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The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

We present an automated data analysis procedure for clinical spectral-domain OCT, capable of correcting instrument-related hyperreflective artifacts, and its application to normal corneas. Quantitative parameters related to corneal transparency are extracted from 85 eyes.

International audience; Loss of corneal transparency, as occurs with various pathologies, infections, immune reactions, trauma, aging, and surgery, is a major cause of visual handicap worldwide. However, current means to assess corneal transparency are extremely limited and clinical and eye-bank practice usually involve a subjective and qualitative observation of opacities, sometimes with comparison against an arbitrary grading scale, by means of slit-lamp biomicroscopy. Here, we describe a novel objective optical data analysis-based method that enables quantifiable and standardized characterization of corneal transparency from depth-resolved corneal images, addressing the demand for such a means in both the laboratory and clinical ophthalmology setting. Our approach is based on a mathematical analysis of the acquired optical data with respect to the light attenuation from scattering processes in the corneal stroma. Applicable to any depth-resolved corneal imaging modality, it has been validated by means of full-field optical coherence tomographic microscopy (FF-OCT or FF-OCM). Specifically, our results on ex-vivo corneal specimens illustrate that 1) in homogeneous tissues, characterized by an exponential light attenuation with stromal depth (z), the computation of the scattering mean-free path (ls) from the rate of exponential decay allows quantification of the degree of transparency; 2) in heterogeneous tissues, identified by significant deviations from the normal exponential z -profile, a measure of exponential-decay model inadequacy (e.g., by computation of the Birge ratio) allows the estimation of severity of stromal heterogeneity, and the associated depth-dependent variations around the average ls enables precise localization of the pathology.

Lack of corneal transparency is a major cause of blindness worldwide. However, means to assess corneal transparency are limited and in current clinical and eye-bank practice usually involve a subjective and qualitative observation of opacities, sometimes with comparison against an arbitrary grading scale, by means of slit-lamp biomicroscopy. To address this unmet need, we have developed a method for corneal transparency assessment based on a new optical data analysis-based approach. Our method allows the objective extraction of quantitative parameters (including the scattering mean-free path, ls, a major indicator of scattering extent and thus of transparency of a medium) based on a physical model of corneal transparency and has been validated by laboratory experiments, using high-resolution, ex-vivo “full-field” optical coherence tomography (FF-OCT). Here, we apply our algorithm to depth-resolved spectral domain OCT (SD-OCT) images of in-vivo corneas and demonstrate the feasibility of our approach by means of four representative clinical cases. Specifically, we illustrate its potential in discriminating between the four clinical cases and, if applicable, deriving the scattering mean-free path as a quantitative measure of corneal transparency from objective analysis of stromal light backscattering (attenuation of the coherent mean) with SD-OCT. This measure may be related to, or expressed as, Strehl ratio reduction and thus retinal PSF broadening. As such, our approach not only has the potential to supply the demand for an objective means to quantify corneal transparency in the clinical setting, but also to create an association with visual function.

We demonstrate the feasibility of deriving quantitative measures of corneal transparency from objective analysis of stromal light backscattering with full-field optical coherence tomography (FF-OCT), addressing an unmet need in ophthalmology.

We uncover the significance of a previously unappreciated structural feature in corneal stroma, important to its biomechanics. Vogt striae are a known clinical indicator of keratoconus, and consist of dark, vertical lines crossing the corneal depth. However we detected stromal striae in most corneas, not only keratoconus. We observed striae with multiple imaging modalities in 82% of 118 human corneas, with pathology-specific differences. Striae generally depart from anchor points at Descemet’s membrane in the posterior stroma obliquely in a V-shape, whereas in keratoconus, striae depart vertically from posterior toward anterior stroma. Optical coherence tomography shear wave elastography showed discontinuity of rigidity, and second harmonic generation and scanning electron microscopies showed undulation of lamellae at striae locations. Striae visibility decreased beyond physiological pressure and increased beyond physiological hydration. Immunohistology revealed striae to predominantly contain collagen VI, lumican and keratocan. The role of these regions of collagen VI linking sets of lamellae may be to absorb increases in intraocular pressure and external shocks.

