Viewpoint: Single Molecules at 31: What’s Next?

Abstract

Optical studies of single molecules have taught us much over the past three decades, but these individual quantum-mechanical objects continue to have promise as probes of nanoscale structure and dynamics in complex systems.

In the more than three decades since the first optical detection and spectroscopy of a single molecule in a condensed phase host in 1989 at low temperature¹ and in 1990 at room temperature,² a wide array of scientific developments have occurred. There is something particularly fascinating about pumping just one molecule at a time with light, and then detecting the signal from that molecule. The single molecule itself is typically 1–3 nm in size, so learning what this molecule is doing by detecting the fluorescence it emits provides an admittedly indirect but useful route to information at the nanoscale. Complex hosts such as cells, polymers, or functional materials feature heterogeneity in local environments, and single molecules such as enzymes or photosynthetic antennas can be in a diversity of chemical or photophysical states, so the removal of ensemble averaging is a wonderful approach to observe molecular mechanisms in deeper detail.³ This Viewpoint highlights some recent focus areas and speculates what might happen in future work.

The number of interesting physical processes that have been explored using single-molecule optical studies is quite astounding. Measurements of emission brightness (emitted photons per second), emission spectrum, excited state lifetimes, orientation of the molecule from polarization studies, and so on all can be used to infer what is happening with the molecule and its nanoenvironment.⁴ Similarly, sensing the degree of energy transfer (FRET, or Förster resonant energy transfer) between two fluorophores or between an emitter and quencher continues to be an excellent readout of molecular motions, orientations, and distances between the emitters on the 3–5 nm spatial scale.^5,6 These measurements can be performed as a function of time to sense changes (depending upon the photon rate to define the time scale), and they can all be performed on immobilized single molecules as well as ones trapped in solution to avoid surface perturbations.⁷ The position of the single molecule is another useful variable to measure because time-dependent trajectories from single-molecule tracking⁸ can lead to insight about diffusion, local viscosity, and the presence of non-Brownian processes on the one hand, or on the other can be used to infer hydrodynamic radius and/or the mobility of the single object.⁹ Even quantum-mechanical effects can be observed such as photon antibunching,¹⁰ single-photon emission,¹¹ Rabi splittings,¹² and others. The single molecule interacts with the local electromagnetic field, and thus, can sense alterations in this field produced by metallic nanoantennas,¹³ or its scattering can be used to develop ways to detect the molecule without fluorescence.¹⁴ All these approaches have been used for ultrasensitive digital detection of biomolecules for biomedical applications¹⁵ including genetic sequencing with single molecules¹⁶ and exploration of the fibril structures of amyloid diseases.¹⁷

Of course, single molecules can also be used to achieve super-resolution microscopy,¹⁸ where optical detail beyond the optical diffraction limit of ~250 nm is derived from using molecular switching or blinking to control the emitting concentration at a low level during any imaging frame. Determining positions of single molecules as samples of locations on an extended structure then can lead to a reconstruction with extremely fine detail. This concept has been implemented in both 2D and 3D imaging,¹⁹ leading to new insights for cell biology from bacteria²⁰ to eukaryotes,²¹ and a Perspective addressing this area has recently appeared.²²

Turning to the future, the author proceeds with some trepidation since as Yogi Berra has said, “It’s tough to make predictions, especially about the future,” and it is dangerous to predict that a particular experiment cannot be done! Given the amazing creativity of researchers worldwide, that prediction would simply be an invitation to be proven wrong. Certainly we can expect to see further expansion into other areas of chemistry such as catalysis or materials and polymers when proper assays are designed, whether they be fluorescence-based²³ or not.²⁴ Computational data analysis of single-molecule behaviors will continue to drive innovations in statistical analysis.²⁵ Combinations of single-molecule imaging with other high resolution microscopies will provide useful ways to offset the advantages and disadvantages of complementary methods: an example would be using single-molecule positions from optical microscopy to annotate the locations of specific biomolecules in a cryo-electron tomography image of a cell.²⁶ New emitting molecules with enhanced emissive properties will certainly lead to more information, precision, and new readout variables,²⁷ and the DNA-PAINT binding-based detection strategy promises to expand.²⁸ Fascinating new insights can be expected when single molecules interact with novel electromagnetic structures such as resonators, metallic plasmonic nanoobjects,^29,30 or metamaterials. The ability of a single molecule in a solid to absorb light in a similar way to a Na atom in a low density gas will likely be used for quantum mechanical applications if the decoherence caused by decay through multiple vibrational states can be controlled. The possibilities are limited only by our imagination as to what can be learned by probing one individual nanoscale molecule, one at a time, by tickling it with light.