Two-Photon Fluorescence Polarization Anisotropy Decay on Highly Diluted Solutions by Phase Fluorometry

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Abstract:

We report here the phase-sensitive measurements of two-photon fluorescence polarization anisotropy (FPA) on highly diluted solutions. We first describe the characterization of the response of the two-photon microscope to the light polarization and its test by means of measurements of the FPA on rhodamine 6G as a function of the viscosity. Further, we report the study of the FPA at high dilutions on a globular protein, the beta-lactoglobulin B (BLG) labeled with Alexa 532. The number of molecules (from 0.4 to 17 molecules per excitation volume) is measured by means of fluorescence correlation spectroscopy (FCS). The average rotational and translational diffusion coefficients measured with the FPA and FCS methods are in good agreement with the protein size and do not show a substantial dependence on the protein concentration, despite the very low signal (≅3 times the background) observed for highly diluted solutions.

Keywords: FLUORESCENCE CORRELATION SPECTROSCOPY; FLUORESCENCE POLARIZATION ANISOTROPY; MULTIFREQUENCY PHASE FLUOROMETER; TWO-PHOTON EXCITATION

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/0003702011951713

Affiliations: Istituto Nazionale per la Fisica della Materia, Universitàdi Milano Bicocca, Via Cozzi 52, I-20126, Milano, Italy.

Publication date: March 1, 2001

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