Single-molecule experiment

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Single polymer molecules (0.4 nm thick chains) recorded under aqueous media at different pH using an AFM. Drastic change of polymer chain conformation is observed at a small change of pH^[1].

A single-molecule experiment investigates the properties of a single individual molecule that can be isolated or 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 behaviour can not be distinguished, and only average characteristics can be measured. Although most measurement techniques are not sensitive enough to observe single molecules, single-molecule fluorescence has emerged as a useful tool for probing various processes which cannot be fully understood on the bulk level, such as the movement of myosin on actin filaments in muscle tissue or the details of individual local environments in solids. Subtle 3D details of polymer molecule conformation can be observed using an atomic force microscopy (AFM). Another crucial single-molecule technique is single molecule force spectroscopy, where single molecules (or couples of interacting molecules), usually polymers, are mechanically stretched and their elastic response recorded in real time.

[edit] 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 Lothar Kador and W. E. Moerner^[2]. One year later Michel Orrit and Jacky Bernard were able to show also the detection of the absorption of single molecules by their fluorescence ^[3].

Many techniques have the ability to observe one molecule at a time, most notably mass spectroscopy, 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 technique by Erwin Neher and Bert Sakmann (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 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 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 have also been used with success.

[edit] Theory

Single molecule fluorescence spectroscopy uses the fluorescence of a molecule to record information pertaining to its environment, structure, and position. The technique affords the ability to obtain information otherwise not available due to ensemble averaging of a bulk material.

[edit] Applications

[edit] 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 motor proteins move along myosin strands in muscle cells.

[edit] Single Molecule Fluorescence Resonance Energy Transfer

[edit] Single Molecule vs Ensemble

[edit] 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 until 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.

[edit] Single-molecule effects

• single-molecule magnet

[edit] Single-molecule techniques

• Microscopy
• Fluorescence
• Fluorescence resonance energy transfer
• Force spectroscopy
• Magnetic tweezers
• Optical tweezers
• Raman spectroscopy (in particular Surface Enhanced Raman Spectroscopy)
• Scanning probe microscopy (including Atomic Force Microscopy)
• electron microscopy
• Single-molecule spectroscopy
• Tethered particle motion (TPM)
• Super-resolution microscopy

[edit] External links

• Single Molecule Online Forums

[edit] References

1. ^ Y. Roiter and S. Minko, AFM Single Molecule Experiments at the Solid-Liquid Interface: In Situ Conformation of Adsorbed Flexible Polyelectrolyte Chains, Journal of the American Chemical Society, vol. 127, iss. 45, pp. 15688-15689 (2005)
2. ^ W. E. Moerner and L. Kador, Optical detection and spectroscopy of single molecules in a solid, Phys. Rev. Lett. 62, 2535 - 2538 (1989)
3. ^ M. Orrit and J. Bernard, Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal, Phys. Rev. Lett. 65, 2716 - 2719 (1990)