It was 28 years ago that the first optical experiments to detect a single molecule took place in the Moerner Laboratory at IBM Research, with relatively esoteric, low-temperature, high-resolution spectral measurements. Since then, room-temperature demonstrations and research efforts of many scientists all over the world have led to a true explosion of the method. New applications and different ways to characterize individual molecules appear almost on a weekly basis. “Single-molecule optical spectroscopy now has broad applications, ranging from basic science to shining light on the fundamental properties of single molecules, all the way over to more applied applications in biology and materials science,” said William Moerner, now a professor of chemistry at Stanford University. “While the first single-molecule experiments were performed on fluorescent impurities in solids, the power of the method quickly became apparent in biological settings.” A photo of actin filaments in diffraction-limited (background) and in superresolution (inset) mode, taken on the Nanoimager. Courtesy of Achillefs Kapanidis and Bo Jing, Department of Physics, University of Oxford. The potency of single-molecule techniques is their capability to attain the ultimate limit of sensitivity — a single emitter. No averaging over a large number of molecules is required, since the properties of just one molecule are measured at a time. The main purpose of microscopy is to observe detail that cannot be seen by the naked eye, but spectroscopy is used to understand how matter responds to light-revealing spectral information; however, both methods can cooperate effectively with each other. The major advance in the past decade has combined single-molecule optical imaging with the ability to control the fraction of the labeled molecules that are actually emitting to overcome the diffraction limit in microscopy. If it is known that the detected signal on the camera originates from a single emitter, its position can be determined with precision, a process called high-precision localization. By recording the positions of many superlocalized single emitters discharging at different times, a complete structure with very fine detail can be reconstructed in a pointillistic fashion. This is the basis of superresolved fluorescence microscopy, for which Eric Betzig, Stefan Hell and Moerner were recognized with the Nobel Prize in chemistry in 2014. But Hell used an alternative approach. Superresolution fluorescence microscopy coupled with single-molecule tracking has already entered the medical/clinical world. In cardiovascular research, tracking enables study of the pathways of individual viruses and the anchoring of proteins. This research is fundamental for analyzing certain neurodegenerative diseases, such as Alzheimer’s, where the polymerization of amyloid plaques and their structures need to be understood on a molecular level. Images taken with a Carl Zeiss ELYRA PS.1 superresolution platform using a 100×/1.46 oil objective. Individual frames from the original time series, in which the emission of individual molecules are seen as diffraction-limited patterns. The molecules switch between emissive and nonemissive states and the patterns are perceived to be “blinking” (top row). Reconstructed localization microscopy image revealing details on a scale below 30 nm (bottom row, left). The same area using conventional fluorescence imaging with conventional resolution of the order of 300 nm (bottom row, right). Courtesy of Niwa and N. Hirokawa, Tokyo University, Japan. “In my opinion, single-molecule spectroscopy is one of the finest detail-level tools that can be applied to life sciences or in fluorescence microscopy,” said Christian Hellriegel, specialist for superresolution techniques at Carl Zeiss Microscopy GmbH in Jena, Germany. He notes a recent trend in the younger generation of researchers taking a chance with single-molecule-based techniques thanks to the availability of more sensitive instruments. “This was not the case, say, 20 years ago when the instruments themselves needed substantial modifications in order to be single-molecule sensitive,” he said. In Berlin, for example, researchers at the optoelectronics development company PicoQuant GmbH are focusing on improving and extending the range of single-molecule detection techniques. PicoQuant researchers are also investigating challenging applications of the techniques that range from chemical analysis to biophysics, biological and biomedical research, medical diagnostics and even materials research. “Over the last [few] years, single-molecule spectroscopy and microscopy have become established tools in a wide range of scientific fields,” said Uwe Ortmann, head of sales and marketing and specialist for spectroscopy and microscopy at PicoQuant. “I would expect the method to become also established in materials science, where the analysis of luminescence behavior of nanoparticles (quantum dots) plays an important role in display technologies or for diagnostic purposes.” Using nanoparticles as luminescent probes in biological or biomedical applications could provide several advantages over organic molecules — namely, photo-stability, tunable surface and upconversion properties. And they are often not very sensitive to quenching by oxygen. Unlike organic molecules, nanoparticles are not usually bleached by repeated or intense exposure to light. This helps greatly when performing imaging, as there is no risk of losing the probe’s signal when scanning repeatedly or for longer time periods, which is often needed to obtain good signal-to-noise ratios. The goal of any powerful diagnostic tool is the early detection of a biological species at low concentration, which can indicate the presence of a certain disease. Early detection is critical for early treatment and better patient health. As scientists grapple with this challenge, many believe single-molecule methods will be the key technique thanks to their extreme detection sensitivity. Although there is still a long way to go to improve selectivity and reduce interfering backgrounds, experts are convinced that single-molecule techniques, combined with other new ways to enrich the signals of interest, will further develop and provide the framework to tackle these demanding problems. Another exciting avenue of interest is an improved understanding of protein localization and transport in cells. Professor Bianxiao Cui, who leads the Cui Laboratory at Stanford University, is currently taking