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

We have combined a confocal laser scanning head modified for TPE (two-photo n excitation) microscopy with some spectroscopic modules to study single mo lecules and molecular aggregates. The behavior of the TPE microscope unit h as been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-pho ton mode can be simply accomplished by switching from a mono-mode optical f iber (one-photon) coupled to conventional laser sources to an optical modul e that allows IR laser beam (two-photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-phot on microscope for spectroscopic applications: fluorescence correlation, lif etime and fluorescence polarization anisotropy measurements. We describe th e measurement of the response of the two-photon microscope to the light pol arization 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 ave rage rotational and translational diffusion coefficients measured with fluo rescence polarization anisotropy and fluorescence correlation methods are i n good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules. (C) 2001 Society o f Photo-Optical Instrumentation Engineers.