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Title: Polarization spectroscopy of single fluorescent molecules

Abstract

Polarization spectroscopy of single fluorescent molecules is used to probe their rotational dynamics. When a molecule is immobilized on a dry surface, its in-plane dipole orientation is precisely determined by utilizing its transition dipole moment. An angular offset between the absorption and the emission dipoles was detected from a single fluorophore revealing its binding geometry to the surface. In an aqueous environment, DNA-tethered fluorophores display dynamics that are well-described by a hindered rotational diffusion model. A detailed description of the model is given, including calculations to estimate depolarization effects resulting from the high numerical aperture objective used to collect fluorescence photons. Protein-conjugated fluorophores display very distinct dynamics with continuous evolution of the rotational profile, possibly reflecting fluctuations in the polypeptide chain. When protein-conjugated fluorophores are allowed to freely diffuse in solution, it is possible to determine the fluorescence polarization anisotropy of each molecule that traverses the laser beam. The anisotropy values could, in principle, be used to identify conformational states of single molecules without the potential artifacts associated with surface immobilization.

Authors:
;  [1]; ;  [2]
  1. Lawrence Berkeley National Lab., CA (United States)
  2. Lawrence Berkeley National Lab., CA (United States). Material Sciences Div.|[Univ. of California, Berkeley, CA (United States). Physics Dept.
Publication Date:
Sponsoring Org.:
USDOE, Washington, DC (United States); Office of Naval Research, Washington, DC (United States)
OSTI Identifier:
691302
DOE Contract Number:  
AC03-76SF00098
Resource Type:
Journal Article
Journal Name:
Journal of Physical Chemistry B: Materials, Surfaces, Interfaces, amp Biophysical
Additional Journal Information:
Journal Volume: 103; Journal Issue: 33; Other Information: PBD: 19 Aug 1999
Country of Publication:
United States
Language:
English
Subject:
40 CHEMISTRY; 55 BIOLOGY AND MEDICINE, BASIC STUDIES; SPECTROSCOPY; FLUORESCENCE SPECTROSCOPY; ANISOTROPY; LASERS; DNA; MOLECULES

Citation Formats

Ha, T., Weiss, S., Laurence, T.A., and Chemla, D.S. Polarization spectroscopy of single fluorescent molecules. United States: N. p., 1999. Web. doi:10.1021/jp990948j.
Ha, T., Weiss, S., Laurence, T.A., & Chemla, D.S. Polarization spectroscopy of single fluorescent molecules. United States. doi:10.1021/jp990948j.
Ha, T., Weiss, S., Laurence, T.A., and Chemla, D.S. Thu . "Polarization spectroscopy of single fluorescent molecules". United States. doi:10.1021/jp990948j.
@article{osti_691302,
title = {Polarization spectroscopy of single fluorescent molecules},
author = {Ha, T. and Weiss, S. and Laurence, T.A. and Chemla, D.S.},
abstractNote = {Polarization spectroscopy of single fluorescent molecules is used to probe their rotational dynamics. When a molecule is immobilized on a dry surface, its in-plane dipole orientation is precisely determined by utilizing its transition dipole moment. An angular offset between the absorption and the emission dipoles was detected from a single fluorophore revealing its binding geometry to the surface. In an aqueous environment, DNA-tethered fluorophores display dynamics that are well-described by a hindered rotational diffusion model. A detailed description of the model is given, including calculations to estimate depolarization effects resulting from the high numerical aperture objective used to collect fluorescence photons. Protein-conjugated fluorophores display very distinct dynamics with continuous evolution of the rotational profile, possibly reflecting fluctuations in the polypeptide chain. When protein-conjugated fluorophores are allowed to freely diffuse in solution, it is possible to determine the fluorescence polarization anisotropy of each molecule that traverses the laser beam. The anisotropy values could, in principle, be used to identify conformational states of single molecules without the potential artifacts associated with surface immobilization.},
doi = {10.1021/jp990948j},
journal = {Journal of Physical Chemistry B: Materials, Surfaces, Interfaces, amp Biophysical},
number = 33,
volume = 103,
place = {United States},
year = {1999},
month = {8}
}