The two-photon-mediated autofluorescence and second harmonic generation (SHG) are acting as a novel diagnostic tool to perform tissue optical tomography with submicron resolution. The three-dimensional corneal ultrastructure of whole depth can be probed 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 multiphoton microscopy based on nonlinear absorption of femtosecond laser pulses at the wavelength of 715-930nm emitted from solid-state Ti: sapphire system is being used as a precise non-invasive monitoring tool to determine the interest of region, to visualize and to verify the outcomes in the invivo intrastromal laser nanosurgery. More interesting, the activated keratocytes have been also observed in-vivo 24 hours after the laser nanosurgery with this system. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric properties of the microstructure of the corneal tissue and for assessing the intrastromal nanosurgery. With the help of the multiphoton-mediated imaging, the next generation of laser refractive surgery approaches based on the nonamplified femtosecond lasers with higher precision and less complications are being evaluated systematically.
The intratissue multiphoton autofluorescence imaging (MAI) and the second harmonic generation (SHG) based on nonlinear process of femtosecond nanojoule laser pulses at wave length of 750-850 nm emitted from solid-state Titanium: Sapphire Chameleon have been used as a highly precise non-destructive tool to realize the in-vivo differentiation of corneal layers with the assistance of intratissue optical tomography and to visualize the keratocyte structures and collagen lamellas with submicron resolution. Multiphoton nonlinear imaging occurs only with high light intensity on an order of MG-GW/cm2 and photon flux density of more than 1024 photons cm-2s-1 in a 0.1femtoliter intrastromal focus volume obtained by diffraction-limited focussing with high-numerical objectives. This technique, acting as a novel diagnostic tool, proved to be essential for femtosecond (fs) nanojoule (nJ) cornea surgery to determine the interest of region preoperation, to visualize and verify the outcomes immediately after the laser surgery and has potential to become a powerful tool in advancing understanding of corneal biomechnics and cellular reactions after laser induced lesion.
We report on the histological results of in-vivo animal follow-up studies on refractive femtosecond laser surgery. Non-invasive flap-free intrastromal ablation as well as flap generation has been performed with MHz nanojoule near infrared femtosecond laser pulses. In particular, the dynamics of corneal wound healing have been studied. Wound-healing effects could be detected up to 90 days post-operation in the case of lasermediated flap generation. The flap-free intrastromal cavity was identified until the 28th day post-treatment. Interestingly, eosinophil granulocytes were observed. The follow-up studies confirmed that the near infrared femtosecond laser at near-nanojoule pulse energy is a highly precise and an attractive tool for intraocular refractive surgery, especially for flap-free intrastromal surgery.
We report on a method for refractive laser surgery based on low-energy femtosecond laser pulses provided by ultracompact turn-key non-amplified laser systems. An additional excimer laser is not required for ablation of the stroma. The novel method has the potential to be used for (i) optical flap creation as well as stroma ablation and (ii) for non-invasive flap-free intrastromal ablation. In addition, 3D multiphoton imaging of the cornea can be performed. In particular, we used sub-nanojoule near infrared 80 MHz femtosecond laser pulses for multiphoton imaging of corneal structures with ultrahigh resolution (< 1μm) as well as for highly precise intraocular refractive surgery. Imaging based on two-photon excited cellular autofluorescence and SHG formation in collagen structures was performed at GW/cm2 intensities, whereas destructive optical breakdown for nanoprocessing occurred at TW/cm2 light intensities. These high intensities were realized with sub-nJ pulses within a subfemtoliter intrastromal volume by diffraction-limited focussing with high NA objectives and beam scanning 50 to 140 μm below the epithelial surface. Multiphoton tomography of the cornea was used to determine the target of interest and to visualize intraocular post-laser effects. Histological examination with light- and electron microscopes of laser-exposed porcine and rabbit eyes reveal a minimum intratissue cut size below 1 μm without destructive effects to surrounding collagen structures. LASIK flaps and intracorneal cavities could be realized with high precision using 200 fs, 80 MHz, sub-nanojoule pulses at 800 nm. First studies on 80 MHz femtosecond laser surgery on living rabbits have been performed.
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