Corneal multiphoton microscopy and intratissue optical nanosurgery by nanojoule femtosecond near-infrared pulsed lasers

Multiphoton microscopy including multiphoton autofluorescence imaging (MAI) and second-harmonic generation (SHG) is being used as a novel diagnostic tool to perform tissue nonlinear optical tomography with submicron resolution. The three-dimensional corneal ultrastructure of whole depth has been viewed without any staining or mechanical slicing. Compared with photodisruptive surgical effects occurring at TW/cm2 light intensity, multiphoton imaging can be induced at MW-GW/cm2 photon intensity. The intratissue surgical effect including nanojoule (nJ) femtosecond laser ablation and flap generation was induced through multiphoton nonlinear absorption at a wavelength of 800 nm and ascertained by the histological outcomes. More interesting, the multiphoton microscopy based on nonlinear absorption of femtosecond laser pulses at the wavelength of 715-930 nm emitted from solid-state Ti:sapphire system is acting as a precise non-invasive monitoring tool to determine the interest of region, to visualize and verify the outcomes in in vivo intrastromal laser nanosurgery. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric and biomechanical properties of biological tissues and that the nJ ultrashort lasers can be used as a ultra-precise nanoscalpel for performing intratissue surgery.