Single-molecule experiments have become an essential technique for understanding biomolecular structure and dynamics. By studying one molecule at a time, scientists can discover key details that are hidden in bulk experiments. In recent decades, advances in microscopy, spectroscopy, microfluidics, and nanotechnology have enabled researchers to probe ever more complex biological questions at the individual molecule level.
Early Foundations in Biophysics
The conceptual foundations for observing single molecules were laid in the early 20th century within the field of biophysics. Increasingly sensitive instruments were allowing scientists to measure tiny biological specimens and subtle molecular properties. This set the stage for the future possibility of isolation and examination of single molecules.
Einstein’s Theoretical Basis for Quantifying Small Numbers of Molecules
In 1905, Albert Einstein published a paper describing how to quantify small numbers of molecules by observing their random motion in liquid. This theoretical framework, known as Brownian motion, statistically relates the behavior of single molecules to measurements made on an ensemble of molecules. The concept gave scientists a tool for talking about single molecule dynamics decades before having instruments that could directly observe them.
Early Advances in Microscopy
The early 20th century saw substantial improvements to light microscopes, with better optics, illumination, contrast methods, and preparation techniques. This allowed visualization of smaller structures. For example, in 1873, a microscope was built with sufficient magnification and resolving power to see bacteria. These capabilities started to bring single cells and large organelles into view, paving the pathway toward imaging even smaller molecular complexes.
Ultracentrifugation Isolates Single Macromolecules
In the 1920s, Theodor Svedberg developed the analytical ultracentrifuge to study proteins and other macromolecules in solution. By spinning samples at very high forces, he could separate particles by size and density. This was used to get purified samples of single molecular species - an essential step toward analyzeing them individually.
Development of Single-Molecule Fluorescence Microscopy
The latter part of the 20th century saw the development of instruments and techniques to directly observe the behavior of individual molecules, made possible largely by fluorescence microscopy. Only a few decades after the first crude fluorescence microscopes, researchers were stunning the world with movies of dynamic biochemical reactions occurring at the single-molecule scale.
Contrast for Resolving Nanoscale Objects
A challenge for imaging anything smaller than the wavelength of light is generating sufficient contrast to see it. Various approaches were developed to make single molecules visible with optical microscopes. In 1989, W.E. Moerner showed laser-induced fluorescence could clearly distinguish individual dye molecules embedded in a crystal at room temperature. Others greatly improved imaging by maximizing signal above background.
Minimizing Photobleaching
One limitation in viewing single fluorophores was their tendency to permanently photobleach from excitation light. Clever techniques minimized this effect so molecules could be observed longer before destruction. For example, reducing illumination intensity and exposure time, scanning quickly, and using reducing and oxygen scavenging systems.
Introduction of Total Internal Reflection Fluorescence (TIRF) Microscopy
In 1985, Daniel Axelrod demonstrated TIRF microscopy, where an evanescent wave excites only fluorophores within about 100 nm of the coverslip surface. This inefficient excitation reduces photobleaching and background interference from solution fluorescence. Its thin illumination field also creates an ideal condition for single-molecule analysis at surfaces.
Imaging Advances with CCD Cameras and Scanning Confocals
Charged-coupled device (CCD) cameras dramatically improved digital fluorescence imaging. Unlike photographic film, a CCD could detect single fluorophores. Confocal scanning microscopes also helped by rejecting out of focus light for better contrast. These technologies turned crude single molecule signals into clearly defined images and measurable data.
Early Landmark Discoveries (1980s-90s)
After conceptual, technical, and instrumentation advances through the early 20th century, single molecule experiments took off in the 1980s and 90s. The combination of tunable lasers, sensitive cameras, and microscopy methods for reducing background enabled researchers to answer fundamental questions by watching one molecule at a time.
Observation of Discrete Enzymatic Turnover Steps
In 1987, John White observed discrete quantal jumps in resonance energy transfer that corresponded to single catalytic cycles of an individual enzyme molecule. This allowed enzyme mechanisms to be correlated with dynamic trajectories instead of just ensemble averages.
Direct Measurement of Molecular Diffusion
Using fluorescence photobleaching recovery, individual dye molecules were tracked in biology in 1993. This allowed measuring diffusion and flow in membranes and porous gels at the single particle level for the first time.
Motor Protein Mechanisms Characterized
Also during the 1990s, kinesin motor proteins were fluorescently labeled to visualize walking along microtubules. This led to characterization of discrete steps and coordination between motor domains during transport. It was now possible to see how nanomachines function in real time.
Single-Molecule Enzymology Becomes a Distinct Discipline
As increasingly intricate biochemistry was recorded from single enzymes in the 1990s, Single-molecule enzymology emerged as a distinct discipline. It was now possible to unambiguously determine mechanisms, define short-lived states, reveal dynamic disorder, quantify heterogeneity, and discover rare events that are hidden in ensemble studies.
Engineering Tools to Trap, Manipulate, and Sequence Single Molecules
With single molecules visible under microscopes, researchers built microfluidic tools in the 1990s-2000s to physically capture them and observe processes in isolated environments. Molecular manipulation advanced to probe properties using mechanical, electrical, thermal, chemical, and electromagnetic forces and signals. DNA sequencing and polymerase reaction dynamics were also revolutionized by single-molecule analysis.
Microfluidic Traps Enable Long-Term Observation
Early single-molecule imaging had detection times limited to a few seconds before diffusion or photodestruction. Various microscale traps were then created to capture molecules. These extended the observation window from seconds to hours, enabling quantification of ultra-rare or slow kinetics. Molecular confinement also allowed controlled stimulation and precise environmental changes.
