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Frontiers of Molecular Science

December 4-7, 2001
Friiberghs Manor, Örsundsbro and Stockholm University

Single-Molecule Spectroscopy - from Low Temperature Physical Chemistry to Biophysics

by W. E. Moerner
Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA

By the late 1980's, it had long been a seemingly impossible dream to be able to "see" a single molecule, as essentially all chemical experiments operated on ensembles of millions to billions of copies of the same molecule. Today, more than ten years have elapsed since the first optical detection and spectroscopy of a single molecule in a solid in 1989(1). We have subsequently witnessed an explosion of interest in the optical detection and spectroscopy of single molecules in complex condensed phases, such as solids, liquids, or proteins. One primary motivation for the study of individual molecules in such hosts derives from their complexity – local environments in complex systems are often heterogeneous, so that the behavior of individuals may not be fully represented by the standard (large N) ensemble average. With single-molecule techniques, however, the actual distribution of behavior can be probed yielding additional information normally hidden by ensemble averaging. Each single molecule acts as an exquisitely sensitive probe or reporter of the immediate local environment around it (the "nanoenvironment"). In addition, if there are time-dependent dynamical states involved, large N measurements average over all the individual systems in a variety of conformational or configurational states. In the single-molecule regime, however, the time dependence of each individual can be followed in time. Thus, for cases where there is either static or dynamic complexity, single-molecule observations have opened a new frontier of molecular science aimed at understanding the diversity of microscopic ways in which physical, chemical, and biological processes involving molecules actually take place.

The early years of single-molecule spectroscopy concentrated on studies of aromatic hydrocarbons like pentacene or perylene in organic crystals like p-terphenyl at liquid helium temperatures. The first optical spectrum of a single molecule in a solid(1) was recorded with a sophisticated laser frequency-modulation technique for a pentacene molecular impurity in a crystal of p-terphenyl at 1.8 K. Later, fluorescence excitation techniques(2) were shown to provide good signal-to-noise for samples and excitation geometries with low background. Optical spectroscopy of individual molecules at low temperatures has allowed the observation many fascinating physical effects(3), for example, the shifts in resonance frequency of a single molecule arising from nearby two-level-system transitions in the solid (spectral "diffusion"), light-driven frequency shifts ("hole-burning" for a single molecule), the resonance Raman spectrum of an individual molecule, quantum optical effects such as photon antibunching, and magnetic resonance of a single molecular spin.

In the mid-90's, the field branched out to include room temperature, as a result of a demonstration by Betzig et al. using near-field excitation techniques to reduce the potential scattering volume(4). Later experiments by several researchers showed that far-field, total internal reflection, and confocal microscopy methods also yield acceptable single-molecule images. Many experiments followed the earlier low-temperature studies to explore what can be learned from fluctuations, vibrational spectroscopy, lifetime measurements, polarization studies, and so on under ambient conditions.

While the initial room-temperature studies concentrated on dye molecules on surfaces, single biomolecules in native environments provide a fertile ground for the use of single-molecule techniques. The currently expanding area of single-molecule biophysics has arisen because many biological processes (DNA/RNA transcription, regulation, signaling, antigen recognition, enzymatic activity, etc.) occur at the single- or few-copy level, where heterogeneity in local environment or conformational state is common. The reporter biomolecule termed the green fluorescent protein (GFP) has become a critical cellular reporter of gene expression and protein localization; surprisingly at the single-molecule level a blinking and switching effect occurs(6). In a related experiment, the behavior of a dual-GFP construct designed to detect calcium ion concentrations by fluorescence resonant energy transfer was examined(7). In a recent experiment, measurement of the polarization properties of a single fluorophore bound to the kinesin cellular motor allowed discovery of a new and unexpected highly mobile state of kinesin the presence of the ADP nucleotide(8). In new experiments, single copies of major histocompatibility complexes of type II (MHCII) labeled with a single small fluorophore can now be observed diffusing in the membrane of live CHO cells. All these experiments illustrate the breadth of possible applications of single-molecule optics to the understanding of biology.

Finally, in the area of quantum optics, several researchers have explored the unique quantum mechanical nature of the light emitted by a single molecule, both at low temperatures and at room temperature. The high stability of some aromatic hydrocarbons in crystals has allowed the creation of a novel room-temperature light source of single photons on demand based on a single-molecule emitter(9).

The advent of single-molecule spectroscopy is now having a broad interdisciplinary impact, from physical/analytical chemistry, to biophysics, the spectroscopy of defects in solids, materials science, quantum optics, and possibly even to future nanotechnology, where small numbers of molecules may be used to build molecular machines.

1. W. E. Moerner and L. Kador, Phys. Rev. Lett. 62, 2535 (1989).

2. M. Orrit and J. Bernard, Phys. Rev. Lett 65, 2716 (1990).

3. For reviews, see W. E, Moerner, Science 265, 46 (1994); W. E. Moerner, Acc. Chem. Res. 29, 563 (1996); Th. Basche, W. E. Moerner, M. Orrit, and U. P. Wild, eds., Single Molecule Optical Detection, Imaging, and Spectroscopy, (Wiley-VCH, Munich, 1997).

4. E. Betzig and R. J. Chichester, Science 262, 1422 (1993).

5. See the recent special issue of Science on Single Molecules, March 12, 1999; W. E. Moerner and M. Orrit, Science 283, 1670 (1999).

6. R.M. Dickson, A.B. Cubitt, R.Y. Tsien, and W.E. Moerner, Nature, 388, 355 (1997).

7. S. Brasselet, S. Brasselet, E. J. G. Peterman, A. Miyawaki, and W. E. Moerner, J. Phys. Chem. B 104, 3676-3682 (2000).

8. H. Sosa, E. Peterman, W. E. Moerner, L. S. B. Goldstein, Nature Structural Biology 8, 540-544 (2001).

9. B. Lounis and W. E. Moerner, Nature 407, 491-493(2000)


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