|diffraction limit||nominal lower bound on feature sizes resolved with scattered particles or radiation; in general, 200 nm for visible light and less than 0.1 nm for high-energy electrons|
|conformational change||a change of the orientation (geometry) of a given chemical group relative to others in a single molecule or macromolecule|
|real-space measurement||in this context, a measurement based on positions of individual molecules, rather than by diffracting radiation through a regular lattice; advantageous since it allows heterogeneous structures to be measured|
|localization||ability to determine the location of a single molecule in space|
|resolution||ability to distinguish two closely-spaced molecules; in general, for far-field measurements, resolution is proportional to the wavelength of probe radiation used|
The ability to measure the structure and behavior of single molecules has evolved in parallel in fields ranging from physics to biology. Molecules studied vary widely: from small molecules less than 1 nm in diameter to DNA molecules with lengths of several thousand nanometers. Some measurements are performed at cryogenic temperatures (often near 1 K) and ultrahigh vacuum (10−9 Torr), others in liquid at room temperature. Some experiments measure the molecule directly, while others require attachment of a high-contrast label, which may be orders of magnitude larger than the molecule itself.
Broadly, the field has evolved from two complementary perspectives that may be understood in analogy to the “top-down vs
bottom-up” dichotomy that has driven nanotechnology. The bottom-up approach starts from single atoms and certain classes of simple molecules, observed under rigorously controlled conditions (often cryogenic temperatures and ultrahigh vacuum). Such experiments have their roots in the early development of field emission and field ionization microscopy1,2
and have evolved and expanded to include such techniques as single-molecule fluorescence(3
) and transmission electron and scanning tunneling microscopies,(4
) some with resolutions better than 0.1 nm.
The top-down approach starts from large macromolecules such as DNA, sometimes with lengths as great as several micrometers.5−7
Although such molecules natively function in complex, three-dimensional cellular environments, early experiments required simplifications in order to observe single molecules. Typically this meant fixing molecules to surfaces and/or operating in simple solutions such as aqueous buffers. A number of experimental techniques have developed in parallel: optical measurements based on attachment of single fluorophores or other optical tags;8−10
force measurements using optical traps,(11
) magnetic beads,5,12
) and patch clamp(16
) and other techniques(17
) based on the application of external fields.
Both bottom-up and top-down approaches have developed increasingly sophisticated single-molecule measurements. For bottom-up measurements, this typically means understanding larger, more complex molecules in less restricted environments (frequently under atmospheric conditions and sometimes in liquids).8,18−21
For top-down measurements, this often means understanding more details about an already complex molecule, either by measuring at shorter length scales or by working in less simplified environments (e.g.
, live cells).22−27
Bottom-up experiments typically provide more detailed information on molecular behavior, and top-down experiments still deal in more complicated molecules and environments.
As these approaches begin to converge in terms of both length scales and target complexity, it becomes important to develop a joint understanding that exploits the measurement capabilities of each. Across the spectrum of tools and targets, experiments have localized and measured the topography of molecules in space, tracked their motion, and quantified their physical properties. Imaging ranges from tracking single molecules in live cells(23
) to mapping the chemical structure of single molecules on surfaces.(28
) Molecular motion can also be measured, from internal conformational changes to nanometer-scale rotation and translation,11,29
as well as the forces required for these motions.(30
) Single-molecule spectroscopic measurements include molecular vibrations,(31
) and electronic spins,(34
) and differences in enzymatic activity.35,36
To create a more unified perspective, we select seminal reviews and experimental examples from across the breadth of the single-molecule literature, grouped broadly by probe type. We discuss electron-based measurements,(18
) optical measurements,8,37
and force-based methods11,12
(), with a primary focus on work in the condensed phase. Each probe type has unique measurement advantages, which we first discuss briefly to provide context for understanding how the physical properties of the sample and length scales to be measured influence the choice of probes.
Figure 1 Quantitative analysis of single-molecule measurements based on photons, force, and electrons. Photonic measurements (left) are usually based on one or more fluorescent labels (either small molecules or fluorescent proteins) or a larger nanoparticle label. (more ...)
