Evaluation of optical properties and micropatterning capabilities of a TPE microscope based on a compact confocal scanning head.

We have set-up a two-photon excitation fluorescence microscope based an a compact commercial confocal laser scanning head and a mode-locked Ti:Sapphire infrared pulsed laser, directly coupled in air to the scanning head We report about the architecture and the imaging capabilities of this microscope using high numerical aperture objectives, in terms of its spatial resolution and excitation volume. 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 path in air guiding the IR laser beam (two-photon/ TPE). In both cases, an optical fiber delivers the emitted fluorescence to the control unit. Under TPE regime, the resolution (Full Width at Half Maximum intensity - FWHM) was 210 nm (x-y) and 700 nm axially at 720 nm excitation. Due to the inherent spatial selectivity of the TPE we are evaluating the utilization of the instrument for micro patterning using the property of photobleaching and the characteristics of photobleaching in comparison with the single-photon mode. Moreover, attention is given to pulse broadening as function of penetration depth. The system is mainly used for biological applications related to cell and tissue imaging and dynamic cellular processes.