Dargestellt wird eine Zusammenfassung von Studien uber die Bedeutung der Laserwellenlange fur die Ultrakurzpulslaser-assistierte Keratoplastik. Der Beitrag bietet eine Zusammenfassung der wesentlichen fur die Transparenz der Hornhaut verantwortlichen physikalischen Mechanismen und berichtet uber Transparenzmessungen sowie Laborexperimente mit Lasern in Gewebe bei verschiedenen Wellenlangen. Die Transparenz gesunder Hornhaut hangt stark mit ihrer regelmasigen Struktur auf mikrometrischen und nanometrischen Grosenskalen zusammen. Viele Indikationen fur die Keratoplastik gehen mit einer Storung dieser Struktur und daher einer teilweise stark reduzierten Gewebetransparenz einher. Dies erklart die oft unbefriedigenden Ergebnisse beim Einsatz von Ultrakurzpulslasern fur diese Prozedur. Theoretische Uberlegungen und Laborversuche zeigen, dass die die Strahlqualitat des Lasers vermindernden Lichtstreuprozesse stark von der Lichtwellenlange abhangen und bei Verwendung von langeren als den derzeit genutzten Wellenlangen praktisch vollstandig unterdruckt werden konnen. Es bietet sich hier die Nutzung eines spektralen Transparenzbereichs bei 1,65 µm an. Die Verwendung von Laserwellenlangen nahe 1,65 µm stellt eine interessante Alternative zur Verbesserung der Ultrakurzpulslaser-assistierten Keratoplastik in stark pathologischem Hornhautgewebe dar.

International audience; Purpose : Clinical surgical laser systems based on ultrashort pulse lasers have become increasingly widespread in corneal surgery. Some forms of keratoplasty require performing laser cuts close to the endothelium, using “safe” values concerning incision depth and pulse energy in order to avoid damaging the endothelium.We present a study on the viability of endothelial cells of porcine corneas exposed to ultrashort laser pulses. The thresholds for cell death in terms of laser pulse energy and closeness of the impacts to the endothelium were compared to numerical simulations of the maximum shock wave amplitude at the corneal endothelium.Methods : Experiments were performed with a laboratory set-up based on a laser emitting 730 fs pulses at a wavelength of 1030 nm in corneas from eyes of pigs aged of about 6 months. Lamellar cuts were induced, first, using pulse energies of 16.5 µJ at distances from the endothelium from 50 µm to 400 µm, and then, at a fixed distance of 50 µm from the endothelium with pulse energies from 2 µJ to 14 µJ. The triple Hoechst 33342-Ethidium-Calcein staining protocol was used to determine the viability of endothelial cells after the laser cut. Numerical simulations of the Euler equations for compressible fluids provided pressure level estimates at the corneal endothelium. The broadening of the laser spot in the volume of the cornea was taken into account for the calculations.Results : Ninety percent of the endothelial cells survived when using 16.5 µJ pulses not closer than 200 µm to the endothelium, or pulses not exceeding 2 µJ at a distance of 50 µm.The comparison of the observed cell viability percentage with the calculated estimates of the shock wave amplitudes generated by the laser pulses yielded cell death thresholds at amplitudes in the Megapascal range.Conclusions : Our results permit to define limits in terms of pulse energy and distance of the incision to the endothelium within which the viability of the endothelial cells is preserved. The parameters used for current forms of corneal surgery assisted by ultrashort pulse lasers are compatible with these limits. However, these limits will need to be taken into account for the development of future laser routines working closely to the endothelium as cell death threshold may be reached under those conditions.

We present an ultrahigh resolution spectral-domain optical coherence tomography imaging system using a broadband superluminescent diode light source emitting at a center wavelength of 1.3 μm. The light source consists of two spectrally shifted superluminescent diodes that are coupled together into a single mode fiber. The effective emission power spectrum has a full width at half maximum of 200 nm and the source output power is 10 mW. The imaging system has an axial resolution of 3.9 μm in air (

Resume Nous presentons un bilan du projet NOUGAT qui a ete finance dans l’edition 2008 du programme TecSan de l’Agence nationale de la recherche. Le projet vise a la conception et a la realisation d’un dispositif innovant de chirurgie oculaire par laser a impulsions ultrarapides pour la chirurgie du glaucome assistee par un systeme d’imagerie in situ. Les avancees significatives obtenues au cours du projet sont entre autres la realisation d’une source laser a impulsions ultrabreves puissante et compacte dont la longueur d’onde d’emission est adaptee a la transparence de la sclere et le developpement d’un systeme d’imagerie optimise base sur la tomographie par coherence optique.

