ABSTRACT

Single-molecule fluorescence spectroscopy addresses biological mechanisms and enables ultrasensitive diagnostics. We describe a new family of single-molecule fluorescence methods that uses alternating-laser excitation (ALEX) of diffusing or immobilized biomolecules to study their structure, interactions, and dynamics. This is accomplished using ratios that report on the distance between and the stoichiometry of fluorophores attached to the molecules of interest. The principle of alternation is compatible with several time scales, allowing monitoring of fast dynamics or simultaneous monitoring of a large number of individual molecules.

Achillefs N. Kapanidis received his B.S. in chemistry (1991) from the Aristotelian University of Thessaloniki, Greece, and his M.Sc. (food chemistry, 1993) and Ph.D. (biological chemistry, 1999) from Rutgers University, New Jersey. Following postdoctoral research at Lawrence Berkeley National Laboratory, he became an Assistant Researcher in the Department of Chemistry and Biochemistry at UCLA. In late 2004, he joined the University of Oxford, United Kingdom, as a University Lecturer in Biological Physics. He studies protein-nucleic acid transactions using single-molecule spectroscopy and biochemistry.

Ted A. Laurence received his B.S. (1997) from California Institute of Technology. He was a National Science Foundation Graduate Fellow (1997-2000) and obtained his Ph.D. (2002) from the University of California, Berkeley. After postdoctoral research at UCLA, he became a fellow in the Physical Biosciences Institute at Lawrence Livermore National Laboratory. His interests include biological applications of single-molecule spectroscopy and surface-enhanced Raman spectroscopy.

Nam Ki Lee received his B.S. (1998) and M.Sc. (2000) from Seoul National University, South Korea. As part of his Ph.D., he joined UCLA (2002-2003) to study single-molecule spectroscopy and biochemistry. He continues this work as a Ph.D. candidate at Seoul National University.

Emmanuel Margeat received his B.S. in physical chemistry (1996) from the University Paul Sabatier in Toulouse, France, and his Ph.D. in biochemistry and molecular biology (2001) from the University of Montpellier, France. He next joined the group of Shimon Weiss at UCLA, studying gene transcription using singlemolecule fluorescence methods. Since 2004, he has been a CNRS researcher, working on protein-nucleic acid interactions at the Centre de Biochimie Structurale, Montpellier.

Xiangxu Kong received his B.S. from Beijing University, China (2001). He is presently a Ph.D. candidate in physical chemistry at UCLA. He studies protein folding and protein-protein interactions using single-molecule fluorescence methods. Shimon Weiss received his B.S. (1984) and D.Sc. (1989) in electrical engineering from Technion, the Israel Institute of Technology. He did his postdoctoral research at AT&T Bell Laboratories, New Jersey. In 1990, he became a Staff Scientist at Lawrence Berkeley National Laboratory, California. In 2001, he joined UCLA as a Professor of Chemistry and Biochemistry and of Physiology. His research interests span the biophysics-chemistry-biology-medicine-nanotechnology interfaces.

Introduction

Room-temperature, real-time observation of individual molecules is revolutionizing several areas of physics, chemistry, and biology by eliminating the ensemble averaging present in conventional analysis and by uncovering static and dynamic heterogeneities.1,2 Single-molecule methods monitor dynamics of biomolecules under equilibrium conditions and complex reaction kinetics under nonequilibrium conditions,3 capturing views that are inaccessible to ensemble methods owing to the lack of synchronization of biomolecules.

Single-molecule detection is characterized by three major attributes. First, the properties of ensembles of billions of molecules can be described reliably by observations of a few single molecules (due to ergodicity). Second, single-molecule observations carry far more information than do ensembles (when ergodicity breaks down). Third, the amount of sample necessary to capture the new information is miniscule (<10-16 mol), several orders of magnitude lower than the amount needed for ensemble methods. Such properties are well aligned with modern science, which moves toward smaller scales, higher throughputs, and higher-complexity systems, the descriptions of which require monitoring of several observables. However, to mine the wealth of information displayed by single molecules, one has to pose the “right” questions to the molecules; in the case of single-molecule fluorescence spectroscopy (SMFS),4,5 this requirement led to novel fluorescent probes and labeling strategies6 and complex excitation/detection schemes. SMFS is applied to either diffusing or immobilized molecules, accessing different time scales of information. Several excellent reviews exist on many aspects of SMFS.7-15 Here, we describe a new family of fluorescence methods that uses alternating-laser excitation of single molecules to study their structure, interactions, and dynamics through measurements of distances and stoichiometries.

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