Within the strategic framework of a national project of the National Institute of the Physics of Matter (INFM), Section B, we have realised a two-photon excitation fluorescence microscope, part of a multipurpose architecture including also lifetime imaging and fluorescence correlation spectroscopy modules.

The core of the architecture is a mode-locked Ti:Sapphire infrared pulsed laser (Tsunami 3960, Spectra Physics Inc., CA), pumped by a high-power (5W 532 nm) solid state laser (Millennia V, Spectra Physics Inc., CA). The system is endowed of a Lok-to-clock module (model 3930, Spectra Physics) Inc., CA).

The features of infrared pulsed laser are high average power, 80 MHz high repetition rate, and 100 fs short pulse width. Tsunami has a tuning range from 650 nm to 1050 nm and in the actual configuration is optimized in a range from 680 nm to 830 nm delivering an output average power from 200 mW to 900 mW.

The scanning and acquisition system for microscopic imaging is based on a modified commercial scanning head Nikon PCM2000 (Nikon Instr., Florence, Italy).

The scanning head operates in the "open pinhole" condition and de-scanned mode. To characterise the spatial resolution of the TPE system, blue fluorescent carboxylate modified microspheres 0.1 mm diameter (F-8797, Molecular Probes, OR, USA) were used. The radial and axial resolution have been measured at 720 nm and fit well with theoretical predictions (210 nm lateral, 700 nm axial).

Two-photon excitation imaging can be performed with all the common fluorescent molecules and also exploiting some autofluorescence of the samples themselves. Second harmonic generation measurements are also in progress. The next picture shows the behaviour of the point-spread-function (left) both axial (circle-line) and radial (square-dotted).

Here is an example of triple fluorescence emission using a single excitation wavelength. Endotelial cells from bovine lungs have been stained with DAPI (for DNA visualization), with mitotracker red (for the mitocondria) and with BODIPY (for the actin filaments). The excitation wavelength is 720 nm. Due to the wide absorption bands of the two-photon process, we are abe to simultaneously excite all these three components.