Polarization spectroscopy of single fluorescent molecules
- Lawrence Berkeley National Lab., CA (United States)
- Lawrence Berkeley National Lab., CA (United States). Material Sciences Div.
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.
- Sponsoring Organization:
- USDOE, Washington, DC (United States); Office of Naval Research, Washington, DC (United States)
- DOE Contract Number:
- AC03-76SF00098
- OSTI ID:
- 691302
- Journal Information:
- Journal of Physical Chemistry B: Materials, Surfaces, Interfaces, amp Biophysical, Journal Name: Journal of Physical Chemistry B: Materials, Surfaces, Interfaces, amp Biophysical Journal Issue: 33 Vol. 103; ISSN 1089-5647; ISSN JPCBFK
- Country of Publication:
- United States
- Language:
- English
Similar Records
Hindered Rotational Diffusion and Rotational Jumps of Single Molecules
Clay Nanoparticle-Supported Single-Molecule Fluorescence Spectroelectrochemistry