24 April 1998 Fluorescence spectroscopy of single-molecule dynamics and its biological applications
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Abstract
The advances in room temperature single molecule detection (SMD) and single molecule spectroscopy (SMS) by laser induced fluorescence provide new tools for testing semi- classical and quantum mechanical theories of light-matter interaction and molecular interactions. They also provide the tools for the study of individual synthetic and biological macromolecules under physiological conditions. Two properties of a single fluorescent probe attached to a macromolecule can be exploited to provide local structural information. The first, its the very high sensitivity of the fluorophore to its immediate local environment, including the sensitivity to the presence of other fluorophores and quenchers near-by. The second is its unique absorption and emission transition dipoles, which can be interrogated by polarized light. Recently, we have developed 'molecular rulers' which use SMD/SMS to measure nanometer distances and distance changes of macromolecules. We also demonstrated the ability to measure orientations and orientation changes of macromolecules with very high accuracy. The spectroscopic signature of the fluorescent light coming from two different color labels which are attached to two sites on the macromolecule is a sensitive measure for dynamic conformations. Distance changes between two sites on the macromolecule are measured via single-pair fluorescence resonance energy transfer and orientation changes are measured by detecting changes in the dipole orientation of a rigidly attached probe or changes in the degree of rotational diffusion of a tethered probe via single molecule fluorescence polarization anisotropy.
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Taekjip Ha, Alice Ting, Joy Liang, Jennifer L. Glass, Daniel S. Chemla, Pete G. Schultz, Shimon Weiss, "Fluorescence spectroscopy of single-molecule dynamics and its biological applications", Proc. SPIE 3273, Laser Techniques for Condensed-Phase and Biological Systems, (24 April 1998); doi: 10.1117/12.306136; https://doi.org/10.1117/12.306136
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