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Journal of Biomedical Optics

Two-photon microscopy and spectroscopy based on a compact confocal scanning head
Author(s): Alberto Diaspro; Giberto Chirico; Federico Federici; Fabio Cannone; Sabrina Beretta; Mauro Robello; Francesca Olivini; Paola Ramoino
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Paper Abstract

We have combined a confocal laser scanning head modified for TPE (two-photon excitation) microscopy with some spectroscopic modules to study single molecules and molecular aggregates. The behavior of the TPE microscope unit has been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical module that allows IR laser beam (two-photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-photon microscope for spectroscopic applications: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the response of the two-photon microscope to the light polarization and discuss fluorescence polarization anisotropy measurements on Rhodamine 6G as a function of the viscosity and on a globular protein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coefficients measured with fluorescence polarization anisotropy and fluorescence correlation methods are in good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules.

Paper Details

Date Published: 1 July 2001
PDF: 11 pages
J. Biomed. Opt. 6(3) doi: 10.1117/1.1382809
Published in: Journal of Biomedical Optics Volume 6, Issue 3
Show Author Affiliations
Alberto Diaspro, Univ. degli Studi di Genoa (Italy)
Giberto Chirico, National Institute for the Physics of Matter (Italy)
Federico Federici, Univ. degli Studi di Genoa (Italy)
Fabio Cannone, Istituto Nazionale per la Fisica della Materia (Italy)
Sabrina Beretta, Istituto Nazionale per la Fisica della Materia (Italy)
Mauro Robello, Univ. degli Studi di Genova (Italy)
Francesca Olivini, Istituto Nazionale per la Fisica della Materia (Italy)
Paola Ramoino, Univ. degli Studi di Genova (Italy)

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