Basics

Single-Molecule Probes of Nanoenvironments in Condensed Matter, Explorations in Solids or Cells Using a Nanometer-Sized Probe, or Probing Local Environments Using Single-Molecule Spectroscopy and Microscopy.


Figure 1: Imaging Single Molecules of Pentacene in a p-Terphenyl Crystal

Most chemical experiments in condensed matter measure the average behavior of a huge number, N, of molecules, where N may range from millions to billions to Avogadro's Number. At the same time, most theoretical models are intended to describe the behavior of a single molecule interacting with its surroundings, and averaging over the number of molecules N is normally required to compute an observable. Using precision laser spectroscopic techniques, we have been detecting and probing the detailed properties of individual impurity molecules hidden deep inside a solid, a protein, or even in a liquid, i.e., the ultimate limit of N=1. This was first done in the Moerner Lab in 1989, and has since expanded dramatically to include many groups around the world. A key reason for doing this is to explore heterogeneity that is normally obscured by ensemble averaging.

Studying one individual molecule in a solid means we are working with an extremely small number of moles of material. You might be aware that the international standards organization, IUPAC, has defined several new prefixes: zepto- for 1E-21, and yocto- for 1E-24. Thus 1 molecule is equivalent to 1.66 yoctomoles. But we think this is unwieldy. Thus we define a new prefix guaca- so that (with apologies to Prof. Avogadro)

1 guacamole = 1 / ( Avocado's Number) of moles.

More seriously, it is worth recalling that each molecule we are probing is only 1 or 2 nanometers in size. This means that when we use a laser to select one probe molecule, we can sense details of the immediate local environment of a truly nanoscopic probe.

To achieve this extreme reduction of the concentration and reach the single-molecule level, we use either (a) low-temperature high-resolution laser spectroscopy to select individual molecules by their resonance frequencies, or (b) near field optical excitation to pump sample volumes much smaller than the diffraction limit, or (c) extremely low concentrations and diffraction-limited confocal or far-field microscopy. By studying a large number of individual molecules one at a time, we are able not only to observe how the usual ensemble average behavior is formed, but also to see unexpected, surprising behavior normally hidden by the usual ensemble averaging.

The phenomena under study include spontaneous changes in resonant frequency caused by host matrix dynamics (spectral diffusion) as well as light-induced resonant frequency changes, which may lead to future optical storage on a single molecule level. Since a very narrow optical transition is extremely sensitive to perturbations, the details of the local environment may be studied by the application of electric, strain, or magnetic fields. By dispersing the emitted light, even the vibrational mode spectrum of a single molecule may be measured! By measuring correlations in the emitted photon stream, fast dynamics including environmental fluctuations, or the purely quantum-mechanical behavior termed photon antibunching may be probed. In biomolecules, we observe fascinating differences in behavior due to conformational states, local environments, or enzymatic cycle, all of which are obscured in large N experiments.

Selected Bibliography

  • "Optical Detection and Spectroscopy of Single Molecules in a Solid," by W. E. Moerner and L. Kador, Phys. Rev. Lett. 62, 2535 (1989). This is the first report of single-molecule detection in condensed phases.
  • "Fluorescence Spectroscropy and Spectral Diffusion of Single Impurity Molecules in a Crystal," by W.P. Ambrose and W. E. Moerner, Nature 349, 225 (1991).
  • "Spectroscopy of Single Impurity Molecules in Solids," by W. E. Moerner and Th. Basche', Angew. Chem. 105, 537 (1993); Angew. Chem. Int. Ed. Engl. 32, 457 (1993).
  • "Examining Nanoenvironments in Solids on the Scale of a Single, Isolated Impurity Molecule," by W. E. Moerner, Science 265, 46 (1994).
  • "High-Resolution Optical Spectroscopy of Single Molecules in Solids, by W. E. Moerner, in "Single Molecules and Atoms," Special Issue of Accounts of Chemical Research, December 1996.
  • "Fundamentals of Single-Molecule Spectroscopy in Solids," Chapter 1 of Single Molecule Optical Detection, Imaging, and Spectroscopy, T. Basche, W. E. Moerner, M. Orrit, and U. P. Wild, eds. (Verlag Chemie, Munich, 1997).
  • W. E. Moerner, "Those Blinking Single Molecules," Science 277, 1059 (1997).
  • W. E. Moerner and M. Orrit, "Illuminating Single Molecules in Condensed Matter," Science 283, 1670-1676 (1999).
  • B. Lounis and W. E. Moerner, "Single Photons on Demand from a Single Molecule at Room Temperature," Nature 407, 491-493 (2000).
  • H. Sosa, E. J. G. Peterman, W. E. Moerner, and L. S. B. Goldstein, "ADP-Induced Rocking of the Kinesin Motor Domain Revealed by Single-Molecule Fluorescence Polarization Microscopy," Nature Structural Biology 8, 540-544 (2001).
  • W. E. Moerner, "A Dozen Years of Single-Molecule Spectroscopy in Physics, Chemistry, and Biophysics," J. Phys. Chem. B 106, 910-927 (2002).
  • W. E. Moerner, "Single-Molecule Optical Spectroscopy of Autofluorescent Proteins," J. Chem. Phys. 117, 10925 (2002).
  • W. E. Moerner and D. P. Fromm, "Methods of Single-Molecule Fluorescence Spectroscopy and Microscopy," Rev. Sci. Instrum. 74, 3597-3619 (2003).
  • E. J. G. Peterman, H. Sosa, and W. E. Moerner, “Single-Molecule Fluorescence Spectroscopy and Microscopy of Biomolecular Motors,” invited review, Ann. Rev. Phys. Chem. 55, 79-96 (2004).
  • P. J. Schuck, D. P. Fromm, A. Sundaramurthy, G. S. Kino, and W. E. Moerner, “Improving the Mismatch Between Light and Nanoscale Objects with Gold Bowtie Nanoantennas,” Phys. Rev. Lett. 94, 017402 (2005).
  • K. A. Willets, S. Y. Nishimura, P. J. Schuck, R. J. Twieg, and W. E. Moerner, "Nonlinear Optical Chromophores as Nanoscale Emitters for Single-Molecule Spectroscopy," invited review, Accounts Chem. Res. 38, 549-556 (2005)
  • S. Y. Kim, Z. Gitai, A. Kinkhabwala, L. Shapiro, and W. E. Moerner, “Single Molecules of the Bacterial Actin MreB Undergo Directed Treadmilling Motion in Caulobacter crescentus,” Proc. Nat. Acad. Sci. (USA) 103, 10929-10934 (2006)..
  • A. E. Cohen and W. E. Moerner, “Suppressing Brownian Motion of Individual Biomolecules in Solution,” Proc. Nat. Acad. Sci. (USA) 103, 4362-4365 (2006).
  • W. E. Moerner, “New Directions in Single-Molecule Imaging and Analysis,” Invited Perspective, Proc. Nat. Acad. Sci. (USA) 104, 12596-12602 (2007).