Single-molecule optical spectroscopy

Michel Orrit

Michel Orrit's scientific field is the interaction of light with organic molecules in condensed matter. He studied surface excitons in molecular crystals with Ph. Kottis in Bordeaux, then in dye-doped Langmuir–Blodgett films in Göttingen with H. Kuhn and D. Möbius during a post-doc stay. With J. Bernard in Bordeaux, he observed the first fluorescence signal from a single molecule in 1990. Orrit moved to Leiden in 2001, where his group applies single-molecule spectroscopy to molecular photophysics, solid-state dynamics, and nonlinear optics. His current interests include gold nanoparticles and molecules as nano-probes of structure and dynamics of soft condensed matter.

Taekjip Ha

In 1996, Taekjip Ha earned a doctorate in physics from the University of California, Berkeley. Prior to joining the University of Illinois at Urbana-Champaign in 2000, he was a postdoctoral fellow at Lawrence Berkeley National Laboratory and a postdoctoral research associate at Stanford University. He is a Gutgsell Professor of Physics and Biophysics and is an investigator with the Howard Hughes Medical Institute. Ha's research is focused on pushing the limits of single-molecule detection methods to study complex biological systems. His group develops state-of-the-art biophysical techniques and applies them to study diverse protein–nucleic acid and protein–protein complexes, and mechanical perturbation and response of these systems both in vitro and in vivo.

Vahid Sandoghdar

Vahid Sandoghdar obtained his BS in physics from the University of California at Davis in 1987 and PhD in physics from Yale University in 1993. After a postdoctoral stay at the Ecole Normale Supérieure in Paris he moved to the University of Konstanz in Germany in 1995, where he started a new line of research to investigate the interaction of light and matter at the nanometer scale, a field that is today known as Nano-Optics. In 2001 he accepted a chair at the Laboratory of Physical Chemistry at ETH in Zurich, Switzerland. In 2011 he became director at the newly founded Max Planck Institute for the Science of Light and Alexander-von-Humboldt Professor at the University of Erlangen-Nuremberg in Germany.

Since its inception more than 20 years ago, optical spectroscopy of single molecules has steadily expanded to an amazing variety of fields of natural science. While the largest expansion arguably took place in the field of molecular biophysics, domains as varied as quantum physics, nanoscience, heterogeneous catalysis, or soft-matter physical chemistry have all benefited from the new, averaging-free insights provided by single molecules. The technique itself has experienced a spectacular breakthrough with super-resolution microscopy, which allows imaging in real time at sub-wavelength scales, down to a dozen of nanometers. Other experimental advances such as ultrafast single-molecule experiments, spectroscopy of individual plasmonic nanoparticles and applications to other new materials continuously open new fields in nanospectroscopy.

The aim of this themed issue is to sample a number of recent conceptual and methodic inroads as well as applications in single-molecule science. The issue combines tutorial reviews with review articles to illustrate the power and versatility of single-molecule optical techniques.

Single molecules can help clarify long-standing problems in physical chemistry, such as the molecular origin of heterogeneous glassy dynamics in supercooled liquids. In the past decade, local probing of polymers and glass formers by single molecules has started to remove ensemble averaging and directly revealed the heterogeneity of glass formers in space and time. In their tutorial review, Paeng and Kaufman (DOI: 10.1039/C3CS60186B) describe rotational diffusion measurements on dye probes dispersed in glass formers, stressing the experimental precautions for reliable measurements and the possible pitfalls in the interpretation of the results. In particular, the choice of the probe and its chemical nature appear to have great consequences on the measured dynamics.

Optical observations of single molecules require repeated excitation by intense laser sources. Therefore, photophysics and photochemistry are central to this technique. This subject is approached in the review by Kozankiewicz and Orrit (DOI: 10.1039/C3CS60165J) under the angle of low-temperature spectroscopy. Concentrating on observations of single molecules embedded in solid matrices, they stress intermolecular charge transfer as a basic mechanism in many blinking processes, in addition to the well-known intersystem crossing transitions. Charge hopping in the vicinity of probe molecules also gives rise to spectral diffusion, together with a broad range of molecular movements of flexible and mobile groups. Methyl groups are particularly active in broadening single-molecule lines down to the lowest temperatures.