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.

In this study we investigate the viability of endothelial cells in porcine corneas after exposure to ultrashort laser pulses under experimental conditions similar to keratoplasty (corneal grafting). Cell death and survival thresholds are determined and compared to estimated amplitudes of the shock waves generated by the laser pulses.

Assessment of stromal light backscattering with full-field optical coherence tomographic microscopy shows promise as objective means to quantify corneal transparency, addressing an unmet yet critical need in anterior segment ophthalmology.

The concept of parallel confocal microscopy with aperture de-correlation has been developed in order to reduce acquisition time and to increase light efficiency over conventional scanning confocal microscopy. It consists in simultaneously observing numerous confocal volumes of a specimen, in lieu of scanning one confocal spot over the region of interest. The cross-talk between neighbouring parallel confocal systems is eliminated by capturing images using a sequence of linearly independent illumination and detection patterns which permit to de-correlate the confocal from the non-confocal image. In this paper, we compare the relative efficiency of parallel and conventional confocal microscopy, using a simple analytical approach, and show that this depends strongly upon the illumination configuration. In particular it is shown that despite its drawbacks in other applications, conventional confocal microscopy still is more efficient than parallel confocal microscopy when the dose, i.e. the total energy onto the sample, must be limited. This may represent a limitation to the advent of parallel confocal microscopy for the imaging of photo-sensitive tissues.

Erosion and deposition at the vessel walls of the main chamber of JET were measured with long term samples during the whole operation period of the Mark I carbon divertor from April 1994 until March 1995. Assuming toroidal symmetry, about 70 g Ni + Cr + Fe was found to be eroded from the inner torus wall and about 55 g Be from the outer torus wall due to sputtering by energetic charge-exchange neutrals. Deposition has been measured on a poloidal section of the Mark I C divertor and on tiles from the inner and outer wall limiters. Eroded material is redeposited in the divertor and in the main chamber where it is found predominantly at the sides of the poloidal limiters and at the outer vessel wall. At these deposition-dominated areas in total about 25 g Ni + Cr + Fe, 28 g Be and 370 g C are found. The carbon originates mostly from the carbon limiters; additionally some may be originating from the divertor plates.

Elastic Recoil Detection Analysis (ERDA) with primary ion beams having energies in the MeV/nucleon range allows the measurement of absolute concentrations and depth profiles especially of low Z elements in the surface layers of a solid. In the present work, we use simple mathematical relations for defining the optimal measurement geometry with respect to measuring large depths with high intensities. Results obtained by the straightforward “surface approximation” are shown and compared to calculations with a more complete model. A parameter discussion is given examining optimal geometries for different experimental set-ups. When using detectors sensitive to very low energies in order to be able to analyse large depths, the intensity loss due to the typically smaller solid angles of these detectors can be compensated by the higher intensity of the recoils in the corresponding geometry.

We have determined the threshold radiant exposure for cell death in the endothelium of porcine cornea exposed to ultrashort laser pulses in the context of keratoplasty and the preparation of endothelial grafts. In this study, by progressively increasing the radiant threshold towards the higher values we have observed a decrease of living corneal endothelial cells. Further study will address the effect of dose and possible mechanism behind cell death.

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 shortwaveinfrared 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 k ¼ 1650 nm rather than the wavelength of k ¼ 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.

