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Polarized fluorescence correlation spectroscopy of DNA‐DAPI complexes
Author(s) -
Barcellona Maria Luisa,
Gammon Seth,
Hazlett Theodore,
Digman Michelle A.,
Gratton Enrico
Publication year - 2004
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.20121
Subject(s) - rotational diffusion , fluorescence correlation spectroscopy , fluorescence anisotropy , microsecond , spectroscopy , autocorrelation , chemistry , molecular physics , polarization (electrochemistry) , rotational correlation time , fluorescence , rotation around a fixed axis , fluorescence spectroscopy , relaxation (psychology) , macromolecule , anisotropy , nuclear magnetic resonance , optics , physics , psychology , social psychology , biochemistry , statistics , mathematics , classical mechanics , quantum mechanics
We discuss the use of fluorescence correlation spectroscopy for the measurement of relatively slow rotations of large macromolecules in solution or attached to other macromolecular structures. We present simulations and experimental results to illustrate the range of rotational correlation times and diffusion times that the technique can analyze. In particular, we examine various methods to analyze the polarization fluctuation data. We have found that by first constructing the polarization function and then calculating the autocorrelation function, we can obtain the rotational motion of the molecule with very little interference from the lateral diffusion of the macromolecule, as long as the rotational diffusion is significantly faster than the lateral diffusion. Surprisingly, for common fluorophores the autocorrelation of the polarization function is relatively unaffected by the photon statistics. In our instrument, two‐photon excitation is used to define a small volume of illumination where a few molecules are present at any instant of time. The measurements of long DNA molecules labeled with the fluorescent probe DAPI show local rotational motions of the polymers in addition to translation motions of the entire polymer. For smaller molecules such as EGFP, the viscosity of the solution must be increased to bring the relaxation due to rotational motion into the measurable range. Overall, our results show that polarized fluorescence correlation spectroscopy can be used to detect fast and slow rotational motion in the time scale from microsecond to second, a range that cannot be easily reached by conventional fluorescence anisotropy decay methods. Microsc. Res. Tech. 65:205–217, 2004. © 2005 Wiley‐Liss, Inc.