Comparison of morphological changes of corneal collagen fibers treated with collagen crosslinking agents using second harmonic generation images.

Corneal cross-linking (CXL) is a common surgical procedure used to modify corneal biomechanics and stabilize keratoconus progression which is still under discussion. Its side effects, which are mostly related to anatomical unpredictability and stromal exposure, are the reason for the search for new CXL agents. In this work we have quantitatively evaluated the porcine corneal stroma architecture treated with collagen crosslinking agents such as riboflavin solutions and açai extract, using second harmonic generation microscopy. Aimed at evaluating the morphological changes in the corneal stroma after collagen crosslinking under a CXL chemical agent, a tubeness filter based Hessian matrix to obtain a 3D fiber characterization of the SHG images was applied. The results showed a curling effect and shortening of the collagen fibers treated with açai as compared to the control. They also showed a higher degree of clustering of the collagen fibers with larger empty spaces when compared to the other two groups. We believe that studies such as these presented in this paper are a good direct nondestructive and free labeling evaluation technique that allows the observation of morphologic features of corneas treated with new CXL agents.

Methacrylated gelatin hydrogels as corneal stroma substitutes: in vivo study.

Methacrylated gelatin (GelMA) hydrogels were prepared to serve as corneal stroma equivalents. They were highly transparent (ca. 95% at 700 nm), mechanically strong and withstood handling and had high human corneal keratocyte viability (98%) after 21 days of culture period. In order to test the in vivo performance of the cell free GelMA hydrogels a pilot in vivo study was carried out using eyes of two white New Zealand rabbits. Hydrogel was implanted in a mid-stromal pocket created and without suture fixation, and observed for 8 weeks under a slit lamp. No edema, ulcer formation, inflammation or infection was observed in both the control (sham) and hydrogel implanted corneas. Corneal vascularization on week 3 was treated with one dose of anti-VEGF application. Hematoxylin and Eosin staining showed that the hydrogel was integrated with the host tissue with only a minimal foreign body reaction. Results demonstrated some degradation in the construct within 8 weeks as evidenced by the decrease of the diameter of the hydrogel from 4 mm to 2.6 mm. High transparency, adequate mechanical strength, biocompatibility and well integration with the host tissue, indicates that this hydrogel is a viable alternative to the current methods for the treatment of corneal blindness and deserves testing on larger number of rabbits and more extensively using microscopy, histology and immune histochemistry.

Compressed collagen intermixed with cornea-derived decellularized extracellular matrix providing mechanical and biochemical niches for corneal stroma analogue.

Compressed collagen is a promising scaffold for corneal stroma analogue due to its facile incorporation of keratocytes while mimicking the mechanical niche of a native cornea with dense collagen fibrillar structures. However, it does not offer the sufficient biochemical niche crucial for in vivo-like quiescent keratocyte phenotype. In this study, we engineered a scaffold for a corneal stroma analogue that mimics both the mechanical and biochemical niches of the corneal stroma by introducing cornea-derived decellularized extracellular matrix (Co-dECM) to the collagen compression process. The compressed collagen intermixed with Co-dECM (COLEM; Co-dECM content, <50 wt%) maintained a uniform structure and showed an elastic modulus and tensile strength on the order of 100 kPa, which is comparable with that of conventional compressed collagen. The COLEM with the 50 wt% Co-dECM content was found to possess 2-fold higher amount of the glycosaminoglycans as compared to the compressed collagen. The biochemical components of Co-dECM in the COLEM were verified to significantly promote the expression of quiescent keratocyte-specific genes, i.e., KERA and ALDH3A1, while improving the optical transmittance of the COLEM by reducing the diameter of collagen fibrils. The ability of the COLEM to construct multicellular in vitro corneal tissue was demonstrated by an additional corneal epithelial cell culture. The results support the hypothesis that COLEM has strong potential use in the development of corneal equivalent for in vitro models and tissue transplantation.

Potential role of stromal collagen in cystine crystallization in cystinosis patients.

