Donald A. Peyrot

Ultrashort pulse laser surgery of the cornea and the sclera

The strongly localized interaction process of ultrashort laser pulses with tissue makes femtosecond lasers a powerful tool for eye surgery. These lasers are now routinely used in refractive surgery and other forms of surgery of the anterior segment of the eye. Several clinical laser systems also offer options for corneal grafting and the potential use of ultrashort pulse lasers in glaucoma surgery has been the object of several recent studies which have shown promising results. While devices aimed for interventions in clear tissue may be based on available solid state or fibre laser technology, the development of tools for surgery in more strongly scattering tissue has to account for the compromised tissular transparency and requires the development of optimized laser sources. The present paper focuses on surgery of clear and pathological cornea as well as sclera. It aims to give an overview over typical medical indications for ultrashort pulse laser surgery, the optics of the tissues involved, the available laser technology, the laser–tissue interaction process, and possible future developments.

Effect of incident light wavelength and corneal edema on light scattering and penetration: laboratory study of human corneas.

PURPOSE The outcome of ultrashort pulse laser surgery of the cornea is strongly influenced by the light scattering properties of the tissue, for which little data are available. The purpose of the present study is to provide quantitative values for light scattering and its relation to the degree of edema. METHODS An experimental optical measuring setup based on confocal geometry was used to measure the unscattered and scattered fractions of light transmitted by eye bank corneas presenting various degrees of edema. From these measurements, the effective light penetration depth in the cornea was calculated as a function of wavelength. RESULTS Corneal transparency depends on the pathological state of the cornea and on wavelength. It may be predicted as a function of corneal thickness, ie, the degree of edema. In healthy and edematous cornea, the percentage of scattered light decreases with increasing wavelength. The total penetration depths at the wavelengths of ~1050 nm (which is used in typical clinical systems) and 1650 nm (which is recommended for future devices) are comparable; however, the former is limited by scattering, which degrades the laser beam quality, whereas the latter is only limited by optical absorption, which may be compensated for. CONCLUSIONS The use of longer wavelengths should help improve the surgical outcome in ultrashort pulse laser surgery of the cornea when working on pathological tissue. A wavelength of approximately 1650 nm appears to be a good compromise, as it allows for reduced light scattering while keeping optical absorption reasonably low.

Wavelength optimization in femtosecond laser corneal surgery.

PURPOSE To evaluate the influence of wavelength on penetration depth and quality of femtosecond laser corneal incisions in view of optimizing procedures in corneal surgery assisted by ultrashort pulse lasers. METHODS We performed penetrating and lamellar incisions on eye bank corneas using several ultrashort pulse laser sources. Several wavelengths within the near-infrared and shortwave-infrared wavelength range were used and the pulse energy was varied. The corneas were subsequently analyzed using light microscopy as well as transmission and scanning electron microscopy. RESULTS We found higher penetration depths and improved incision quality when using wavelengths close to λ = 1650 nm rather than the wavelength of λ = 1030 nm typical in current clinical systems. Optical micrographs show an improvement of the penetration depth by a factor of 2 to 3 while maintaining a good incision quality when using the longer wavelength. These results were confirmed with micrographs obtained with transmission and scanning electron microscopy. CONCLUSIONS A wavelength change from the standard 1030 nm to 1650 nm in corneal surgery assisted by ultrashort pulse laser considerably reduces light scattering within the tissue. This results in a better preservation of the laser beam quality in the volume of the tissue, particularly when working at depths required for deep lamellar or penetrating keratoplasty. Using this wavelength yields improved penetration depths into the tissue; it permits use of lower energies for any given depth and thus reduces unwanted side effects as thermal effects.