The most important shortcoming of single-molecule detection has been the limited photostability of fluorescent molecules, leading to blinking and bleaching. The article by Blanchard et al. (DOI: 10.1039/C3CS60237K) considers some of the fundamental photophysical processes that are responsible for these phenomena and provides an informative and valuable review of strategies for fighting them. Recent developments of protective agents that can be added to solutions for quenching the triplet state have attracted a lot of attention. However, the most promising approach seems to be based on covalent conjugation of protective agents directly to the fluorophore of interest. The authors present the progress and remaining challenges of this novel strategy.

The photophysics and photochemistry of the most current dye molecules used for fluorescent labeling in biophysics and cell biochemistry are reviewed by Levitus and colleagues (DOI: 10.1039/C3CS60211G). Many processes can affect fluorescence signals, from isomerization to the population of dark states leading to blinking or bleaching. Two important classes of dyes are cyanines and rhodamines. A good understanding of the molecular photophysics and photochemistry of these dyes is crucial, not only to avoid interpretation pitfalls, but, even more appealingly, to exploit them in new super-resolution microscopy schemes.

Single-molecule localization-based super-resolution microscopy requires probes that can be switched on and off on demand such that at most one probe is fluorescently active in a diffraction-limited imaging volume. The review by Van de Linde and Sauer (DOI: 10.1039/C3CS60195A) elaborates on the application of reversible photochemical reactions to switching, and the uses of switches in various schemes for super-resolution imaging and single-molecule tracking. They examine the mechanisms of switching as well as the design, development, requirements and improvement of photochemical molecular switches. The role of redox reactions is emphasized. Photochemical reactions can lead to activatable, convertible, and fully controllable switching.

The review by Nienhaus and Nienhaus (DOI: 10.1039/C3CS60171D) focuses on photoactivatable or switchable fluorescent proteins, which are highly desirable for super-resolution applications because they can be genetically encoded and used for live cell imaging. Nienhaus and Nienhaus report recent progress in engineering such fluorescent proteins with emphasis on the optimization of their photophysical properties required for various modes of super-resolution microscopy.

Another important and difficult problem into which single molecules have provided new insight is heterogeneous catalysis and enzymatic reactions. The review by Chen and colleagues (DOI: 10.1039/C3CS60215J) focuses on heterogeneous catalysis by metal nanoparticles, mostly gold and platinum, investigated via the single molecules produced in fluorogenic reactions. In the time domain, activity fluctuations can be understood with a thermodynamic model and assigned to dynamical surface restructuration events. Those events are more frequent for the softer metal gold than for platinum. The activity also varies spatially, not only according to the crystal facet studied, but also along gradients inside facets. The authors show how fluorescent signals can become models for more general reactions and detection methods such as surface-enhanced Raman scattering.

The tutorial review by Hofkens and colleagues (DOI: 10.1039/C3CS60245A) discusses single-molecule investigations of catalytic activities, in the biochemical realm as well as in the inorganic realm of heterogeneous catalysis on solid surfaces. Heterogeneous catalysis can be followed by single-molecule fluorescence in various materials such as porous silica, zeolites, metal–organic frameworks, or gold nanoparticles. Single molecule measurements have revealed that enzymes are much more complex than the standard textbooks lead us to believe. The experiments discussed here on lipase enzymes reveal “lazy and busy” periods in enzymatic reactions. This feature is very general, as it is found again and again in a broad variety of enzymatic reactions.