Electrons have a number of features that make them useful probes at the very short length scales relevant for single-molecule measurements. Their small mass means they exhibit substantial quantum mechanical tunneling behavior, which allows measurement of distances up to a few nanometers with sub-Ångström sensitivity. Coupling tunneling with inelastic processes enables measurement of vibrational and other energy levels.38,39
The sub-Ångström wavelength of high-energy electrons means they are able to resolve atomic-scale features in diffraction experiments.(40
) Since electrons are responsible for molecular bonding, measuring electronic conductance through a molecule can in some cases also probe single-molecule conformations.(41
The excellent spatial resolution achieved in electron-based single-molecule measurements comes at a cost. Observed areas are typically quite small (often much less than 1 μm2). Thus, while measuring the behavior of a single molecule relative to its immediate environment is straightforward, relating it to micro- to macroscopic features can be more difficult. Electron-based measurements also place fairly stringent requirements on sample preparation: scanning tunneling microscopy generally requires samples no more than a few nanometers thick on conductive substrates, and transmission electron microscopy requires samples to be electron-transparent (usually less than 100 nm thick and composed of low-atomic-number materials).
Photons in the visible and near-visible ranges have much longer wavelengths and, consequently, are used in different ways to quantify single-molecule behavior. The longer length scales typically probed under photonic illumination make such measurements especially useful in quantifying the relationships between single molecules and micro- to macroscopic features in their environment, such as in biological samples. Measuring the behavior of a single molecule requires that the molecule display a unique optical signature to distinguish it from up to trillions of background molecules; almost universally this is achieved by covalently binding a fluorescent emitter or other optical tag to the molecule of interest.(42
) Fluorophores can be chosen to be sensitive to pH, electric fields, ionic strength, and other factors, providing a probe of the target molecule’s immediate environment.(43
) Fluorescence polarization measurements can be used to determine fluorophore orientation, which correlates with target molecule orientation.(44
The diffraction limit would appear to restrict photonic measurements to features of hundreds of nanometers and larger. However, subdiffraction optical methods, such as stimulated emission depletion and selective photoactivation, are beginning to allow single fluorophores to be localized down to tens of nanometers, usually at a cost to measurement time and thus the ability to probe dynamics. Measurements of energy transfer efficiency between two fluorescent dyes or plasmonic probes can be used to measure the distance between the probes enabling measurements of the dynamics of protein conformation changes on millisecond time scales.
In addition to diffraction-based limitations on spatial resolution, fluorescence measurements are constrained by the need to add a label to the molecule of interest and by the fact that fluorescent dyes eventually bleach, losing their ability to fluoresce after 104−106 excitations. Nanoparticle probes are less sensitive to bleaching, but are often larger than the molecule being measured.
Forces between a sharp cantilever and a surface can be used to measure the topography of single molecules on a surface. Typical lateral resolution is 1−10 nm (depending on the radius of curvature of the cantilever tip), with vertical resolution better than 1 nm. However, careful instrumental design (often including ultrahigh vacuum and cryogenic temperatures) and noncontact imaging based on frequency shifts can provide subnanometer lateral resolution.
Force-based methods are also useful for understanding force-induced conformational changes in single molecules. Such measurements typically involve a molecule tethered to a surface and to a probe (such as an AFM tip, magnetic bead, or nanoparticle suitable for optical trapping). Forces on the order of 0.1−1000 pN are applied to the probe, resulting in measured displacements on the order of nanometers, usually corresponding to protein unfolding or motion of a molecular motor.(45
In comparison to optical traps and magnetic beads, AFM allows the application and measurement of larger forces, usually with lower spatial resolution due to surface drift relative to the probe. Often AFM measurements use nonspecific binding between target and probe, which can impact reproducibility. Optical traps work in a lower force regime (<100 pN) but can provide better spatial resolution (<1 nm), especially if the target is bound to two traps rather than a trap and a surface.(46
) Targets are usually bound to the trap bead using specific covalent strategies, increasing reproducibility, but photodamage of the target is a concern. Magnetic beads work in an even lower force regime (<20 pN) and provide reduced spatial resolution relative to optical traps but eliminate the concern of photodamage and allow the probe to be rotated controllably.6,45
Forces can also be applied to many magnetically labeled targets in parallel.(47