A single-molecule experiment investigates the properties of an individual molecule

Molecule

A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...

 that can be distinguished for the purpose of an experiment or analysis. Single-molecule studies may be contrasted with measurements on an ensemble or bulk collection of molecules, where the individual behavior can not be distinguished, and only average

Average

In mathematics, an average, or central tendency of a data set is a measure of the "middle" value of the data set. Average is one form of central tendency. Not all central tendencies should be considered definitions of average....

 characteristics can be measured. Since many measurement techniques in biology, chemistry and physics are not sensitive and cannot observe single molecules, single-molecule fluorescence techniques (that have emerged since the 90s for probing various processes on the level of individual molecules) caused a lot of excitement, since these supplied many new details on the measured processes that were not accessible in the past. Indeed, since the 90s, many techniques for probing individual molecules were developed. Examples for systems and such experimental’s techniques include: the movement of myosin on actin filaments in muscle tissue or the details of individual local environments in solids studied using spectroscopy. Biological polymers' conformations measured using an atomic force microscopy (AFM). Using force spectroscopy

Force spectroscopy

Force spectroscopy is a dynamic analytical technique that allows the study of the mechanical properties of single polymer molecules or proteins, or individual chemical bonds. It is performed by pulling on the system under scrutiny with controlled forces...

, single molecules (or couples of interacting molecules), usually polymer

Polymer

A polymer is a large molecule composed of repeating structural units. These subunits are typically connected by covalent chemical bonds...

s, can be mechanically stretched and their elastic response recorded in real time. Recordings of ion channels (patch clamp

Patch clamp

The patch clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in cells. The technique can be applied to a wide variety of cells, but is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle...

 experiments) were the first example for single molecule experiments (these were performed since the 70s), yet these were limited for the particular system of an ion-channel.

History

In the gas phase at ultralow pressures, single-molecule experiments have been around for decades, but in the condensed phase only since 1989 with the work by W. E. Moerner

W. E. Moerner

William Esco Moerner , was born in 1953 in Pleasanton, California, and grew up in San Antonio, Texas. He received his B.S. in Physics with Top Honors, B.S. in Electrical Engineering with Top Honors, and his A.B. in Mathematics summa cum laude from Washington University in St. Louis in 1975...

 and Lothar Kador. One year later Michel Orrit and Jacky Bernard were able to show also the detection of the absorption of single molecules by their fluorescence.

Many techniques have the ability to observe one molecule at a time, most notably mass spectrometry

Mass spectrometry

Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...

, where single ions are detected. In addition one of the earliest means of detecting single molecules, came about in the field of ion channels with the development of the patch clamp

Patch clamp

The patch clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in cells. The technique can be applied to a wide variety of cells, but is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle...

 technique by Erwin Neher

Erwin Neher

Erwin Neher is a German biophysicist.Erwin Neher studied physics at the Technical University of Munich from 1963 to 1966. In 1966, He was awarded a Fulbright Scholarship to study in the US...

 and Bert Sakmann

Bert Sakmann

-External links:*...

 (who later went on to win the Nobel prize for their seminal contributions) However, the idea of measuring conductance to look at single molecules placed a serious limitation on the kind of systems which could be observed.

Fluorescence

Fluorescence

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

 is a convenient means of observing one molecule at a time, mostly due to the sensitivity of commercial optical detectors, capable of counting single photons. However, spectroscopically, the observation of one molecule requires that the molecule is in an isolated environment and that it emits photons upon excitation, which owing to the technology to detect single photons by use of photomultiplier tubes (PMT) or avalanche photodiodes (APD), enables one to record photon emission events with great sensitivity and time resolution.

More recently, single molecule fluorescence is the subject of intense interest for biological imaging, through the labeling of biomolecules such as proteins and nucleotides to study enzymatic function which cannot easily be studied on the bulk scale, due to subtle time-dependent movements in catalysis and structural reorganization. The most studied protein has been the class of myosin/actin enzymes found in muscle tissues. Through single molecule techniques the step mechanism has been observed and characterized in many of these proteins.

Nanomanipulators such as the atomic force microscope

Atomic force microscope

Atomic force microscopy or scanning force microscopy is a very high-resolution type of scanning probe microscopy, with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit...

 are also suited to single molecule experiments of biological significance, since they work on the same length scale of most biological polymers. Besides, atomic force microscopy (AFM) is appropriate for the studies of synthetic polymer molecules. AFM provides a unique possibility of 3D visualization of polymer chains. For instance, AFM tapping mode is gentle enough for the recording of adsorbed polyelectrolyte molecules (for example, 0.4 nm thick chains of poly(2-vinylpyridine)) under liquid medium. The location of two-chain-superposition correspond in these experiments to twice the thickness of single chain (0.8 nm in the case of the mentioned example). At the application of proper scanning parameters, conformation of such molecules remain unchanged for hours that allows the performance of experiments under liquid media having various properties (Roiter and Minko, 2005). Optical tweezers

Optical tweezers

Optical tweezers are scientific instruments that use a highly focused laser beam to provide an attractive or repulsive force , depending on the refractive index mismatch to physically hold and move microscopic dielectric objects...

 have also been used with success.

Concept

Single molecule fluorescence spectroscopy uses the fluorescence of a molecule for obtaining information on its environment, structure, and position. The technique affords the ability of obtaining information otherwise not available due to ensemble averaging (that is, a signal obtained when recording many molecules at the same time represents an average property of the molecule's dynamics). The results in many experiments of individual molecules are two-state trajectories.

