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

We have combined a confocal laser scanning head modi- fied for TPE (two-photon excitation) microscopy with some spectro- scopic 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 demonstra- tion 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 char- acterization of the two-photon microscope for spectroscopic applica- tions: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the re- sponse 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 pro- tein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coeffi- cients measured with fluorescence polarization anisotropy and fluo- rescence 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