Expanding single-molecule fluorescence spectroscopy to capture complexity in biology

Abstract

Fundamental biological processes are driven by diverse molecular machineries. In recent years, single-molecule fluorescence spectroscopy has matured as a unique tool in biology to study how structural dynamics of molecular complexes drive various biochemical reactions. In this review, we highlight underlying developments in single-molecule fluorescence methods that enable deep biological investigations. Recent progress in these methods points toward increasing complexity of measurements to capture biological processes in a living cell, where multiple processes often occur simultaneously and are mechanistically coupled.

Figure 1

Different modes of single-molecule fluorescence spectroscopy. Fluorescent signals from molecules tethered to a surface are recorded in real time. The number of simultaneously monitored molecules are limited by the size of the camera chip. Structural dynamics within the molecular complex can be probed using smFRET, where Förster energy transfer between two fluorescent probes report their distance, typically in 20–80 Å range. Discrete quantized steps in fluorescent intensity correspond to changes in the distance between the fluorescent donor and acceptor dye molecules. Compositional dynamics can be monitored as colocalization of different molecules within the same complex, a technique referred to as colocalization single-molecule spectroscopy (CoSMoS). High background caused by direct excitation of diffusing fluorophores can be suppressed using zero-mode waveguides (ZMWs), which limit the excitation volume within the nano-fabricated structures.

Figure 2

Reconstitution of physiological conditions in single-molecule fluorescence experiments. Interaction between the molecule of interest and cellular components is reproduced via: (a) embedding molecules such as a membrane protein into a local environment that mimics the lipid bilayer (e.g. a nanodisc), (b) supplementing all biological factors needed for the mechanism studied, such as the addition of a cell lysate, and (c) fluorescent labeling of molecules within living cells and observing them in vivo. Figure of bilayer and membrane protein adapted from [20].

Figure 3

Combining single-molecule fluorescence measurements with other spectroscopic or microscopic methods. (a) Fluorescence spectroscopy can be combined with a force spectroscopy method, such as rotor bead tracking shown here (figure adapted from [51••]). One end of the complex is tethered to the magnetic bead to either induce mechanical force into the system or confine the molecular movements within the convenient axis, which are detected as scattering light from the rotor bead in addition to the fluorescence signal. (b) Fluorescence tagging of a molecular complex can be used with electron cryotomography in tandem, where the fluorescence imaging of a frozen cell reveals broad localization of specific molecules, providing additional information for the electron tomography.