Cystinosis is a genetic disease that leads to the accumulation of intracellular cystine crystals in all organs including cornea due to the loss of cystine efflux transporters in the lysosome of the cells. While the mechanism for formation of intracellular cystine is well understood for most organs, it does not explain many observations for crystal accumulation in the cornea of cystinosis patients. First, the crystals in cornea are extracellular and needle-like with several hundred microns length which is in sharp contrast with the rectangular or hexagonal crystals found throughout other organs. Second, these crystals are arranged parallel to the stromal collagen, which is a unique to the cornea. Third, crystal growth in the cornea reaches a saturation point after where no further crystallization occurs. We propose a hypothesis supported by in vitro and ex vivo data to explain these observations. We hypothesize that the stroma crystals form extracellularly due to the ionic interactions between the cystine diffusing into the eye and collagen fibrils present in the stroma. We examine cystine crystal growth both with in vitro polymer solutions and ex vivo in rabbit cadaver eyes to show that negatively charged polymers lead to the formation of more cystine precipitation in aqueous solution and that needle-like cystine crystal formation can be observed only in presence of certain polyelectrolytes including collagen. This proposed mechanism explains many of the yet unanswered questions but it needs further support from in vivo studies. The improved understanding could lead to improved treatment of corneal cystinosis.

Measuring the depth of crosslinking demarcation line in vivo: Comparison of methods and devices.

PURPOSE: To assess the interrelationship of different methods of measuring the demarcation line depth after corneal collagen crosslinking (CXL). SETTING: University eye clinic, Oslo, Norway. DESIGN: Prospective case series. METHODS: Eyes having CXL for progressive keratoconus were evaluated 1 month after CXL by in vitro confocal microscopy (IVCM), optical coherence tomography (OCT), and Scheimpflug imaging. When applying IVCM, the depth of the CXL demarcation line was measured with 2 methods; that is, IVCM keratocyte disappearance and IVCM intensity increase. With OCT, the evaluations were made by measuring the depth of the corneal stromal demarcation line. Scheimpflug imaging was used with 2 depth-measuring methods; that is manual Scheimpflug and objective Scheimpflug intensity change. The demarcation line depths in the central cornea were compared by the intraclass correlation coefficient (ICC) and pairwise comparison of the measured treated depth. If acceptable correlations (ICC > 0.7) were found, Bland-Altman analysis was performed. RESULTS: Twenty eyes of 20 patients were evaluated. Acceptable correlations were found between depth measurements using OCT-IVCM keratocyte disappearance (ICC = 0.80), OCT-IVCM intensity increase (ICC = 0.75), and IVCM intensity increase-IVCM keratocyte disappearance (ICC = 0.91). The Bland-Altman plots of these 3 pairs showed sufficient levels of agreement. Using pairwise comparison of these pairs, the measured depths were in the same level by the OCT-IVCM intensity increase only (P = .529). CONCLUSIONS: Scheimpflug images were inaccurate for measuring the CXL demarcation line depth. The 2 confocal microscopy methods and OCT images showed good correlation. Of these 3 pairs, only measurements with OCT and IVCM intensity increase depths were in the same level.

Nonlinear optical corneal collagen crosslinking of ex vivo rabbit eyes.

PURPOSE: To determine whether riboflavin-induced collagen crosslinking (CXL) could be precisely achieved in the corneal stroma of ex vivo rabbit eyes using nonlinear optical excitation with a low numerical aperture lens and enlarged focal volume. SETTING: Gavin Herbert Eye Institute, University of California Irvine, Irvine, California, USA. DESIGN: Experimental study. METHODS: The corneal epithelium was removed and the corneas were soaked in 0.5% riboflavin solution. Using a 0.1 numerical aperture objective, a theoretical excitation volume of 150 µm × 3 µm was generated using 1 W of 760 nm femtosecond laser light and raster scanned with 4.4 µm line separation at varying effective speeds over a 4.50 mm × 2.25 mm area. Corneal sections were examined for collagen autofluorescence. RESULTS: Collagen autofluorescence was enhanced 2.9 times compared with ultraviolet-A (UVA) CXL. Also, increasing speed was linearly associated with decreasing autofluorescence intensity. The slowest speed of 2.69 mm/s showed a mean of 182.97 µm ± 52.35 (SD) long autofluorescent scan lines axially in the central cornea compared with 147.84 ± 4.35 µm for UVA CXL. CONCLUSIONS: Decreasing dwell time was linearly associated with decreasing autofluorescence intensity, approaching that of UVA CXL at a speed of 8.9 mm/s. Using an effective speed of 8.9 mm/s, nonlinear optical CXL could be achieved over a 3.0 mm diameter area in fewer than 4 minutes. Further development of nonlinear optical CXL might result in safer, faster, and more effective CXL treatments. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.

