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.

Keywords: single‐molecule fluorescence spectroscopy; protein conformation; protein–protein interactions; protein dynamics; live‐cell experiments; molecular heterogeneities; single‐pair Förster resonance energy transfer; fluorescence correlation spectroscopy; single‐molecule/particle tracking

Figure 1. Wide‐field and point‐scanning fluorescence microscopes provide different information about a sample. (a) Illumination and detection scheme for wide‐field fluorescence microscopy. Excitation light (green) is focussed onto the back focal plane (BFP) on the optical axis of the objective. A collimated excitation light beam enters the sample and excites fluorophores at different distances from the coverslip. Detection of emitted light (red) occurs from the focal plane. (b) Illumination and detection scheme for TIRFM. The excitation light beam is focussed on the BFP of the objective far away from the optical axis of the objective resulting in total internal reflection at the coverslip–sample interface. Fluorophores in the sample are excited by an evanescent wave close to the interface. Emitted light is detected from the focal plane close to the coverslip. (c) Illumination and detection scheme for point‐scanning confocal microscopy. The excitation light is focussed onto a point within the sample by the objective. Light emitted within the focal plane is collected by the objective and passed towards a point detector. Light from outside the focal plane is rejected by a pinhole inserted in the light path behind the objective (not shown). (d) Performance comparison of important imaging parameters (arrow heads) and accessible sample parameters (boxes). Green: better performance, red: worse performance.
Figure 2. Size comparison of fluorescent labels commonly applied in single‐molecule fluorescence microscopy. To scale sizes of a fluorescent protein (EGFP), SNAP‐tag protein (crystal structure of hAGT – human alkylguanine‐DNA alkyltransferase – is shown), a synthetic fluorophore (tetramethylrhodamine – TMR), a semiconductor quantum dot (Qdot) with streptavidin coating and an IgG‐type antibody. Note that the size of the synthetic fluorophore is enlarged by a factor of 10 for illustration purposes. Only one quarter of the Qdot is shown for illustrative purposes. For EGFP, SNAP‐tag, streptavidin and antibody renderings from crystal structures are shown. PDB accession numbers for crystal structures: EGFP – 2Y0G, streptavidin – 3RY1, hAGT – 1EH6, IgG – 1HZH.
Figure 3. Surface immobilisation and passivation strategies for observing individual molecules over extended time spans. (a) Surface passivation can be achieved by coating the glass surface with BSA protein (green). Specific attachment of target molecules is achieved by addition of low concentrations of biotinylated BSA exploiting the tight biotin – Streptavidin (blue/yellow) linkage. Biotinylated target molecules carrying a fluorophore bind to surface‐bound Streptavidin. (b) BSA can be replaced by the organic polymer PEG which effectively passivates surfaces against many types of biomolecules. Specific immobilisation of substrate molecules (dark blue) is achieved by deposition of substrate‐specific antibodies within the PEG‐surface. (c) Covalent immobilisation of substrate molecules can be achieved using maleimid‐functionalised PEG surfaces which can form a covalent bond with thiol moieties in the substrate molecule. (d) Spatial confinement of nonfunctionalised target molecules can be achieved by incorporation into surface‐tethered lipid vesicles.
Figure 4. Single‐molecule light microscopy techniques provide a range of different readouts and measurable parameters. (a) The number of fluorescent labels in a labelled protein complex can be determined by observing individual fluorophore bleaching events. A simulated intensity trace of a trimeric protein complex with initially three fluorescent labels (green) is shown. (b) Changes in the proximity of two fluorescently labelled moieties within a biomolecule or a biomolecular complex can be measured by single‐pair (ALEX) FRET. A 2D FRET efficiency – stoichiometry plot with corresponding FRET efficiency and stoichiometry histograms is shown (left). Simulated donor (green) and acceptor (red) fluorophore intensity traces for a protein complex switching between a no FRET and a high FRET state are shown (right). (c) Tracking the position of a particle in space over time yields information about the mobility of the particle and its interactions with surrounding structures (left). The statistical analysis of the mean squared displacement at different time intervals yields insight into the type of motion a particle exhibits (right). Colour coding of the trajectory from red (start) to blue (end) of particle observation time. (d) Autocorrelation analysis of intensity fluctuations in a intensity time trace can be performed to obtain information about particle numbers and mobilities. Autocorrelation curves (grey) and corresponding fits for a fast species at low concentration (red fit) and a slow species at high concentration (green fit) are shown. Inset: Exemplary intensity time trace from an FCS experiment.
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Further Reading

Liu Z , Lavis LD and Betzig E (2015) Imaging live‐cell dynamics and structure at the single‐molecule level. Molecular Cell 58: 644–659.

Selvin PR and Ha T (2007) Single‐Molecule Techniques: A Laboratory Manual, 1st edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. ISBN: 978-0879697754.

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How to Cite close
Yserentant, Klaus, and Herten, Dirk‐Peter(Mar 2016) Single‐Molecule Light Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002997.pub3]