Biomolecule labeling

Single fluorophores can be chemically attached to biomolecules, such as proteins or DNA, and the dynamics of individual molecules can be tracked by monitoring the fluorescent probe. Spatial movements within the Rayleigh limit can be tracked, along with changes in emission intensity and/or radiative lifetime, which often indicate changes in local environment. For instance, single-molecule labeling has yielded a vast quantity of information on how kinesin

Kinesin

A kinesin is a protein belonging to a class of motor proteins found in eukaryotic cells. Kinesins move along microtubule filaments, and are powered by the hydrolysis of ATP . The active movement of kinesins supports several cellular functions including mitosis, meiosis and transport of cellular...

 motor proteins move along myosin

Myosin

Myosins comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes. They are responsible for actin-based motility. The term was originally used to describe a group of similar...

 strands in muscle cells.

Single-molecule fluorescence resonance energy transfer

Here, the molecule is labeled in (at least) two places. A laser beam is focused on the molceule exciting the first probe. When this probe relaxes and emits a photon, it has a chance of exciting the other probe. The efficiency of the absorption of the photon emitted from the first probe in the second probe depends on the distance among these probes. Since the distance changes with time, this experiemnt probes the internal dynamics of the molecule.

Theoretical notes

• Single-molecule versus ensemble When looking on individual molecules, one usually can construct propagators, and jumping time probability density functions, of the first order, the second order and so on, where from bulk experiments, one usually obtain the decay of a correlation function. From the information contained in these unique functions (obtained from individual molecules), one can extract a relatively clear picture on the way the system behaves; e.g. its kinetic scheme
  Kinetic scheme
  In physics and chemistry and related fields, a kinetic scheme is a network of states and connections among the states representing the scheme of a dynamical process. Usually, a kinetic scheme represents a Markovian process, where when the process is not Markovian, the scheme is a generalized...
  
  , or its potential of activity, or its reduced dimensions form
  Reduced dimensions form
  In biophysics and related fields, reduced dimension forms are unique on-off mechanisms for random walks that generate two-state trajectories...
  
  . In particular, one can construct (many properties of) the reaction pathway of an enzyme when monitoring the activity of an individual enzyme The problem with the analysis of single molecule data is filtering the noise.

Impact

Single-molecule techniques impacted optics, electronics, and biology. In the biological sciences, the study of proteins and other complex biological machinery was limited to ensemble experiments that nearly made impossible the direct observation of their kinetics. For example, it was only after single molecule fluorescence microscopy was used to study kinesin-myosin pairs in muscle tissue that direct observation of the walking mechanisms were understood. These, experiments, however, have for the most part been limited to in vitro studies, as useful techniques for live cell imaging have yet to be fully realized. The promise of single molecule in vivo imaging, however, brings with it an enormous potential to directly observe bio-molecules in native processes. These techniques are often targeted for studies involving low-copy proteins, many of which are still being discovered.

Single-molecule techniques

• Microscopy
  Microscopy
  Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...
  
  
• Fluorescence
  Fluorescence
  Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
  
  
• Fluorescence resonance energy transfer
  Fluorescence resonance energy transfer
  Förster resonance energy transfer , also known as fluorescence resonance energy transfer, resonance energy transfer or electronic energy transfer , is a mechanism describing energy transfer between two chromophores.A donor chromophore, initially in its electronic excited state, may transfer energy...
  
  
• Force spectroscopy
  Force spectroscopy
  Force spectroscopy is a dynamic analytical technique that allows the study of the mechanical properties of single polymer molecules or proteins, or individual chemical bonds. It is performed by pulling on the system under scrutiny with controlled forces...
  
  
• Magnetic tweezers
  Magnetic tweezers
  A magnetic tweezer is a scientific instrument for exerting and measuring forces on magnetic particles using a magnetic field gradient. Typical applications are single-molecule micromanipulation, rheology of soft matter, and studies of force-regulated processes in living cells. Forces are typically...
  
  
• Optical tweezers
  Optical tweezers
  Optical tweezers are scientific instruments that use a highly focused laser beam to provide an attractive or repulsive force , depending on the refractive index mismatch to physically hold and move microscopic dielectric objects...
  
  
• Raman spectroscopy
  Raman spectroscopy
  Raman spectroscopy is a spectroscopic technique used to study vibrational, rotational, and other low-frequency modes in a system.It relies on inelastic scattering, or Raman scattering, of monochromatic light, usually from a laser in the visible, near infrared, or near ultraviolet range...
  
   (in particular Surface Enhanced Raman Spectroscopy
  Surface Enhanced Raman Spectroscopy
  Surface enhanced Raman spectroscopy or surface enhanced Raman scattering is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces...
  
  )
• Scanning probe microscopy
  Scanning probe microscopy
  Scanning Probe Microscopy is a branch of microscopy that forms images of surfaces using a physical probe that scans the specimen. An image of the surface is obtained by mechanically moving the probe in a raster scan of the specimen, line by line, and recording the probe-surface interaction as a...
  
   (including Atomic Force Microscopy
  Atomic force microscope
  Atomic force microscopy or scanning force microscopy is a very high-resolution type of scanning probe microscopy, with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit...
  
  )
• electron microscopy
• Single-molecule spectroscopy
• Tethered particle motion
  Tethered particle motion
  Tethered particle motion is a biophysical method that is used for studying various polymers such as DNA and their interaction with other entities such as proteins....
  
   (TPM)
• Super-resolution microscopy
• Voltage clamp
  Voltage clamp
  The voltage clamp is used by electrophysiologists to measure the ion currents across the membrane of excitable cells, such as neurons, while holding the membrane voltage at a set level. Cell membranes of excitable cells contain many different kinds of ion channels, some of which are voltage gated...
  
   and patch clamp
  Patch clamp
  The patch clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in cells. The technique can be applied to a wide variety of cells, but is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle...
  
  experiments