Direct Mechanical Measurement and Manipulation of Single Molecules
Instruments such as atomic force microscopes and optical/magnetic tweezers were soon used to physically touch individual molecules. This allowed applying and measuring pico-newton scale forces to study molecular elasticity, adhesion, mechanochemistry, work cycles, and force-induced binding/rupture/unfolding. Positioning stages also enabled real-time manipulation of single molecules.
Advances in Automated Single Cell Analysis at High Throughput
As tools for single molecule examination improved, there was a parallel drive toward high throughput automated analysis. Flow cells, microfluidic chips, and microarrays brought massively parallel investigation of single cells and molecules. Combined with advances in microscopy, sample handling, computing, digital data processing, and machine learning, massive datasets of individual molecular behaviors could now be acquired and mined in depth.
Decades of DNA Sequencing Advancements
Many quintessential single molecule techniques arose from innovations in DNA sequencing. Methods evolved from tagging chain terminators in the 1970s, to polymerase immobilization and step-by-step imaging in the 80s-90s, to present day non-terminated monitoring of hundreds of thousands of DNA polymerases simultaneously replicating anchored DNA molecules while their fluorescently labeled nucleotides are observed in real time as they are incorporated.
Single-Molecule Techniques Mature into Indispensable Tools (2000s)
After its initial debut with specialized ultrasensitive instrumentation, single-molecule analysis graduated into an accessible wide-spread methodology employing standardized robust commercial instrumentation. Turnkey single-molecule systems now offer advanced capabilities to general researchers. Novel discoveries continue as single-molecule techniques address new questions and are applied in broader interdisciplinary contexts.
Super-Resolution and Single-Molecule Localization Microscopy (SMLM)
In the mid 2000s, multiple groundbreaking techniques gave conventional diffraction-limited fluorescence microscopy 10-100X higher spatial resolution to create nanoscale images of single molecules inside cells. This includes photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and related methods. Researchers could now quantify protein spatial patterning, clustering, and interactions throughout cells in amazing molecular-scale detail.
DNA Curtains Offer New Insights on Single DNA-Binding Proteins
An impactful technique introduced in the mid 2000s was DNA curtains, which uses hydrodynamic force to uniformly stretch and align hundreds of DNA molecules on a fluid lipid bilayer, leaving them anchored at one end. This enables clear visualization of fluorescently tagged DNA-binding proteins interacting with the DNA over long time periods.
Widespread Application of Single-Molecule FRET (smFRET)
Single-pair Förster resonance energy transfer became a widely applied single-molecule technique in the 2000s for detecting molecular proximity, conformation, composition order, and binding interactions. Unlike previous biochemical methods that time-averaged ensembles, smFRET offers real-time quantal photonic signals from each individual dual-labeled bimolecular complex.
Commercial Single-Molecule Systems Enable Routine Use
Standardized commercial instruments have brought advanced single-molecule capabilities to the masses. Total internal reflection fluorescence (TIRF) microscopy systems, combined with microfluidic platforms and user-friendly software, have made routine single-molecule imaging, trapping, force mechanics, kinetics, and data quantification broadly accessible.
Outlook: New Frontiers in Single-Molecule Research (2020s and Beyond)
Single-molecule techniques continue advancing into new research frontiers, enabled by ongoing improvements in imaging methods, nanofabrication, chemistries, automation, and data science. As barriers to directly studying molecular behaviors one by one are removed, unexpected discoveries will catalyze scientific progress worldwide.
Novel Non-Optical Single-Molecule Imaging Modalities Emerge
While fluorescence microscopy has been the dominant approach for detecting single molecules, new modalities are emerging including electron, tunneling, ion conductance, acoustic, magnetic, plasmonic, electrochemical, Raman, mass spectrometry, and force microscopies, as well as label-free optical imaging. These expand the range of molecular properties and environments that can be probed at the single entity scale.
Integrating Orthogonal Single-Molecule Measurements
Correlating distinct simultaneous single-molecule observations, by combining imaging, sequencing, force probing, current or potential sensing, and other modalities into integrated instruments will provide multidimensional understanding of intricate molecular processes. Data can be unified and analyzed to model causal mechanisms that are ambiguous from any one signal alone.
Artificial Intelligence and Machine Learning Boost Discovery from Complex Single-Molecule Datasets
Advanced algorithms augment human discovery by discerning patterns within enormous high-dimensional single-molecule datasets that are impossible to mentally process or visually intuit. Neural networks can help segment, classify, cluster, and predict experimental observations. This will undoubtedly uncover emergent phenomena and molecular behaviors that may have otherwise escaped human detection.
So from basic origins in mathematical concepts and early microscopic observations, to landmarks in visualizing purified enzymes and motor proteins, to explosive technological growth in measurement instrumentation, to present-day turnkey commercial systems and emerging future opportunities, the discipline of studying one molecule at a time has cemented itself as a fundamental pillar of the biological sciences. Ongoing single-molecule advances continuously provide an awe-inspiring window into the dynamic nanoscale world that dictates cellular life itself.
FAQs
When did single-molecule experiments first emerge?
The foundations were laid in the early 20th century, but single-molecule experiments truly emerged in the 1980s and 90s with advances in laser technology, sensitive cameras, and microscopy techniques to reduce background noise.
How are single molecules made visible?
Fluorescence microscopy techniques label molecules with dyes. Clever methods maximize the signal while reducing background noise and photobleaching effects.
How are single molecules trapped?
Microfluidic devices and optical/magnetic tweezers can capture and isolate individual molecules for prolonged observation periods.
What breakthroughs did single-molecule analysis enable?
It revealed key insights on enzyme mechanisms, molecular motors, DNA-protein interactions, sequencing, super-resolution microscopy within cells, and more.
What does the future hold for single-molecule science?
Novel techniques and integrating data from multiple measurements on one molecule will uncover new discoveries. AI will help find patterns in massive datasets.