this a step further by using single-molecule imaging to study the long-distance vesicle transport in neurons. “Single-molecule optical spectroscopy is very useful in dissecting out protein-protein, protein-DNA interaction and super localization,” Cui said. “In recent years, it has been applied to more complex biological systems.” Work at the lab of Cui’s Stanford colleague Moerner is one example of the use of SMS for the study of complex systems. Currently, Moerner and postdoctoral student Leonhard Moeckl are using the technique to observe the structures formed in mammalian cancer cells with resolution down to a few tens of nanometers. Other researchers in the Moerner Lab are following the motions of signaling proteins in the primary cilium of mammalian cells, and they’re exploring the organization of proteins, DNA and RNA in the nucleus. Using a special experimental setup known as the anti-Brownian electrokinetic (ABEL) trap, for example, Allison Squires in the Moerner Lab is performing single-molecule optical spectroscopy over extended times in solution. By counteracting Brownian motion, she is able to disentangle the photophysics of single trimeric C-phycocyanin proteins. The deeper understanding of the light-harvesting process in cynobacteria may help with the design of artificial photosynthetic systems. Researchers at the Moerner Lab are also using SMS to investigate protein-DNA interactions. Probing how proteins are involved in DNA development reveals a better understanding of DNA code and how it applies to gene transcription. This will enable researchers to investigate the discrete steps necessary to turn on individual genes and examine how the process goes wrong in cancer and other diseases. Multicolor single-molecule fluorescence is also an excellent vehicle for developing new assays for DNA sequencing and pathogen detection. Such assays are facilitated by the use of highly automated and stable microscopes that are now available from all major microscope manufacturers and microscope startups. Adjustment of optical elements of a microscope setup in the Moerner Lab. Courtesy of William Moerner, Stanford University. One example is the Nanoimager, released in 2016 by an Oxford University spinout, Oxford Nanoimaging. The company was founded by professor Achillefs Kapanidis and Ph.D. student Bo Jing and arose out of their biophysics research on protein-DNA interactions at the Clarendon Laboratory at Oxford University. The Nanoimager is a compact microscope module (the size of a shoebox) powered by a desktop-tower-size module comprising lasers and electronics. “[The Nanoimager] can be used on regular laboratory benches and office desks, allowing single-molecule fluorescence to ‘escape’ from physics laboratories and reach chemists and biologists in academia and industry,” Kapanidis said. He added that instruments such as the Nanoimager will open up SMS to new users and assay developers. This in turn should lead to a large array of potential applications in biotechnology and in the clinic. As experiments become easier to perform, new applications will emerge that were not previously possible or were thought to be too complex or too arduous to pursue. “More automation in sample preparation [and] higher throughput aided by both microfluidic structures and robust, automated data analysis should bring the field to the next level,” Kapanidis said. “This may enable the type of step change achieved by sequencing companies when they moved from first- to second-generation sequencing that has really revolutionized not only genomics, but many fields including molecular biology and diagnostics.” The Why and How of Single-Molecule Spectroscopy As its name suggests, single-molecule spectroscopy (SMS) looks at the behavior and properties of individual molecules as opposed to the average value obtained from trillions of molecules. In contrast to bulk techniques, which measure a certain parameter averaged over many signals originating from single emitters, single-molecule techniques look at each emitter one at a time. Only with a single-molecule measurement can subtle features of the system be detected, such as events that occur with lower frequency that would otherwise be “hidden” in the data set. Particular factors that stem from subpopulations or local variations, for example, would also otherwise be lost in the averaged view. “All of these single observations pooled together can, of course, reconstitute the bulk response of the system, but the other way around is not possible — one cannot infer the underlying single-molecule response from a bulk measurement,” explained Stanford’s William Moerner, the Nobel Prize-winning pioneer in single-molecule imaging. “When single molecules are used for superresolution microscopy, nanoscale structures can be observed, which were not visible optically before.” Compared with bulk measurements, signals of single molecules are rather weak, which means highly sensitive detectors must be used. This sensitivity, however, introduces a problem: It not only picks up the signal from the species under investigation, but it is also affected by any contaminating background emission from sample imperfections. “The first step in any single-molecule experiment is hence to prepare the sample as cleanly as possible to exclude impurities,” Moerner said. “The actual single-molecule measurement then consists of exciting the single molecule, typically by a laser, followed by detection of the emitted signal.” A simple setup needs a single-pulsed or continuous laser and a detector capable of finding the photons emitted from a single molecule. More sophisticated experiments may require sophisticated cameras or several fast detectors and a highly stable piezo scanning system. The main applications derive from one important aspect: Mechanistic details become visible and quantifiable. These include the motion of individual molecules in cells, between cells or organelles, or within complex fluids. As a result, SMS reveals subpopulations of behaviors, and the individual molecule can relay information from its immediate nanoscopic environment — for example, the way the environment interacts with this molecule or the presence of another molecule with which it can interact. “In life sciences, a lot of attention is being devoted to molecule-molecule interactions, as this is the basis of all cellular machinery, fundamental to understand how cells, drugs, genes or proteins interact with each other,” said Christian Hellriegel of Carl Zeiss Microscopy.