Decellularization of human stromal refractive lenticules for corneal tissue engineering.

Small incision lenticule extraction (SMILE) becomes a procedure to correct myopia. The extracted lenticule can be used for other clinical scenarios. To prepare for allogeneic implantation, lenticule decellularization with preserved optical property, stromal architecture and chemistry would be necessary. We evaluated different methods to decellularize thin human corneal stromal lenticules created by femtosecond laser. Treatment with 0.1% sodium dodecylsulfate (SDS) followed by extensive washes was the most efficient protocol to remove cellular and nuclear materials. Empty cell space was found inside the stroma, which displayed aligned collagen fibril architecture similar to native stroma. The SDS-based method was superior to other treatments with hyperosmotic 1.5 M sodium chloride, 0.1% Triton X-100 and nucleases (from 2 to 10 U/ml DNase and RNase) in preserving extracellular matrix content (collagens, glycoproteins and glycosaminoglycans). The stromal transparency and light transmittance was indifferent to untreated lenticules. In vitro recellularization showed that the SDS-treated lenticules supported corneal stromal fibroblast growth. In vivo re-implantation into a rabbit stromal pocket further revealed the safety and biocompatibility of SDS-decellularized lenticules without short- and long-term rejection risk. Our results concluded that femtosecond laser-derived human stromal lenticules decellularized by 0.1% SDS could generate a transplantable bioscaffold with native-like stromal architecture and chemistry.

An Ultra-thin Amniotic Membrane as Carrier in Corneal Epithelium Tissue-Engineering.

Amniotic membranes (AMs) are widely used as a corneal epithelial tissue carrier in reconstruction surgery. However, the engineered tissue transparency is low due to the translucent thick underlying AM stroma. To overcome this drawback, we developed an ultra-thin AM (UAM) by using collagenase IV to strip away from the epithelial denuded AM (DAM) some of the stroma. By thinning the stroma to about 30 µm, its moist and dry forms were rendered acellular, optically clear and its collagen framework became compacted and inerratic. Engineered rabbit corneal epithelial cell (RCEC) sheets generated through expansion of limbal epithelial cells on UAM were more transparent and thicker than those expanded on DAM. Moreover, ΔNp63 and ABCG2 gene expression was greater in tissue engineered cell sheets expanded on UAM than on DAM. Furthermore, 2 weeks after surgery, the cornea grafted with UAM based cell sheets showed higher transparency and more stratified epithelium than the cornea grafted with DAM based cell sheets. Taken together, tissue engineered corneal epithelium generated on UAM has a preferable outcome because the transplanted tissue is more transparent and better resembles the phenotype of the native tissue than that obtained by using DAM for this procedure. UAM preserves compact layer of the amniotic membrane and maybe an ideal substrate for corneal epithelial tissue engineering.

Supra-molecular assembly of a lumican-derived peptide amphiphile enhances its collagen-stimulating activity.

C16-YEALRVANEVTLN, a peptide amphiphile (PA) incorporating a biologically active amino acid sequence found in lumican, has been examined for its influence upon collagen synthesis by human corneal fibroblasts in vitro, and the roles of supra-molecular assembly and activin receptor-like kinase ALK receptor signaling in this effect were assessed. Cell viability was monitored using the Alamar blue assay, and collagen synthesis was assessed using Sirius red. The role of ALK signaling was studied by receptor inhibition. Cultured human corneal fibroblasts synthesized significantly greater amounts of collagen in the presence of the PA over both 7-day and 21-day periods. The aggregation of the PA to form nanotapes resulted in a notable enhancement in this activity, with an approximately two-fold increase in collagen production per cell. This increase was reduced by the addition of an ALK inhibitor. The data presented reveal a stimulatory effect upon collagen synthesis by the primary cells of the corneal stroma, and demonstrate a direct influence of supra-molecular assembly of the PA upon the cellular response observed. The effects of PA upon fibroblasts were dependent upon ALK receptor function. These findings elucidate the role of self-assembled nanostructures in the biological activity of peptide amphiphiles, and support the potential use of a self-assembling lumican derived PA as a novel biomaterial, intended to promote collagen deposition for wound repair and tissue engineering purposes.

Size-Dependent Diffusion of Dextrans in Excised Porcine Corneal Stroma.

