A new method for in vivo assessment of corneal transparency using spectral-domain OCT.

Corneal transparency is essential to provide a clear view into and out of the eye, yet clinical means to assess such transparency are extremely limited and usually involve a subjective grading of visible opacities by means of slit-lamp biomicroscopy. Here, we describe an automated algorithm allowing extraction of quantitative corneal transparency parameters with standard clinical spectral-domain optical coherence tomography (SD-OCT). Our algorithm employs a novel pre-processing procedure to standardize SD-OCT image analysis and to numerically correct common instrumental artifacts before extracting mean intensity stromal-depth (z) profiles over a 6-mm-wide corneal area. The z-profiles are analyzed using our previously developed objective method that derives quantitative transparency parameters directly related to the physics of light propagation in tissues. Tissular heterogeneity is quantified by the Birge ratio Br and the photon mean-free path (ls) is determined for homogeneous tissues (i.e., Br~1). SD-OCT images of 83 normal corneas (ages 22-50 years) from a standard SD-OCT device (RTVue-XR Avanti, Optovue Inc.) were processed to establish a normative dataset of transparency values. After confirming stromal homogeneity (Br <10), we measured a median ls of 570 µm (interdecile range: 270-2400 µm). By also considering corneal thicknesses, this may be translated into a median fraction of transmitted (coherent) light Tcoh(stroma) of 51% (interdecile range: 22-83%). Excluding images with central saturation artifact raised our median Tcoh(stroma) to 73% (interdecile range: 34-84%). These transparency values are slightly lower than those previously reported, which we attribute to the detection configuration of SD-OCT with a relatively small and selective acceptance angle. No statistically significant correlation between transparency and age or thickness was found. In conclusion, our algorithm provides robust and quantitative measurements of corneal transparency from standard SD-OCT images with sufficient quality (such as 'Line' and 'CrossLine' B-scan modes without central saturation artifact) and addresses the demand for such an objective means in the clinical setting.

Full-field Optical Coherence Microscopy for Histology-like Analysis of Stromal Features in Corneal Grafts.

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.

Quantitative measures of corneal transparency, derived from objective analysis of depth-resolved corneal images, demonstrated with full-field optical coherence tomographic microscopy.

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.

Stromal striae: a new insight into corneal physiology and mechanics.

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.

Cell viability and shock wave amplitudes in the endothelium of porcine cornea exposed to ultrashort laser pulses.

PURPOSE: Some forms of keratoplasty assisted by ultrashort-pulse lasers require performing laser cuts close to the endothelium, which requires the knowledge of "safe" values concerning incision depth and pulse energy preserving endothelial cell viability. Our study aims to determine the thresholds for cell death in porcine corneas exposed to ultrashort laser pulses, in terms of laser pulse energy and nearness of the impacts to the endothelium. METHODS: Using a laboratory laser set-up, lamellar cuts were induced while varying pulse energies and distances from the endothelium. A fluorescent staining protocol was used to determine the percentage of surviving endothelial cells. Numerical simulations of the Euler equations for compressible fluids provided pressure level and axial and radial pressure gradient estimates at the endothelium. RESULTS: Ninety percent of the endothelial cells survived when using 16.5 µJ pulses no closer than 200 µm to the endothelium, or pulses not exceeding 2 µJ at a distance of 50 µm. The comparison of the observed percentage of surviving cells with the estimates of the shock wave amplitudes and gradients generated by the laser pulses yielded cell death thresholds at amplitudes in the megapascal range, or gradients of the order of 108 Pa/m. CONCLUSIONS: Our results provide limits in terms of pulse energy and distance of the incision from the endothelium within which endothelial cell viability is preserved. Current forms of corneal laser surgery are compatible with these limits. However, these limits will need to be considered for the development of future laser routines working in close proximity to the endothelium.

Multimodal Highlighting of Structural Abnormalities in Diabetic Rat and Human Corneas.

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.

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.

Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy.

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.

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.

Self-focusing and spherical aberrations in corneal tissue during photodisruption by femtosecond laser.

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.

Harmonic microscopy of isotropic and anisotropic microstructure of the human cornea.

In this study we present combined third-harmonic generation (THG) and second-harmonic generation (SHG) microscopy images of intact human corneas, and we analyze experimentally and theoretically the origin of the THG signal. Multiharmonic microscopy provides detailed images of the cornea microstructure over its entire thickness. A component of the THG signal originates from cellular structures and another one originates from anisotropy changes between successive collagen lamellae in the stroma. This anisotropy-related signal can be specifically detected using circular incident polarization, and provide contrasted images of the stacking and tissue-scale heterogeneity of stromal lamellae. Forward-radiated THG and SHG signals are generally anticorrelated, indicating that maximum THG is obtained from lamellar interfaces whereas maximum SHG is obtained from within lamellae. Polarization-resolved THG imaging reflects the a ernate anisotropy directions of the lamellae. We present a model for THG imaging of layered anisotropic samples and numerical calculations that account for our observations.

Multimodal nonlinear imaging of the human cornea.

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.

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.

Deposit of glass fragments during femtosecond laser penetrating keratoplasty.

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 microJ, and the "depth into glass" parameter at 50 microm, 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 incisions were obtained with an experimental laser source with 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 mum 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 stopping the procedure at the lens-cornea interface.

Towards new indices of arterial stiffness using systolic pulse contour analysis: a theoretical point of view.

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) x ST] / (SV x Deltat), 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 Deltat 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.

In situ monitoring of second-harmonic generation in human corneas to compensate for femtosecond laser pulse attenuation in keratoplasty.

The application of femtosecond lasers in corneal transplant surgery requires high pulse energies to compensate for the strong optical scattering in pathological corneas. However, excessive energies deteriorate the quality of the incisions. The aim of this study is to demonstrate the dependence of side effects on local radiant exposure, numerical aperture, and tissue properties, to quantify the penetration depth of the laser for individual corneas, and to provide a method for optimizing the energy in the volume of the cornea. We examine histological and ultrastructural sections of clear and edematous corneas with perforating and lamellar incisions performed at different pulse energies. We demonstrate that the augmented energies in edematous corneas may result in unwanted side effects even when using high numerical apertures. The dependence of the laser beam penetration depth on pulse energy is evaluated by histology and an exponential decrease is observed. We show that the penetration length can be determined by evaluating the backscattered second-harmonic emission associated with the nonlinear optical properties of the tissue. This approach represents a noninvasive method for the in situ quantification of the laser beam attenuation, enabling us to adapt the pulse energy accordingly. Experiments using adapted energies show that the side effects are minimized.

Quasi-spherical focal spot in two-photon scanning microscopy by three-ring apodization.

We present a beam-shaping technique for two-photon excitation (TPE) fluorescence microscopy. We show that by inserting a properly designed three-ring pupil filter in the illumination beam of the microscope, the effective optical sectioning capacity of such a system improves so that the point spread function gets a quasi-spherical shape. Such an improvement, which allows the acquisition of 3D images with isotropic quality, is obtained at the expense of only a small increase of the overall energy in the axial sidelobes. The performance of this technique is illustrated with a scanning TPE microscopy experiment in which the image of small beads is obtained. We demonstrate an effective narrowing of 12.5% in the axial extent of the point spread function, while keeping the 82% of the spot-fluorescence efficiency.