Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture

Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. Th e core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960 , Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been mea sured to define spatial resolution performances. The TPE microscope has bee n used with a wide range of excitable fluorescent; molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spec tral range from W to green. An example is reported on 3D imaging of the hel ical structure of the sperm head of the Octopus Eledone cirrhosa labelled w ith an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode. (C) 1999 Wiley-Liss, In c.