Delivery of therapeutic agents to the eye requires efficient transport through cellular and extracellular barriers. We evaluated the rate of diffusive transport in excised porcine corneal stroma using fluorescently labeled dextran molecules with hydrodynamic radii ranging from 1.3 to 34 nm. Fluorescence correlation spectroscopy (FCS) was used to measure diffusion coefficients of dextran molecules in the excised porcine corneal stroma. The preferential sensitivity of FCS to diffusion along two dimensions was used to differentially probe diffusion along the directions parallel to and perpendicular to the collagen lamellae of the corneal stroma. In order to develop an understanding of how size affects diffusion in cornea, diffusion coefficients in cornea were compared to diffusion coefficients measured in a simple buffer solution. Dextran molecules diffuse more slowly in cornea as compared to buffer solution. The reduction in diffusion coefficient is modest however (67% smaller), and is uniform over the range of sizes that we measured. This indicates that, for dextrans in the 1.3 to 34 nm range, the diffusion landscape of corneal stroma can be represented as a simple liquid with a viscosity approximately 1.5 times that of water. Diffusion coefficients measured parallel vs. perpendicular to the collagen lamellae were indistinguishable. This indicates that diffusion in the corneal stroma is not highly anisotropic. Our results support the notion that the corneal stroma is highly permeable and isotropic to transport of hydrophilic molecules and particles with hydrodynamic radii up to at least 34 nm.

Quantitative analysis of collagen lamellae in the normal and keratoconic human cornea by second harmonic generation imaging microscopy.

PURPOSE: To characterize the structural properties of collagen lamellae in the normal and keratoconic human corneal stroma, we measured their width and angle relative to Bowman's layer (BL). METHODS: Thirteen normal and four keratoconic corneas were examined. Collagen lamellae in tissue blocks from the central cornea were visualized by second harmonic generation imaging microscopy. Images obtained in 1-µm steps from BL to Descemet's membrane (DM) were subjected to three-dimensional reconstruction. The reconstructed data sets were divided into 10 layers of equal depth (L1-L10) for analysis. The width of lamellae adherent to BL (L0) was also determined. RESULTS: For the normal cornea, the width (mean ± SD) of collagen lamellae was 6.5 ± 1.7 µm at L0, decreased to 4.3 ± 1.3 µm at L1, and then increased gradually with progression toward DM to 122.2 ± 34.5 µm at L10, whereas the angle of lamellae was 20.9° ± 5.4° at L1 and decreased initially to 10.6° ± 3.2° at L2 before declining gradually to 2.7° ± 2.2° at L10. The width and angle of collagen lamellae in the keratoconic cornea were significantly larger and smaller, respectively, relative to those in the normal cornea. CONCLUSIONS: In the normal human cornea, collagen lamellae adjacent to BL are narrow and form a steep angle with BL, whereas they increase in width and their angle relative to BL flattens with progression toward DM. These properties of collagen lamellae are altered in keratoconus and are likely related to abnormalities of corneal shape.

An association between corneal inflammation and corneal lymphangiogenesis after keratoplasty.

PURPOSE: To examine the relationship between corneal inflammation and corneal lymphangiogenesis after keratoplasty. METHODS: Rat corneal lymphangiogenesis was examined by lymphatic vessel endothelial receptor (LYVE-1) immunohistochemistry and whole mount immunofluorescence at 1, 3, 7, 10, and 14 days after corneal transplantation. Corneal inflammation was evaluated by inflammation index (IF) grading and NF-κB immunohistochemistry at the same time points. The association between lymphatic vessel counting (LVC) and the IF scores was then examined. RESULTS: LYVE-1 positive lymphatic vessels occurred in the corneal stroma on day 3, developed throughout days 7 and 10, and peaked in number at day 14 after keratoplasty. Corneal inflammation was strong on day 3, and then resolved gradually, but increased again from days 7 to 14 after the transplantation. LVC was strongly and positively correlated with IF after keratoplasty (r = 0.41; P < 0.05). However, changes in IF scores and LVC were not parallel. CONCLUSION: A close, but not parallel, relationship was found between corneal lymphangiogenesis and corneal inflammation after corneal transplantation.

Imaging the intact mouse cornea using coherent anti-stokes Raman scattering (CARS).

