Single‐Molecule Light Microscopy

Abstract The complexity of biological processes requires experimental techniques which are able to resolve events on appropriate temporal and spatial scales. As all biological processes are ultimately driven by the dynamics and interactions of individual molecules, studies on the single‐molecule level provide important insights about a large variety of parameters at thermodynamic equilibrium and without ensemble averaging. In the life sciences, single‐molecule experiments are preferentially performed using fluorescence light microscopy owing to its high sensitivity, its temporal resolution and its ability to address live and thus dynamic specimen. By today, a range of single‐molecule techniques such as single‐pair Förster resonance energy transfer ( FRET ), single‐molecule tracking and different counting techniques are readily available to characterise molecular interactions, conformational dynamics, complex stoichiometries and translational mobilities in biological systems both in vitro and in situ . Key Concepts Single‐molecule analysis of biological processes allows to observe the behaviour of individual molecules rather than ensemble averages. Owing to its sensitivity, fluorescence microscopy is the dominating single‐molecule technique used in the life sciences. Detection of individual fluorescently labelled biomolecules requires dilution of the sample and spatial restriction of the observation volume. A variety of single‐molecule fluorescence microscopy techniques is now being routinely applied in many laboratories. The recent introduction of single‐molecule DNA sequencers illustrates the commercial impact of single‐molecule techniques in the life sciences. The absolute stoichiometry of individual macromolecular complexes can be determined by different counting methods such as photobleaching step analysis. Single‐pair Förster resonance energy transfer (spFRET) can be used to measure molecular interactions and intramolecular distances at the nanometre level. Tracking of individual molecules in their native context helps understanding how their behaviour is influenced by interactions with other molecules and by cellular processes. Among other things, fluorescence correlation spectroscopy (FCS) can be used to investigate the diffusion of individual molecular species and the interaction of multiple species with each other.