Development of a nonlinear fiber-optic spectrometer for human lung tissue exploration

Several major lung pathologies are characterized by early modifications of the extracellular matrix (ECM) fibrillar collagen and elastin network. We report here the development of a nonlinear fiber-optic spectrometer, compatible with an endoscopic use, primarily intended for the recording of second-harmonic generation (SHG) signal of collagen and two-photon excited fluorescence (2PEF) of both collagen and elastin. Fiber dispersion is accurately compensated by the use of a specific grism-pair stretcher, allowing laser pulse temporal width around 70 fs and excitation wavelength tunability from 790 to 900 nm. This spectrometer was used to investigate the excitation wavelength dependence (from 800 to 870 nm) of SHG and 2PEF spectra originating from ex vivo human lung tissue samples. The results were compared with spectral responses of collagen gel and elastin powder reference samples and also with data obtained using standard nonlinear microspectroscopy. The excitation-wavelength-tunable nonlinear fiber-optic spectrometer presented in this study allows performing nonlinear spectroscopy of human lung tissue ECM through the elastin 2PEF and the collagen SHG signals. This work opens the way to tunable excitation nonlinear endomicroscopy based on both distal scanning of a single optical fiber and proximal scanning of a fiber-optic bundle.

Histologic and ultrastructural characterization of corneal femtosecond laser trephination.

Purpose: The purpose of this study was to evaluate the quality of femtosecond laser corneal trephination in eye bank eyes by histologic and ultrastructural investigation. Methods: We performed Z-shaped, tophat-shaped, and mushroom-shaped trephinations of swelled corneas from eye bank eyes using an Intralase FS60 system. The corneoscleral discs were fixed immediately after the laser procedure without removing the buttons. Thin and ultrathin tissue sections were examined by light and transmission electron microscopy. Results: Optical micrographs of the corneal tissue revealed that the femtosecond laser was efficient in producing Z-shaped, tophat-shaped, and mushroom-shaped dissections with reproducible high cut regularity. Investigations by transmission electron microscopy demonstrated that cut edges were of good quality devoid of thermal or mechanical damage of the adjacent tissues. However, cellular and collagenous nanometric debris was created by the laser. In the anterior stroma, they formed a layer of several microns in thickness residing on the terminated disrupted collagen fibers, whereas in the posterior stroma, they formed a thinner pseudomembrane running along the edges of the incision. Conclusions: Corneal trephination performed by the femtosecond laser preserves the ultrastructure of the disrupted collagen fibers. In edematous corneas, a layer of cellular and collagenic debris thicker in the anterior stroma and thinner in the posterior stroma runs along the edges of the incision obtained at a constant laser energy density.

Laser parameters, focusing optics, and side effects in femtosecond laser corneal surgery

Nowadays, femtosecond lasers are routinely used in refractive eye surgery. Until recently, commercialised clinical systems were exclusively based on ytterbium or neodymium-doped solid state lasers emitting sub-picosecond pulses at a wavelength of about 1 μm and repetition rates of a few 10 kHz. These systems use pulse energies in the μJ range and focussing optics of NA = 0.3 to 0.5. Recent developments have provided a variety of alternative and equally viable approaches: systems are now available using nJ pulses at high numerical apertures and MHz repetition rates - an approach so far only used for femtosecond cell surgery - and fibre laser technology is now being used for femtosecond laser corneal surgery. Recent research has also provided more insight in side effects occurring in present systems: self focusing phenomena and so far unexplained periodical structures have been observed even at high numerical apertures (NA >> 0.5) and moderate pulse energies. The interaction of femtosecond laser pulses with strongly scattering tissue has been studied in view of extending the application of femtosecond lasers to keratoplasty for opaque corneas and to glaucoma surgery. The use of new laser wavelengths and adaptive optics has been proposed. Despite the reputation of femtosecond surgical systems for their precision, repeatability and the absence of secondary effects or complications, a closer examination reveals the presence of subtle phenomena which merit further investigation. We present three of these phenomena: the influence of optical aberration on the quality of the incision, the occurrence of filamentation effects, and the deposit of microscopic glass fragments when performing penetrating incisions.