International audience; PURPOSE: This study aimed to highlight structural corneal changes in a model of type 2 diabetes, using in vivo corneal confocal microscopy (CCM). The abnormalities were also characterized by transmission electron microscopy (TEM) and second harmonic generation (SHG) microscopy in rat and human corneas. METHODS: Goto-Kakizaki (GK) rats were observed at age 12 weeks (n = 3) and 1 year (n = 6), and compared to age-matched controls. After in vivo CCM examination, TEM and SHG microscopy were used to characterize the ultrastructure and the three-dimensional organization of the abnormalities. Human corneas from diabetic (n = 3) and nondiabetic (n = 3) patients were also included in the study. RESULTS: In the basal epithelium of GK rats, CCM revealed focal hyper-reflective areas, and histology showed proliferative cells with irregular basement membrane. In the anterior stroma, extracellular matrix modifications were detected by CCM and confirmed in histology. In the Descemet's membrane periphery of all the diabetic corneas, hyper-reflective deposits were highlighted using CCM and characterized as long-spacing collagen fibrils by TEM. SHG microscopy revealed these deposits with high contrast, allowing specific detection in diabetic human and rat corneas without preparation and characterization of their three-dimensional organization. CONCLUSION: Pathologic findings were observed early in the development of diabetes in GK rats. Similar abnormalities have been found in corneas from diabetic patients. TRANSLATIONAL RELEVANCE: This multidisciplinary study highlights diabetes-induced corneal abnormalities in an animal model, but also in diabetic donors. This could constitute a potential early marker for diagnosis of hyperglycemia-induced tissue changes.

Hyperglycemia-induced fibrillar collagen formation in the Descemet’s membrane is highlighted in diabetic rat corneas by in vivo confocal microscopy, in vivo SHG microscopy and electron microscopy. Abnormalities are also observed in ex vivo human corneas.

It is generally known that the microstructure of polycrystalline CVD diamond samples has a strong impact on their thermal properties. Despite the fact that nowadays layers can be deposited with macroscopic thermal conductivities or diffusivities rivalling those of type II natural diamonds, the samples are highly thermally inhomogeneous and sometimes show local values differing by up to two orders of magnitude. To examine these phenomena more closely, we present a microscopic photothermal measuring method for the local thermal diffusivity. We demonstrate the feasibility of diffusivity measurements at a sample surface of ca. 20 × 20 μm. We show results obtained on a reference sample of known diffusivity and present measurements on a small single crystal diamond, a local measurement at the substrate side of a CVD diamond layer, and a measurement of the diffusivity inside a microcrystal at the growth side of a CVD diamond layer.

Following constant improvement in the synthesis methods of diamond in metastable conditions by chemical vapour deposition, it is nowadays possible to grow polycrystalline diamond with thermal conductivities of over 20 W K−1 cm−1. This gives rise to applications of this material in the thermal management of components in optronics and microelectronics. Reliable, precise thermal characterization methods are therefore needed. However, measuring the thermal conductivity of CVD diamonds is a non-trivial task: the high conductivity makes it difficult to establish a measurable thermal gradient and furthermore, the different experimental setups are differently sensitive to the particular polycrystalline structure of the layers. A recent round-robin test revealed considerable differences in the values measured using the different techniques. In this paper the principal measuring methods for the thermal conductivity of CVD diamonds are reviewed and compared. Then the influence of the microstructure on the thermal properties as well as the correlations with the presence of impurities and crystal defects detected by Raman and luminescence spectroscopy are discussed. Les methodes de synthese par depǒt chimique en phase vapeur dans des conditions metastables ont fait de tels progres qu'il est aujourd'hui possible de faire pousser des couches de diamant polycristallin dont la conductivite thermique depasse 20 W K−1 cm−1. Cette valeur elevee ouvre un large champ d'applications dans le domaine de L'optronique et de la micro-electronique. Un besoin grandissant de methodes de caracterisation thermique fiables et precises se fait sentir. Cependant, mesurer la conductivite thermique d'un materiau est une tache ardue: il est en effet difficile d'etablir un gradient thermique vu la forte conductivite thermique des echantillons. De plus, la structure polycristalline des couches peut affecter plus ou moins les differents montages experimentaux. Recemment, un test comparatif a revele des differences considerables dans les resultats obtenus par les differentes techniques. Dans ce papier, nous voulons passer en revue et comparer les methodes de mesure utilisees pour la determination de la conductivite thermique du diamant CVD. Nous discuterons L'influence de la microstructure sur les proprietes thermiques et etablirons des correlations avec la presence d'impuretes et de defauts dans le cristal detectes par spectroscopie Raman et de luminescence.

