Most physical, chemical and biophysical experiments in condensed phases measure the average behavior of a huge number of molecules, from millions to billions to Avogadro's Number. At the same time, most theoretical models describe the behavior of a single molecule interacting with its surroundings, and ensemble averaging over the number of molecules N is normally required to compute an observable. To explore what happens when ensemble averaging is removed, we use single-molecule optical spectroscopy, a set of ultrasensitive far-field and near-field laser techniques that allow us to detect and probe the optical properties of individual molecules (N=1). In this way we can explore truly local behavior inside a solid, a liquid, or in a biomolecular system such as a single protein in a living bacterial cell (see Figure).

Why is this new regime, single-molecule nanoscience, of interest? Complex systems such as condensed matter or biomolecules can contain hidden heterogeneity produced by different local environments, different conformational states, or even different protein folds. Single-molecule studies allow us to explore hidden heterogeneity because we measure the distribution of behavior by recording the properties of each member of the ensemble, one by one. There are several specific ways single molecule measurements can provide new information. Photochemistry or other photophysical changes in the immediate local environment can be detected as changes in resonant frequency, lifetime, or emission spectrum of the single molecule (spectral diffusion). We also obtain kinetic information from the time-dependent changes in the brightness of the molecule, or from the polarization changes that occur when the fluorophore rotates, or from the physical motion of the single-molecule label due to diffusion or transport. By measuring energy transfer between two different fluorophores, distance information on the 5-9 nm scale can be obtained. Many functional nanomachines operating within the cell operate one by one, thus the ability to observe single copies provides a new way to try to understand how the system works. In collaboration with the molecular biology and biochemistry communities, we work to discover how much can be learned with such single-molecule biophysical measurements. Recent studies have explored various genetically encoded fluorescent proteins like GFP, kinesin molecular motors, Ca++ ion concentration sensors, transmembrane proteins of the immune system in living cells, and genetic regulatory proteins in bacteria. To enable further single-molecule imaging in cells, we are actively involved in the development of new single-molecule fluorophores.

Single molecules also provide a window into a growing new field, nanophotonics. We have used a single molecule to make a quantum mechanical (non-Poissonian) light source operating at room temperature. In addition, a single molecule can be viewed as a probe of its immediate local nanoenvironment on the scale on the order of the molecular size (~1 nm). Because single molecules are nanoscale emitters, when active control is used to turn molecules on an off, it is possible to build up a superresolution image of the sample, far beyond the diffraction limit. We are also working to use nanoscale metallic electromagnetic structures to locally enhance light and therefore modify the interaction between light and molecules.

Recently, we have built an Anti-Brownian Electrokinetic trap which uses optical microscopy and active electrophoretic/electroosmotic feedback to grab and manipulate single nanoscale objects in solution for detailed optical measurements, without the need to attach the objects to a surface.

I invite you to my group's home page for more information!

1) "New Directions in Single-Molecule Imaging and Analysis," W. E. Moerner, Proc. Nat. Acad. Sci. (USA), 104, 12596 (2007).

2) "Suppressing Brownian Motion of Individual Biomolecules in Solution," A. E. Cohen and W. E. Moerner, Proc. Nat. Acad. Sci. (USA), 103, 4362 (2006).

3) "Improving the Mismatch Between Light and Nanoscale Objects with Gold Bowtie Nanoantennas," P. J. Schuck, D. P. Fromm, A. Sundaramurthy, G. S. Kino, and W. E. Moerner, Phys. Rev. Lett., 94, 017402 (2005).

4) "Methods of Single-Molecule Fluorescence Spectroscopy and Microscopy," W. E. Moerner and D. P. Fromm, Rev. Sci. Instrum., 74, 3597-3619 (2003).

5) "A Dozen Years of Single-Molecule Spectroscopy in Physics, Chemistry, and Biophysics," W. E. Moerner , J. Phys. Chem.B, 106, 910-927 (2002).

6) "Illuminating Single Molecules in Condensed Matter," W.E. Moerner and M. Orrit, Science, 283, 1670-1676 (1999).