Spectral and lifetime domain measurements of rat brain tumours

During glioblastoma surgery, delineation of the brain tumour margins remains difficult especially since infiltrated and normal tissues have the same visual appearance. This problematic constitutes our research interest. We developed a fibre-optical fluorescence probe for spectroscopic and time domain measurements. First measurements of endogenous tissue fluorescence were performed on fresh and fixed rat tumour brain slices. Spectral characteristics, fluorescence redox ratios and fluorescence lifetime measurements were analysed. Fluorescence information collected from both, lifetime and spectroscopic experiments, appeared promising for tumour tissue discrimination. Two photon measurements were performed on the same fixed tissue. Different wavelengths are used to acquire two-photon excitation-fluorescence of tumorous and healthy sites.

Label free multiphoton imaging of human pulmonary tissues through two-meter-long microstructured fiber and multicore image-guide

This work deals with label free multiphoton imaging of the human lung tissue extra-cellular matrix (ECM) through optical fibers. Two devices were developed, the first one using distal scanning associated to a double clad large mode area (LMA) air-silica microstructured fiber, the second one using proximal scanning of a miniature multicore image guide (30000 cores inside a 0.8 mm diameter). In both cases, the main issue has been efficient linear and nonlinear distortion pre-compensation of excitation pulses. By inserting before the delivery fiber a compact (10 cm × 10 cm footprint) grisms-based stretcher (a grating in close contact with a prism) made of readily available commercial components, we achieved as short as 35-femtosecond-duration pulses that were temporally compressed at the direct exit of a 2-meter-long fiber. Interestingly, this femtosecond pulse fiber delivery device is also wavelength tunable over more than 100 nm inside the Ti: Sapphire emission band. With the help of distal scan system, those unique features allowed us to record elastin (through two-photon fluorescence) and collagen (through second harmonic generation) fibered network images. These images were obtained ex-vivo with only 15 mW @ 80 MHz of IR radiation delivered to the alveoli or bronchus tissues. 3D imaging with 400-μm-penetration depth inside the tissue was possible working with a 2-meter-long LMA fiber. With the help of proximal scanning, the miniature image guide allowed us to perform endoscopic real time microimaging of the ECM ex vivo.

Toward nonlinear endomicroscopy for exploration of the pulmonary airways: preliminary spectroscopic study of human lung tissue using a single-mode fibre

We are developing a non-linear fibered endomicroscope for imaging the extracellular matrix collagen and elastin fibrillar networks during bronchoscopy. As a proof of concept, laser pulses at the output of a standard 2 meter long single-mode fibre have been obtained with pulse duration of about 50 fs and pulse energy up to 50 uJ, using a specially designed grism line for the dispersion compensation. With these pulses, we performed a spectroscopic characterization of the non-linear endogenous signal, consisting of two-photon fluorescence and second harmonic generation, and originated from human pulmonary tissue of various thickness, both in forward and backward geometry of signal collection, with excitation at 830 nm.

New compact femtosecond laser source for penetrating keratoplasty at 1.65 µm

Femtosecond laser surgery in the volume of corneal tissue is typically performed wavelengths of about 1 μm, which gives excellent results on transparent corneas. However, the outcome is much worse in the case of oedematous or pathological corneas as the laser beam propagation is disturbed by optical scattering. Our studies suggest that this phenomenon can be greatly reduced by using a better suited laser wavelength. Best results are obtained at 1.65 μm. Currently, no compact femtosecond laser at this wavelength is commercially available. We have developed a new simple, compact and stable laser source consisting of a non linear crystal pumped by a compact commercial solid-state laser emitting at 1.03 μm in a configuration of an Optical Parametric Generation (OPG). The output wavelength of this system can be tuned in the spectral range of 1.45 - 1.8 μm. A series of ex vivo penetrating incisions using energies of the order of a few microjoules on corneal tissues have been performed while varying the wavelengths from 1.45 μm to 1.7 μm. The results have been compared to experiments performed at 0.8 μm and 1 μm. The use of longer infrared wavelengths around 1.65 μm for femtosecond laser keratoplasty significantly improves the quality and the penetration depth of incision in case of pathological tissues, without inducing any additional side effects.