Research Area: Physical chemistry, chemical physics, single-molecule biophysics, super-resolution imaging, nanoparticle trapping
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 employ ensemble averaging over the number of molecules N to compute an observable. To explore what happens when ensemble averaging is removed, we use single-molecule optical spectroscopy and imaging, 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 or enzyme in a living cell (see Figure).
Why is this new area, single-molecule optical nanoscience, of interest? Complex systems including molecules in condensed phases or biomolecules in cells 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 present within cells 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. Our studies have explored various genetically encoded fluorescent proteins like GFP, kinesin molecular motors, Ca++ ion concentration sensors, chaperonins assisting protein folding, transmembrane proteins of the immune system in and out of 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 photoswitchable 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, and we have used nanoscale metallic electromagnetic structures to locally enhance light and therefore modify the interaction between light and single molecules. On a deeper level, 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 object under study, far beyond the optical diffraction limit. Several advanced techniques for obtaining three-dimensional information from single-molecule photoswitching are under development in the Moerner lab, especially the double-helix point spread function.
Recently, we have built an Anti-Brownian ELectrokinetic (ABEL) trap which uses precision 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. We are actively applying this device to measure the optical dynamics of single antenna proteins and single redox enzymes in solution of importance to energy conversion and storage.
I invite you to my group's home page for more information!
1) “Microscopy Beyond the Diffraction Limit Using Actively Controlled Single Molecules,” W.E. Moerner, J. Microsc., 246, 213-220 (2012).
2) “Probing Single Biomolecules in Solution Using the Anti-Brownian ELectrokinetic (ABEL) Trap,” Q. Wang, R.H. Goldsmith, Y. Jiang, S.D. Bockenhauer, and W.E. Moerner, Acc. Chem. Res., 45, 1955-1964 (2012).
3) "Three-dimensional Tracking of Single mRNA Particles in S. cerevisiae Using a Double-Helix Point Spread Function,” M.A. Thompson, J.M. Casolari, M. Badieirostami, P.O. Brown, and W.E. Moerner, Proc. Nat. Acad. Sci. (USA), 107, 17864 (2010).
4) "Superresolution Imaging in Live Caulobacter Crescentus Cells Using Photoswitchable EYFP,” J.S. Biteen, M.A. Thompson, N.K. Tselentis, G.R. Bowman, L. Shapiro, and W.E. Moerner, Nature Meth. (USA), 5, 947-949 (2008).
5) "New Directions in Single-Molecule Imaging and Analysis,” W.E. Moerner, Proc. Nat. Acad. Sci. (USA), 104, 12596 (2007).
6) "Suppressing Brownian Motion of Individual Biomolecules in Solution,” A.E. Cohen and W.E. Moerner, Proc. Nat. Acad. Sci. (USA), 103, 4362 (2006).