A series of highly oriented and textured microwave CVD diamond films, where only the deposition time was varied, was deposited on silicon wafers in order to follow the evolution of the microstructure and defect content with film thickness. SEM, XRD, Raman spectroscopy, luminescence measurements, and ESR were applied to determine the microstructure and the defect density. It is shown that the growth of highly oriented and textured film proceeds in two stages. The first is characterized by marked changes of the microstructure (size, orientation, and shape of the grains) which directly influence the generation of defects. The second stage, which begins as soon as the film surface is covered with {100} tiles, leads to minor changes in the microstructure and defect incorporation. The defect-related part of the Raman spectrum has been decomposed into two contributions, the D-band which mainly responds to grain boundary defects, and the G-band which is more sensitive to volume defects. ESR measures volume defects, grain boundaries provide only a minor contribution. In this respect, ESR and Raman G-band are similar. {111} growth sectors produce defect densities which are three to four times higher than their {100} counterparts. The thermal conductivity is shown to depend not only on the grain boundary density but also on bulk defects. Optical transparency in the visible range is strongly affected by surface roughness, however, using a new method, it has been possible to determine an absorption coefficient for the second stage which is consistent with recently published literature data.

The outstanding thermal conductivity of CVD diamond gives rise to many potential industrial applications, necessitating the availability of reliable, standardized, thermal measurement methods. Although, in general, these measurements are in agreement for traditional materials, for CVD diamond different measurement methods can yield significantly different values for comparable or even identical samples. The aim of this work is to compare the methods and estimate to what extent these variations may be caused by the inhomogeneity and anisotropy of the samples themselves. Using a six-layer model to represent a typical CVD diamond layer, the potential signals for the principal methods are calculated and the results are compared.

Over 60 CVD diamond films with thicknesses in the range 2–600 μm, grown from C/H, C/H/O, C/H/Cl and C/H/N gas mixtures by microwave plasma CVD, combustion flame synthesis and r.f. plasma torch CVD, were compared in terms of their thermal, morphological, Raman and luminescence data. Correlation diagrams reveal that the content of sp2-hybridized carbon is the main factor determining the thermal properties of the films. Other parameters, e.g. thickness, crystallinity and defects, only influence the thermal performance by changing the phase purity. The presence of oxygen and nitrogen in the CVD gas phase restricts the thermal conductivity of the films to values well below the 2200 ± 200 W m−1 K−1 achieved for polycrystalline films, 250 μm thick, grown from methane and hydrogen. Diamond films with thicknesses of less than 4 μm and thermal conductivities of more than 700 W m−1 K−1 were grown from C/H and C/H/O mixtures.

Glaucoma is a disease of the optic nerve that is usually associated with an increased internal pressure of the eye and can lead to a decreased vision and eventually blindness. It is the second leading cause of blindness worldwide with more than 80 million people affected and approximately 6 million blind. The standard clinical treatment for glaucoma, after unsuccessful administration of eyedrops and other treatments, is performing incisional surgery. However, due to post-surgical complications like scarring and wound healing, this conventional method has a global success rate of only about 60%. In comparison, as femtosecond laser surgery may be performed in volume and is a priori less invasive and less susceptible of causing scarring, glaucoma laser surgery could be a novel technique to supplement the conventional glaucoma surgery. We have been working on the development of a new tool for glaucoma treatment that uses an optimized femtosecond laser source centered at 1.65 μm wavelength for making the surgery and an imaging system based on optical coherence tomography (OCT) for guiding the laser surgery. In this proceeding, we present the results obtained so far on the development and utilization of Fourier-domain OCT imaging system working at 1.3 μm center wavelength for guiding the laser incision. Cross-sectional OCT image of pathological human cornea showing the Schlemm's canal, where the surgery is intended to be done, is presented. By coupling OCT imaging system with the laser incision system, we also demonstrate real-time imaging of femtosecond laser incision of cornea.

International audience; BACKGROUND: Second Harmonic Generation (SHG) microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet's membrane, in the posterior cornea. METHODOLOGY/PRINCIPAL FINDINGS: We studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet's membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats. CONCLUSIONS/SIGNIFICANCE: Our study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies.

We present a variant of the traditional photoreflectance microscopy. It allows thermal diffusivity measurements on samples as small as a few tens of microns, possibly of mediocre surface quality.

