Nonlinear laser scanning microscopy is widely used for noninvasive imaging in cell biology and tissue physiology. However, multiphoton fluorescence imaging of dense, transparent connective tissue (e.g., cornea) is challenging since sophisticated labeling or slicing is necessary. High-resolution, high-contrast second harmonic generation (SHG) imaging of corneal tissue based on the intrinsic structure of collagen is discussed. The three-dimensional corneal ultrastructure in depths up to hundreds of microns can be probed noninvasively, without any staining or mechanical slicing. As an important application of second harmonic imaging in ophthalmology, the modification of corneal ultrastructure using femtosecond laser intrastromal ablation is systematically investigated to evaluate next-generation refractive surgical approaches.

Based on the transparency of corneal tissue and on laser plasma mediated non-thermal tissue ablation, near infrared femtosecond lasers are promising tools for minimally invasive intrastromal refractive surgery. Femtosecond lasers also enable novel nonlinear optical imaging methods like second harmonic corneal imaging. The microscopic effects of femtosecond laser intrastromal surgery were successfully visualized by using second harmonic corneal imaging with diffraction limited resolution, strong imaging contrast and large sensing depth, without requiring tissue fixation or sectioning. The performance of femtosecond laser intrastromal surgery proved to be precise, repeatable and predictable. It might be possible to integrate both surgical and probing functions into a single femtosecond laser system.

Refractive surgery in the pursuit of perfect vision (e.g. 20/10) requires firstly an exact measurement of abberations induced by the eye and then a sophisticated surgical approach. A recent extension of wavefront measurement techniques and adaptive optics to ophthalmology has quantitatively characterized the quality of the human eye. The next milestone towards perfect vision is developing a more efficient and precise laser scalpel and evaluating minimal-invasive laser surgery strategies. Femtosecond all-solid-state MOPA lasers based on passive modelocking and chirped pulse amplification are excellent candidates for eye surgery due to their stability, ultra-high intensity and compact tabletop size. Furthermore, taking into account the peak emission in the near IR and diffraction limited focusing abilities, surgical laser systems performing precise intrastromal incisions for corneal flap resection and intrastromal corneal reshaping promise significant improvement over today's Photorefractive Keratectomy (PRK) and Laser Assisted In Situ Keratomileusis (LASIK) techniques which utilize UV excimer lasers. Through dispersion control and optimized regenerative amplification, a compact femtosecond all-solid-state laser with pulsed energy well above LIOB threshold and kHz repetition rate is constructed. After applying a pulse sequence to the eye, the modified corneal morphology is investigated by high resolution microscopy (Multi Photon/SHG Confocal Microscope)

PURPOSE: Currently, refractive surgical excimer laser systems are calibrated by ablating plastic lenses, which are measured by lensometer and analyzed by a technician. The accuracy of this method is approximately 0.25 diopters (D) in sphere and cylinder power. Theoretically, objective calibration using wavefront technology would be significantly more accurate, thereby improving surgical outcomes. This study describes a Shack-Hartmann-based instrument, which has been developed to measure ablated plastic lenses for calibration and quality control of the excimer laser. METHODS: A calibration instrument comprising an LED source at 640 nm, a lenslet array, beam-guiding optics, and a CCD camera was designed to perform full wavefront analysis. The measurement plane is conjugate to the lenslet array plane, and the diameter of the pupil is 5 mm. Accuracy was determined by measuring a set of well-calibrated spherical and cylindrical glass lenses. Plastic lenses were ablated, and high-precision measurements were performed by surface profile scanner. RESULTS: In the power range of -6.00 to +4.00 D, repeatability exceeded 0.01 D, accuracy of measurement exceeded 0.04 D, and 1° for the axis of cylinder lenses. The measurement of excimer-ablated plastic lenses agreed with high-precision surface profile scanner measurements within 0.10 D, and repeatability exceeded 0.01 D. CONCLUSIONS: Wavefront technology-based, high-precision measurement of calibration lenses can more accurately set the energy of the excimer laser, which enhances the accuracy of refractive laser correction. In automating calibration, the new instrument removes operator subjectivity and decreases the time needed for calibration. [J Refract Surg. 2006;22:938-942.]

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