Intraocular multiphoton microscopy with subcellular spatial resolution by infrared femtosecond lasers.

We reported on the in situ nonlinear optical sectioning of the corneal and retinal tissues based on the multiphoton microscopy (MPM) with different excitation wavelengths of infrared femtosecond (fs) lasers. The multiphoton nonlinear processing including two-photon fluorescence (2PF) and second harmonic generation (SHG) was induced under condition of high light intensities on an order of MW-GW/cm2. The laser beams emitted from the solid-state Ti: sapphire systems were focused in a 0.1 femtoliter focus volume of a high numerous aperture diffraction-limited objective (40 x 1.3 N.A., oil). The corneal layers have been visualized using nonlinear optical tomography. In particular, corneal Bowman's layer was optically determined in situ. The cellular and collagen components of tissues were selectively displayed with submicron spatial resolution and high efficiency without any assistance of staining or slicing. The preliminary study on retinal optical tomography is here also reported. MPM is a promising and convenient non-invasive technique by which the tissue layers can be visualized and the selective displaying of the tissue microstructures be realized. The optical biopsy based on intrinsic emission of MPM yields details that provide three-dimensional displaying of the tissue component and even have the potential to be used in clinical diagnostics.