We present measurements of the in-plane thermal diffusivity of free-standing diamond films of thicknesses from 0.3 to 6 μm. The photothermal method used for the measurements is non-destructive and contact-free, and does not require any sample preparation. The measured values lie in the range of 0.1 to 1.0 cm 2 s −1 , which is an order of magnitude below typical results obtained on bulk material. The results show a strong dependence of the thermal diffusivity on the film thickness and morphology. Phonon scattering at grain boundaries was identified as the dominating process limiting the heat diffusion. The availability of sets of films with different characteristics allowed the determination of the spatial dependence of the heat diffusivity even in very thin membranes. The observed effects are in good accordance with the extrapolation of measurements previously performed on much thicker samples. The data are correlated to results gained by Raman spectroscopy and spectroscopic ellipsometry.

The optical properties of the cornea have been a research subject of great interest for many years. Several early theories have been put forward to explain with more or less success the optical transparency of this tissue, but it was not until Maurice demonstrated in a very elegant way during the 50s that this optical transparency could be explained by the regular ultrastructure of the cornea. When becoming edematous, the cornea's ultrastructure is perturbed and the tissue becomes a strongly scattering medium. With the emergence of ophthalmologic surgery by ultrashort pulse lasers in recent years, a regain of interest in the subject of corneal transparency arose. However, relatively little and no recent data of transparency spectra measurements covering a large wavelength range is available in the literature. The purpose of this study is to provide quantitative values for light scattering and its relation to the degree of edema by measuring the spectrum of transmitted light through corneas presenting different degrees of edema. This paper focus on the comparison of laboratory measurements published earlier with a new simple method we propose We also for eye banks to quantitatively measure the degree of transparency of corneal grafts by measuring the modulation transfer function of a Siemens star viewed through a corneal graft. Indeed, there is no current method to determine the transparency of corneal graft but the subjectivity of the laboratory technician or the ophthalmic surgeon.

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.

In the last decade, femtosecond laser applications in the biology and medicine have largely developed. The ultrashort laser has proved to be a strongly accurate and selective cutting tool, by depositing energy in a very confined volume with minimum collateral effects.

Purpose We realized an innovative device for ocular surgery by ultrafast pulse laser optimised for corneal grafting. Methods We constructed a demonstrator device that reproduces surgical conditions of corneal transplant. It is thus possible to realize with the help of an easy handling automatised interface all the kinds of already existing corneal transplants. Also, in order to maximize the spatial quality of the beam, a wavefront correction system using a deformable mirror module has been added. The Demonstrator contains an erbium fiber laser emitting at 1,6 microns. This laser delivers a beam of a few Joule with pulse duration of 700 femtoseconds and a repetition rate of 100-200 KHz. It includes deformable mirrors permitting horizontal displacements and a wavefront sensor. It also contains the administration system of the laser beam. Results The experiments carried out with a surgical tunable source confirmed the initial assumptions: the penetration depth is limited to wavelengths close to 1 microns. When increasing the wavelength, the drop of the scattering compensates the absorption and therefore the penetration depth is slowly varying when increasing the wavelength. The laser does not penetrate near the maximum of the water absorption band located at 1,45 microns. However, the use of a wavelength of 1,6 micros enables an important increasing of penetration depth (factor 3) while conserving the same energy of current technologies. Conclusion The use of a laser source with a wavelength corresponding to the window of transparency of the cornea (1,65 microns) permits to increase both the penetration depth of an ultrafast laser source and the cut quality.

International audience; 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.

International audience; The use of ultrashort pulse lasers is current in refractive surgery and has recently been extended to corneal grafting (keratoplasty). When performing keratoplasty, however, permanent degradation of the optical properties of the patient's cornea compromises the penetration depth of the laser and the quality of the incisions, therefore causing unwanted secondary effects. Additionally, corneal grafting needs considerably higher penetration depths than refractive surgery. Little data are available about the interaction processes of the femtosecond pulses in the volume of pathological corneas—i.e., in the presence of spherical aberrations and optical scattering. We investigate the influence of the focusing numerical aperture on the laser–tissue interaction. We point out that at low numerical apertures (NAs), tissue damage is produced below and above the focal region. We attribute this phenomenon to nonlinear self-focusing effects. On the other hand, at high NAs, spherical aberrations become significant when focusing at high depths for posterior surgeries, which also limit the cutting efficiency. As high NAs are advisable for reducing unwanted nonlinear effects and ensure accurate cutting, particular attention should be paid to aberration management when developing clinical femtosecond lasers.

