Leander Zickler

Second−harmonic imaging of cornea after intrastromal femtosecond laser ablation

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.

Mini−invasive corneal surgery and imaging with femtosecond lasers

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.

Noninvasive evaluation of mini-invasive femtosecond laser refractive surgery

Nd:glass femtosecond laser is promising as next generation mini-invasive eye surgical laser, with the advantages of excellent beam quality, high surgical precision and minimized side effects. However, there are still many open questions concerning the precision, efficiency and collateral effects of femtosecond laser refractive surgery. By non-invasive microscopic imaging methods including confocal, multiphoton, second harmonic and atomic force microscopy, we successfully characterized the three dimensional corneal ultrastructure without applying fixation and slicing. Based on the intrinsic properties of collagen, second harmonic cornea imaging proved to be outstanding to analyze the outcome of femtosecond laser intrastromal ablations. Strong contrast and large sensing depth second harmonic image was obtained without fixation, sectioning or labelling. The three dimensional ultrastructure of porcine cornea after Nd:glass femtosecond laser intrastromal surgery was examined to evaluate the concepts of minimum-invasive all-optical refractive eye surgery. No thermal damages were recognized and the surgical outcome appeared highly predictable. Due to the similarities between the physical principals of nonlinear laser scanning microscopy and femtosecond laser ablations, a setup of the Nd:glass femtosecond laser system integrating both the surgery and probing functions was proposed.

Femtosecond all-solid-state laser for refractive surgery

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 todays 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).

Refractive Surgical Applications of Femtosecond Lasers

Ultrashort pulse lasers have attracted much interest over the past decade and still are a field of vital research. Scientists and engineers initially focused on the laser source itself, with efforts concentrating on the search for reliable pulse-forming processes and the pursuit of novel schemes for pulse amplification. With ultrafast lasers migrating from laboratory setups that require daily “Ph.D. service” to a simple turn-key tool with standalone operation by a nonlaser expert, it is now possible to explore numerous useful applications.

Microscopic Evaluation of Femtosecond Laser Intrastromal Surgery

Diode pumped Nd:glass all-solid-state femtosecond laser is promising for next generation refractive surgery, with the advantages of excellent surgical precision, minimal tissue damage and improved system stability and compactness. The microscopic evaluation of the outcome of femtosecond laser surgery is crucial before clinical applications. By two-photon laser scanning microscopy and non-invasive second harmonic imaging, the three dimensional ultrastructure of the porcine cornea is visualized without requiring slicing or staining. The minimal-invasive corneal flap cutting and non-invasive intrastromal surgery are investigated. Femtosecond laser intrastromal surgery demonstrated high ablation precision and mimimal side effects. However, there are elongated filaments/streaks observed in the cornea stroma, most likely due to the focusing optics and self-focusing.