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SPECIAL SECTION ON FRONTIERS IN MICROSCOPY

Two-photon microscopy and spectroscopy based on a compact confocal scanning head

[+] Author Affiliations
Alberto Diaspro

University of Genoa, National Institute for the Physics of Matter (INFM), Department of Physics, Via Dodecaneso 33, 16146 Genova, Italy

Giberto Chirico

National Institute for the Physics of Matter (INFM) Department of Physics, Piazza delle Scienze, 3, 20126 Milano, Italy

Federico Federici

University of Genoa, National Institute for the Physics of Matter (INFM), Department of Physics, Via Dodecaneso 33, 16146 Genova, Italy

Fabio Cannone, Sabrina Beretta

National Institute for the Physics of Matter (INFM), Department of Physics, Piazza delle Scienze, 3, 20126 Milano, Italy

Mauro Robello

University of Genoa, National Institute for the Physics of Matter (INFM), Department of Physics, Via Dodecaneso 33, 16146 Genova, Italy

J. Biomed. Opt. 6(3), 300-310 (Jul 01, 2001). doi:10.1117/1.1382809
History: Received Mar. 20, 2001; Revised Mar. 24, 2001; Accepted Apr. 11, 2001
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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. © 2001 Society of Photo-Optical Instrumentation Engineers.

© 2001 Society of Photo-Optical Instrumentation Engineers

Citation

Alberto Diaspro ; Giberto Chirico ; Federico Federici ; Fabio Cannone ; Sabrina Beretta, et al.
"Two-photon microscopy and spectroscopy based on a compact confocal scanning head", J. Biomed. Opt. 6(3), 300-310 (Jul 01, 2001). ; http://dx.doi.org/10.1117/1.1382809


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