PURPOSE. To evaluate the potential of third-harmonic generation (THG) microscopy combined with second-harmonic generation (SHG) and two-photon excited fluorescence (2PEF) microscopies for visualizing the microstructure of the human cornea and trabecular meshwork based on their intrinsic nonlinear properties. METHODS. Fresh human corneal buttons and corneoscleral discs from an eye bank were observed under a multiphoton microscope incorporating a titanium-sapphire laser and an optical parametric oscillator for the excitation, and equipped with detection channels in the forward and backward directions. RESULTS. Original contrast mechanisms of THG signals in cornea with physiological relevance were elucidated. THG microscopy with circular incident polarization detected microscopic anisotropy and revealed the stacking and distribution of stromal collagen lamellae. THG imaging with linear incident polarization also revealed cellular and anchoring structures with micrometer resolution. In edematous tissue, a strong THG signal around cells indicated the local presence of water. Additionally, SHG signals reflected the distribution of fibrillar collagen, and 2PEF imaging revealed the elastic component of the trabecular meshwork and the fluorescence of metabolically active cells. CONCLUSIONS. The combined imaging modalities of THG, SHG, and 2PEF provide key information about the physiological state and microstructure of the anterior segment over its entire thickness with remarkable contrast and specificity. This imaging method should prove particularly useful for assessing glaucoma and corneal physiopathologies. (Invest Ophthalmol Vis Sci. 2010;51:2459‐2465) DOI:10.1167/iovs.09-4586

International audience; Nous présentons un premier bilan du projet Greco qui vise la conception et la réalisation d'un dispositif innovateur de chirurgie oculaire par laser à impulsions ultrarapides optimisé pour la greffe de cornée.

The thermal diffusivity of dry human epidermis was determined in vitro by studying thermal wave propagation in thin epidermal layers at frequencies between 10 and 200 Hz. Transmission measurements were performed on samples applied to a plane copper support at the underside of which thermal waves were generated by means of a square voltage controlled power transistor. Additionally, measurements were performed on epidermal layers with metal and air backing, in which thermal waves were generated by the absorption of intensity modulated light in a thin, superficially applied graphite layer (short and open circuit measurements). Thermal waves were detected by means of the laser beam deflection technique which allows the contactless measurement of the oscillatory surface temperature of a sample with respect to amplitude and phase. A critical discussion of methods shows that the thermal diffusivity is most reliably determined by transmission experiments. From experimental data obtained by this method a mean value alpha = (2.8 +/- 0.9) x 10(-4) cm2 s-1 was evaluated for the thermal diffusivity of dry epidermis.

International audience; Femtosecond laser surgery in the volume of corneal tissue is difficult in the case of oedematous or pathological corneas: in those corneas, the propagation of the laser beam is perturbed by the optical scattering. This phenomenon can be greatly reduced by using a better suited laser wavelength. A series of ex vivo surgical experiments has been conducted at wavelengths around 1600 nm. The results have been compared to experiments performed at 800 nm and 1000 nm. We have compared penetration depth and incision quality as a function of wavelength and energy.

We present a femtosecond Optical Parametric Amplificator (OPA) which was conceived to perform surgical interventions on eyes using eye-safe wavelengths. It is pumped by a Ti: sapphire laser emmiting 50 fs pulses at 820 nm and at a repetition rate of 1 kHz. A particular effort was made to obtain a very flat and efficient gain spectrum especially around degeneracy. An OPA scheme using consecutively β-BaB 2 O 4 (BBO) crystal and BiB 3 O 6 (BiBO) provides the required behaviour (figure 1). While most OPA are composed of identical crystals on all stages, the originality of our configuration consists in combining the advantages of two different crystals.

International audience; The specular transmittance spectrum of human corneas is studied using a confocal geometry set-up. The comparison of the obtained spectrum with the total transmittance spectrum permits the determination of the stromal scattering spectrum of pathological corneas. The dependence of the scattering cross section on wavelength dependence is analyzed.

International audience; New, compact, and wavelength-optimized ultrashort-pulse laser systems may facilitate corneal grafting and enable new forms of glaucoma surgery.