PURPOSE: The aim of this study was to image the cellular and noncellular structures of the cornea and limbus in an intact mouse eye using the vibrational oscillation of the carbon-hydrogen bond in lipid membranes and autofluorescence as label-free contrast agents. METHODS: Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Sequential images were collected through the full thickness of the cornea and limbal regions. Line scans along the transverse/sagittal axes were also performed. RESULTS: Analysis of multiple CARS/TPAF images revealed that corneal epithelial and endothelial cells could be identified by the lipid-rich plasma membrane CARS signal. The fluorescent signal from the collagen fibers of the corneal stroma was evident in the TPAF channel. The transition from the cornea to sclera at the limbus was marked by a change in collagen pattern (TPAF channel) and thickness of surface cells (CARS channel). Regions within the corneal stroma that lack collagen autofluorescence coincided with CARS signal, indicating the presence of stromal fibroblasts or nerve fibers. CONCLUSIONS: The CARS technique was successful in imaging cells in the intact mouse eye, both at the surface and within corneal tissue. Multiphoton images were comparable to histologic sections. The methods described here represent a new avenue for molecular specific imaging of the mouse eye. The lack of need for tissue fixation is unique compared with traditional histology imaging techniques.

Quantification of collagen ultrastructure after penetrating keratoplasty - implications for corneal biomechanics.

PURPOSE: To quantify long-term changes in stromal collagen ultrastructure following penetrating keratoplasty (PK), and evaluate their possible implications for corneal biomechanics. METHODS: A pair of 16 mm post-mortem corneo-scleral buttons was obtained from a patient receiving bilateral penetrating keratoplasty 12 (left)/28 (right) years previously. Small-angle x-ray scattering quantified collagen fibril spacing, diameter and spatial order at 0.5 mm or 0.25 mm intervals along linear scans across the graft margin. Corresponding control data was collected from two corneo-scleral buttons with no history of refractive surgery. Wide-angle x-ray scattering quantified collagen fibril orientation at 0.25 mm (horizontal)×0.25 mm (vertical) intervals across both PK specimens. Quantification of orientation changes in the graft margin were verified by equivalent analysis of data from a 13 year post-operative right PK specimen obtained from a second patient in a previous study, and comparison made with new and published data from normal corneas. RESULTS: Marked changes to normal fibril alignment, in favour of tangentially oriented collagen, were observed around the entire graft margin in all PK specimens. The total number of meridional fibrils in the wound margin was observed to decrease by up to 40%, with the number of tangentially oriented fibrils increasing by up to 46%. As a result, in some locations the number of fibrils aligned parallel to the wound outnumbered those spanning it by up to five times. Localised increases in fibril spacing and diameter, with an accompanying reduction in matrix order, were also evident. CONCLUSIONS: Abnormal collagen fibril size and spatial order within the PK graft margin are indicative of incomplete stromal wound remodelling and the long term persistence of fibrotic scar tissue. Lasting changes in collagen fibril orientation in and around PK wounds may alter corneal biomechanics and compromise the integrity of the graft-host interface in the long term.

Rate dependent biomechanical properties of corneal stroma in unconfined compression.

The corneal stroma is a highly ordered extracellular matrix and mainly responsible for the mechanical strength of the cornea. The rate dependent mechanical and rheological properties of the cornea are not completely understood and there is large variation in the reported estimates. In this work, the rate dependent mechanical behavior of the corneal stroma was investigated using experimental studies and theoretical models. Unconfined compression stress-relaxation experiments at different displacement rates and compressive strains were conducted. The unconfined compression material parameters, i.e. corneal out-of-plane modulus, in-plane modulus and permeability coefficient were determined from curve-fitting the experimental data with a transversely isotropic biphasic model. It was found that the maximum force reached during the step loading increased with both increasing magnitude and rate of the compressive strain. It was also observed that at all loading rates the in-plane Young's modulus increased with increasing strain, while the permeability coefficient decayed with increasing compressive strain. At a constant compressive strain, both the in-plane Young's modulus and the permeability coefficient increased with increasing the loading rate. Regardless of loading rates and compressive strains, a range of corneal out-of-plane modulus of 0.6 kPa to 13.8 kPa, in-plane modulus of 0.5 MPa to 4.8 MPa, and permeability coefficient of 1×10⁻¹4 m4/N·s to 7×10⁻¹4 m4/N·s was found.

An x-ray scattering study into the structural basis of corneal refractive function in an avian model.

