweezers, atomic force microscopy, and nanopore-based sensing. These techniques enable direct measurement of forces, conformational changes, binding/unbinding events, and reaction pathways with nanometer spatial and millisecond (or better) temporal resolution. They are widely applied to study nucleic acid–protein interactions, molecular motors, enzyme kinetics, folding energy landscapes, and mechanotransduction at the level of individual molecules.
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