Multiphotonen-Mikroskopie und Laser-Nanochirurgie der Kornea mittels Naher-Infrarot-Nanojoule-Femtosekunden-Laserpulse
Three sub-theses intracorneal multiphoton-mediated optical nanosurgery including flapfree non-invasive intrastromal ablation and flap generation; corneal nonlinear optical histology based on multiphoton microscopy; uses of multiphoton imaging in the femtosecond (fs) laser corneal nanosurgery (nJ) in this work have been studied based on the versatile 80MHz near-infrared (NIR) intense nJ fs lasers emitted from the solid-state Ti: Sapphire laser system with rabbits. The era of laser refractive surgery with the excimer lasers was heralded by the U.S. Food and Drug Administration's approval (FDA) in 1995. Conventional laser corneal refractive surgery (PRK, LASIK) for visual correction based on the high energy ultraviolet (UV) nanosecond excimer laser pulses is now just being challenged due to its disadvantages such as collateral damage outside the focal volume, UV mutation effects and induction of oxidative stress, and microkeratome-related complications. The NIR fs lasers have recently attracted amount attention due to its advantages such as no photon damage outside the focal femtoliter volume and multiphoton absorption without compromising viability. The nanodissection capability of the nJ fs laser pulses has been here confirmed through the histological outcomes of an intratissue fissure without any detrimental effects on overlying layers in the intratissue ablation procedure as well as corneal flap and intrastromal lenticule in the flap generation procedure. The encouraging surgical advances have potential in the treatment of the visual disorders as well as in the ultra-precise ablation of intratissue neoplasia, nanosurgical applications in the subcellular organelle and in developmental biology. Multiphoton microscopy at a high light intensity of MW-GW/cm2 based on the simultaneous absorption of more than one photon is one of the most exciting developments in biomedical imaging. So far, the anatomical micro- and nanostructures of corneal tissue have been extensively studied and reported with conventional histological and electron microscopical methods as well as the confocal one-photon laser scanning microscopy (CLSM). Based on the advantages of multiphoton microscopy such as in-vivo selective displaying of tissue components with subcellular spatial resolution and high contrast, no requirement of cellular and tissue staining or slicing, no out-of-focus photobleaching, improved background discrimination, increased penetration through the biological bulk tissue with NIR light, and no need of pinhole aperture, the excitation of intracellular NAD(P)H/NAD(P)+ in the autofluorescence imaging and the SHG imaging of noncentrosymmetric collagen have been used in this work as novel diagnostic tools for invivo differentiation of corneal layers, for imaging of corneal cells such as epithelial cells, keratocytes and endothelial cells and collagen lamellas. The activated keratocytes (myofibroblasts) have been here as well detected in the wound repair of intrastromal surgery with this nonlinear optical imaging technique. This multiphoton optical imaging technique has potential to become a powerful means in advancing understanding of corneal biomechnics and even cellular reactions in term of laser lesions. The uses of the multiphoton imaging technique has been also proved in this work to be essential for fs nJ cornea surgery such as to determine the surgical interest of region preoperation, to visualize and verify the outcomes of the laser surgery on line.