Avian vision diseases in which eye growth is compromised are helping to define what governs corneal shape and ultrastructural organization. The highly specific collagen architecture of the main corneal layer, the stroma, is believed to be important for the maintenance of corneal curvature and hence visual quality. Blindness enlarged globe (beg) is a recessively inherited condition of chickens characterized by retinal dystrophy and blindness at hatch, with secondary globe enlargement and loss of corneal curvature by 3-4 months. Here we define corneal ultrastructural changes as the beg eye develops posthatch, using wide-angle x-ray scattering to map collagen fibril orientation across affected corneas at three posthatch time points. The results disclosed alterations in the bulk alignment of corneal collagen in beg chicks compared with age-matched controls. These changes accompanied the eye globe enlargement and corneal flattening observed in affected birds, and were manifested as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Progressive remodeling of peripheral stromal collagen in beg birds posthatch may relate to the morphometric changes exhibited by the disease, likely as an extension of myopia-like scleral remodeling triggered by deprivation of a retinal image.

Ultrastructure alteration in the corneal stroma of hydrated camel corneoscleral button and corneal button.

PURPOSE: To investigate the changes in the ultrastructural architecture of hydrated corneoscleral button (HCSB) and hydrated corneal button (HCB). METHOD: Corneoscleral buttons (n = 4) and corneal buttons (n = 4) were hydrated in deionized water for 24 and 48 h and were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy. The tissue was dehydrated and embedded in TAAB 031 resin. Normal CSB and CB were also processed using the same method. Ultrathin sections were stained with uranyl acetate and lead citrate. The sections were observed under JEOL 1400 transmission electron microscope. RESULTS: In both HCSB and HCB, the lamellae were disorganized and proteoglycans (PGs) were degenerated and detached from the collagen fibrils (CFs). In the HCSB samples, interfibrillar spacing increased but the diameter of the CF had not changed compared with those in the normal tissue. Within the HCSB, the interfibrillar spacing in the posterior stroma was significantly higher compared with those in the anterior and middle stroma. In the HCB, both the interfibrillar spacing and CF diameter had significantly increased compared to those observed in both the HCSB and normal tissue. The CFs were severely damaged exhibiting very thin microfilaments within CFs. Remnants of the CFs were present in the stromal matrix. CONCLUSION: Hydration of the cornea in both the HCSB and HCB increases the interfibrillar spacing in the anterior, middle, and posterior stroma. The CF in the HCB was enlarged and had degenerated. This could be due to penetration of water into the intermolecular spacing within the CF.

Yellow corneal ring associated with vitamin supplementation for age-related macular degeneration.

PURPOSE: To report the first described cases of peripheral yellow corneal rings secondary to vitamin supplementation for age-related macular degeneration (ARMD). DESIGN: Retrospective single-center case series. PARTICIPANTS: The eyes of 4 patients taking vitamin supplementation for ARMD were examined at the University of Pittsburgh Medical Center Department of Ophthalmology between January 2010 and April 2011. METHODS: We reviewed the medical records of 4 patients with peripheral corneal rings receiving vitamin supplementation for ARMD. MAIN OUTCOME MEASURES: The presence of peripheral yellow corneal rings, skin findings, and serum carotene levels. RESULTS: Each patient had circumferential, yellow, peripheral corneal rings and exhibited subtle yellowing of the skin most notable on the palms. Serum carotene levels were normal in 2 of the 3 patients and markedly elevated in the last patient in whom it was measured. CONCLUSIONS: It is unclear at this time how to counsel patients with this ocular finding. We suspect that these rings are more common than generally appreciated because they may have a subtle appearance or be misdiagnosed as arcus senilis. We suggest that a formal study be performed on a cohort of patients taking vitamin supplementation for macular degeneration that specifically screens for yellow rings and measures serum carotene levels when they are identified.

Rapid thermal equilibration of differentially heated protein and water in bovine corneal stroma.

We measure and simulate the thermal response of bovine corneal stroma to a picosecond IR heating pulse. A thermal diffusion model is developed for this tissue based on the spatial distribution and properties of protein and water constituents in the stroma. In this idealized model, differentially heated protein and water constituents thermally equilibrate with a thermalization time of 515 ps. Using transient absorption spectroscopy for picosecond protein thermometry, a significantly faster thermalization time of 165 ps is measured. The implications of this faster than expected thermalization for the energy-partition model of short-pulse mid-IR tissue ablation are discussed.