More than 15 years ago, pioneering experiments have shown that single enzymes can have a personality which can also change over time, generally referred to as static and dynamic disorder, respectively. Lu (DOI: 10.1039/C3CS60191A) shows that ingenious single molecule approaches can be designed to extract rich sets of information from fluorescence intensities, lifetimes and energy transfer in order to dissect complex multiple enzymatic reactions. He also paints an exciting future where single-molecule fluorescence spectroscopy is combined with active manipulation of single enzymes via external means.

One of the most powerful methods to study biomolecular dynamics and interactions is fluorescence resonance energy transfer (FRET). Peterman and Prevo (DOI: 10.1039/C3CS60292C) introduce the physical basis of FRET, and show how flexible and powerful this technology can be by applying it to the motor protein kinesin as an example. Site-specific labeling of different parts of a biological molecule or a molecular complex allows us to infer localized conformational changes and movements of interest, and in the case of kinesin, helped researchers gain deep insight into how several chemical reaction steps of ATP hydrolysis are coupled to the mechanical work done by the kinesin while it is walking on its molecular track.

Single molecule FRET (smFRET) can measure conformational changes and intermolecular interactions during biochemical reactions in real time. However, it mainly reports on only one quantity, the distance between the donor and acceptor fluorophores, whereas many physiologically relevant reactions have too high a complexity to dissect by just recording one quantity over time. Hohng and colleagues (DOI: 10.1039/C3CS60184F) review recent progress in extending smFRET to three and four colors so that multiple coordinates of single molecules or single complexes can be followed clearly. They also discuss combination of smFRET with optical and magnetic tweezers so that single molecular response to external forces and torque can be precisely monitored.

FRET is also a powerful probe of near-field interactions between two fluorophores. However, a quantitative analysis of FRET is usually complicated and requires additional information. The article by Hohlbein and colleagues (DOI: 10.1039/C3CS60233H) reviews the basic principles of FRET and a strategy called alternating-laser excitation (ALEX), in which the donor–acceptor unit is alternately excited at wavelengths corresponding to the absorption of the donor and acceptor. The authors present a detailed description of this method and discuss several interesting biophysical applications where ALEX has been successfully employed. They also project on the future development of this technique, especially in combination with protein-induced fluorescence enhancement and camera-based stroboscopic measurements.

Protein folding has been studied at the single-molecule level for almost fifteen years using either fluorescence or mechanical signals. Deniz and Banerjee (DOI: 10.1039/C3CS60311C) review the exciting discoveries enabled by single molecule FRET and by fluorescence correlation spectroscopy regarding the thermodynamics and kinetics of protein folding in isolation, and mediated by chaperone proteins. The focus is increasingly shifting to intrinsically disordered proteins which were shown to have novel intermediate states and to their assemblies and aggregates with strong implications for human health.

Many biochemical reactions require multiple components that are highly orchestrated regarding their timing in participation. For example, two or more different proteins may come and act on a single RNA molecule to carry out ‘splicing’ reactions that remove specific portions of RNA and connect the remaining pieces together. Using multi-color single molecule fluorescence microscopy to observe differently labeled proteins on the same DNA or RNA molecule can reveal their relative order of binding and dissociation as well as their stoichiometry as Hoskins and colleagues (DOI: 10.1039/C3CS60208G) summarize. They also discuss various tools at our disposal regarding protein labeling and for obtaining information-rich signals from transcription, splicing and translation.

A good example of biochemical processes of bewildering complexity requiring multiple components is DNA replication. Researchers have labored in the last fifteen years to bring the clarity and precision of single molecule fluorescence spectroscopy and mechanical manipulation to the field of DNA replication. Van Oijen and Stratmann (DOI: 10.1039/C3CS60391A) provide a survey of the literature showing key examples of technical innovations and scientific discoveries thus enabled.

Although single molecule fluorescence imaging, in particular FRET, has been used to reveal intricate details of biochemical reactions, many studies require specific labeling of proteins which is cost- and time-intensive, and is not scalable to proteome-wide analysis. Myong and Hwang (DOI: 10.1039/C3CS60201J) review an alternative method called protein-induced fluorescence enhancement (PIFE) which does not require protein labeling but yet is effective in revealing the binding and dissociation of protein on single nucleic acids and also led to several interesting discoveries on protein dynamics on nucleic acids such as repetitive translocation, free sliding, and stepwise filament formation and disassembly.