International audience; Background: To evaluate femtosecond laser interaction with the applanation lens during pre-programmed penetrating keratoplasty corneal cuts. Methods: Three different-shaped penetrating keratoplasty dissections were performed on edematous corneas from bank eyes using a clinical femtosecond laser system (Intralase FS60) with energies higher than 2 μJ, and the “depth into glass” parameter at 50 μm, which is defined as the length over which the laser interacts with the glass of the applanation cone in contact with the cornea. Additional full-thickness corneal incisionswere obtainedwith an experimental laser sourcewith technical characteristics similar to the clinical laser. Following cutting, tissue sections were examined by optical microscopy (OM), transmission electron microscopy (TEM), and electron energy loss spectroscopy (EELS). After the procedure, the cones were examined by optical and scanning electron microscopy (SEM). A control was obtained by repeating the procedures and stopping the laser at the cornea–lens interface. Results: OM and TEM analysis of the tissue showed the presence of solid particles of a maximum dimension of 1.5 μm on the epithelium and the anterior stroma, regardless of the laser system used to cut. The EELS technique revealed their composition as silicon dioxide. We believe that the fragments originate from the applanation cone, which is machined by the laser interacting with the glass in contact with cornea. This is consistent with the structures observed on the lens by OM and SEM. Radial and circumferential tracks on the surface of the lens are visible, corresponding to the laser path in penetrating keratoplasty protocols. No particles were found in the control samples. Conclusions: When performing penetrating keratoplasty corneal cuts by infra-red femtosecond laser, the applanation lens in contact with the cornea is machined by the laser depending on the system parameters. As a consequence, microscopic glass fragments are created, which may remain in the tissue. This unwanted effect can be avoided by opping the procedure at the lens-cornea interface.

International audience; Total arterial stiffness plays a contributory role throughout aging and in numerous cardiovascular diseases, including hypertension. Aortic stiffening is responsible for an increased characteristic impedance (ie, the impedance to the left ventricular pulsatile flow), thus increasing the forward pressure-wave amplitude that contributes to pulse pressure elevation. Aortic stiffening also increases pulse wave velocity, and this results in anticipated and enhanced wave reflections, further augmenting central pulse pressure. Unfortunately, there is no simple time-domain estimate of characteristic impedance. Furthermore, recent guidelines have reviewed the limitations of diastolic pulse contour analysis to estimate arterial stiffness in the time domain. The present theoretical article proposes that systolic pulse contour analysis may provide new, simple time-domain indices quantifying pulsatile load in resting humans. Our proposal was mainly based on 2 simple, validated assumptions: (1) a linear aortic pressure-flow relationship in early systole and (2) a triangular aortic flow wave during systole. This allowed us to describe new time-domain estimates of characteristic impedance, pulsatile load (waveguide ratio), total arterial compliance, and total arterial stiffness. It is demonstrated that total arterial stiffness may be estimated by the following formula: [(Pi - DAP) × ST] / (SV × Δt), where Pi is the aortic pressure at the inflection point (peak forward pressure wave), DAP is diastolic aortic pressure, ST is systolic ejection time, SV is stroke volume, and Δt is the time-to-Pi. A mathematical relationship among time intervals and indices of pulsatile load is demonstrated, and the clinical implications are discussed in terms of cardiovascular risk and stroke volume prediction.

International audience; 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.

International audience; Femtosecond lasers start to be routinely used in refractive eye surgery. Current research focuses on their application to glaucoma and cataract surgery as well as cornea transplant procedures. To avoid unwanted tissue damage during the surgical intervention it is of utmost importance to maintain a working energy just above the ablation threshold and maintain the laser energy at this working point independently of the local and global tissue properties. To quantify the attenuation of the laser power density in the tissue by absorption, scattering and modification of the point spread function we monitor the second harmonic radiation generated in the collagen matrix of the cornea when exposed to ultrashort laser pulses. We use a CPA system with a regenerative amplifier delivering pulses at a wavelength of 1.06 µm, pulse durations of 400 fs and a maximum energy of 60 µJ. The repetition rate is adjustable from single shot up to 10 kHz. The experiments are performed on human corneas provided by the French Eye bank. To capture the SHG radiation we use a photomultiplier tube connected to a lockin amplifier tuned to the laser repetition rate. The measured data indicates an exponential decay of the laser beam intensity in the volume of the sample and allows for the quantification of the attenuation coefficient and its correlation with the optical properties of the cornea. Complementary analyses were performed on the samples by ultrastructural histology.