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 (congruent to3 times the background) observed for highly diluted solutions.

Olivini, F., Beretta, S., Chirico, G. (2001). Two-photon fluorescence polarization anisotropy decay on highly diluted solutions by phase fluorometry. APPLIED SPECTROSCOPY, 55(3), 311-317 [10.1366/0003702011951713].

Two-photon fluorescence polarization anisotropy decay on highly diluted solutions by phase fluorometry

CHIRICO, GIUSEPPE
2001

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 (congruent to3 times the background) observed for highly diluted solutions.
Articolo in rivista - Articolo scientifico
fluorescence polarization anisotropy; fluorescence correlation spectroscopy; two-photon excitation; multifrequency phase fluorometer
English
mar-2001
55
3
311
317
none
Olivini, F., Beretta, S., Chirico, G. (2001). Two-photon fluorescence polarization anisotropy decay on highly diluted solutions by phase fluorometry. APPLIED SPECTROSCOPY, 55(3), 311-317 [10.1366/0003702011951713].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/3052
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