Single molecules, being point-like dipoles, can probe near-field optical fields locally. In particular, recent research has shown that optical absorption and emission can be massively enhanced in the vicinity of metallic structures. This new field of plasmonics or nano-optics has opened new vistas for single-molecule measurements.

Although most of the research in single-molecule detection has concerned fluorescence, many groups have worked to extend this ultimate sensitivity to Raman spectroscopy. The small Raman cross sections, however, make this task more than twelve orders of magnitude more difficult than fluorescence detection. The article by Van Duyne and co-workers (DOI: 10.1039/C3CS60187K) provides an overview of the challenges and recent developments for enhancing the Raman signal. In particular, they discuss the use of plasmonic particles (or antennas) and metallic tips for detecting both resonant and nonresonant Raman transitions at the single-molecule level.

Mauser and Hartschuh (DOI: 10.1039/C3CS60258C) present a thorough discussion of the research on the application of apertureless near-field optical microscopy. In this approach a tip is used as a nano-antenna to create and mediate a confined optical field near a molecule or sample. The authors provide a concise theoretical treatment of optical nano-antennas, practical requirements and microscope designs, as well as a thorough review of the recent literature on tip-enhanced Raman, fluorescence, photocurrent and electroluminescence microscopies.

The review by Meixner’s group (DOI: 10.1039/C3CS60357A) presents a general approach of electromagnetic enhancement in optical cavities, such as standard Fabry–Pérot interferometers, and in the vicinity of plasmonic structures. They discuss the influences of fluorescence quantum yield and of saturation on the observed enhancements. Their analysis applies to enhanced Raman scattering as well as to enhanced fluorescence. Several examples illustrate this treatment, from light-harvesting complexes and tip-enhanced spectroscopy on carbon nanotubes and inorganic emitters, to gold nanoparticles themselves considered as emitters.

The tutorial review by Tinnefeld et al. (DOI: 10.1039/C3CS60207A) considers the issue of concentration in single-molecule detection and examines the challenges that have prevented the more widespread use of single-molecule methods in industry. Plasmonics provides a possible solution to this problem. In particular, Tinnefeld and colleagues scrutinize applications where very high concentrations, e.g. where chemical reactions take place, and very low concentrations, e.g. in high-end biosensing, are at play. Methods based on spatial and temporal separation of analyte molecules, concentration amplification, confinement of the electromagnetic field and many especially-conceived experimental designs are reported in various contexts.

Finally, organic molecules are not the only possible optical probes producing large fluorescence or photoluminescence signals. Semiconductor nanocrystals, especially those of the II–VI family, are models of artificial atoms and have been proposed for many applications in quantum optics, labeling, and light detection and harvesting. As these quantum dots (QDs) have proven extremely bright and photostable, they have been an important component in the material toolbox of single-molecule optics. Over the past two decades, scientists have explored various features of the photophysics in these systems and pursued various synthesis strategies to improve them.

The extensive and readable overview by Bawendi and co-workers (DOI: 10.1039/C3CS60330J) describes the developments that have led to our current understanding of the origins of blinking. In particular, the authors present photon-correlation Fourier spectroscopy as an alternative method to time binning for analyzing the temporal behavior of single-particle emission such as spectral linewidths and diffusion.

Lounis and colleagues (DOI: 10.1039/C3CS60209E) present an in-depth review of the spectroscopy of QDs and of the wealth of new knowledge provided by single QD measurements. After summarizing the basic theoretical description of these systems, they discuss a number of spectroscopic properties, including blinking, spectral diffusion, mostly about the most studied material CdSe, for which extensive data are available. The most complete picture arises from spectroscopy at cryogenic temperatures, for which band edge excitons, multi-excitons and charged excitons can all be assigned and understood.

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