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Collagen XIV is poorly characterized in the body, and the current knowledge of its function in the cornea is limited. The aim of the current study was to elucidate the role(s) of collagen XIV in regulating corneal stromal structure and function. Analysis of collagen XIV expression, temporal and spatial, was performed at different postnatal days (Ps) in wild-type C57BL/6 mouse corneal stromas and after injury. Conventional collagen XIV null mice were used to inquire the roles that collagen XIV plays in fibrillogenesis, fibril packing, and tissue mechanics. Fibril assembly and packing as well as stromal organization were evaluated using transmission electron microscopy and second harmonic generation microscopy. Atomic force microscopy was used to assess stromal stiffness. Col14a1 mRNA expression was present at P4 to P10 and decreased at P30. No immunoreactivity was noted at P150. Abnormal collagen fibril assembly with a shift toward larger-diameter fibrils and increased interfibrillar spacing in the absence of collagen XIV was found. Second harmonic generation microscopy showed impaired fibrillogenesis in the collagen XIV null stroma. Mechanical testing suggested that collagen XIV confers stiffness to stromal tissue. Expression of collagen XIV is up-regulated following injury. This study indicates that collagen XIV plays a regulatory role in corneal development and in the function of the adult cornea. The expression of collagen XIV is recapitulated during wound healing.

Polyethylene glycol (PEG)–based conformal coating (CC) encapsulation of transplanted islets is a promising β cell replacement therapy for the treatment of type 1 diabetes without chronic immunosuppression because it minimizes capsule thickness, graft volume, and insulin secretion delay. However, we show here that our original CC method, the direct method, requiring exposure of islets to low pH levels and inclusion of viscosity enhancers during coating, severely affected the viability, scalability, and biocompatibility of CC islets in nonhuman primate preclinical models of type 1 diabetes. We therefore developed and validated in vitro and in vivo, in several small- and large-animal models of type 1 diabetes, an augmented CC method—emulsion method—that achieves hydrogel CCs around islets at physiological pH for improved cytocompatibility, with PEG hydrogels for increased biocompatibility and with fivefold increase in encapsulation throughput for enhanced scalability.

The meniscus is a fibrocartilaginous tissue that plays an essential role in load transmission, lubrication, and stabilization of the knee. Loss of meniscus function, through degeneration or trauma, can lead to osteoarthritis in the underlying articular cartilage. To perform its crucial function, the meniscus extracellular matrix has a particular organization, including collagen fiber bundles running circumferentially, allowing the tissue to withstand tensile hoop stresses developed during axial loading. Given its critical role in preserving the health of the knee, better understanding structure-function relations of the biomechanical properties of the meniscus is critical. The main objective of this study was to measure the compressive modulus of porcine meniscus using Atomic Force Microscopy (AFM); the effects of three key factors were investigated: direction (axial, circumferential), compartment (medial, lateral) and region (inner, outer). Porcine menisci were prepared in 8 groups (= 2 directions x 2 compartments x 2 regions) with n = 9 per group. A custom AFM was used to obtain force-indentation curves, which were then curve-fit with the Hertz model to determine the tissue’s compressive modulus. The compressive modulus ranged from 0.75 to 4.00 MPa across the 8 groups, with an averaged value of 2.04±0.86MPa. Only direction had a significant effect on meniscus compressive modulus (circumferential > axial, p = 0.024), in agreement with earlier studies demonstrating that mechanical properties in the tissue are anisotropic. This behavior is likely the result of the particular collagen fiber arrangement in the tissue and plays a key role in load transmission capability. This study provides important information on the micromechanical properties of the meniscus, which is crucial for understanding tissue pathophysiology, as well as for developing novel treatments for tissue repair.

We report a water-soluble and non-toxic method to incorporate additional extracellular matrix proteins into gelatin hydrogels, while obviating the use of chemical crosslinkers such as glutaraldehyde. Gelatin hydrogels were fabricated using a range of gelatin concentrations (4%-10%) that corresponded to elastic moduli of approximately 1 kPa-25 kPa, respectively, a substrate stiffness relevant for multiple cell types. Microbial transglutaminase was then used to enzymatically crosslink a layer of laminin on top of gelatin hydrogels, resulting in 2-component gelatin-laminin hydrogels. Human induced pluripotent stem cell derived spinal spheroids readily adhered and rapidly extended axons on GEL-LN hydrogels. Axons displayed a more mature morphology and superior electrophysiological properties on GEL-LN hydrogels compared to the controls. Schwann cells on GEL-LN hydrogels adhered and proliferated normally, displayed a healthy morphology, and maintained the expression of Schwann cell specific markers. Lastly, skeletal muscle cells on GEL-LN hydrogels achieved long-term culture for up to 28 days without delamination, while expressing higher levels of terminal genes including myosin heavy chain, MyoD, MuSK, and M-cadherin suggesting enhanced maturation potential and myotube formation compared to the controls. Future studies will employ the superior culture outcomes of this hybrid substrate for engineering functional neuromuscular junctions and related organ on a chip applications.

The mouse lens is frequently used both in vivo and ex vivo in ophthalmic research to model conditions affecting the human lens, such as presbyopia. The mouse lens has a delicate structure which is prone to damage and biomechanical changes both before and after extraction from the whole globe. When not properly controlled for, these changes can confound the biomechanical analysis of mouse lenses. In this study, atomic force microscopy microindentation was used to assess changes in the Young's Modulus of Elasticity of the mouse lens as a function of mouse age and postmortem time. Old mouse lenses measured immediately postmortem were significantly stiffer than young mouse lenses (p = 0.028). However, after 18 h of incubation, there was no measurable difference in lens stiffness between old and young mouse lenses (p = 0.997). This demonstrates the need for careful experimental control in experiments using the mouse lens, especially regarding postmortem time.

Islet transplantation within mechanically stable microcapsules offers the promise of long-term diabetes reversal without chronic immunosuppression. Reinforcing the ionically gelled network of alginate (ALG) hydrogels with covalently linked polyethylene glycol (PEG) may create hybrid structures with desirable mechanical properties. This report describes the fabrication of hybrid PEG-ALG interpenetrating polymer networks and the investigation of microcapsule swelling, surface modulus, rheology, compression, and permeability. It is demonstrated that hybrid networks are more resistant to bulk swelling and compressive deformation and display improved shape recovery and long-term resilience. Interestingly, it is shown that PEG-ALG networks behave like ALG during microscale surface deformation and small amplitude shear while exhibiting similar permeability properties. The results from this report's in vitro characterization are interpreted according to viscoelastic polymer theory and provide new insight into hybrid hydrogel mechanical behavior. This new understanding of PEG-ALG mechanical performance is then linked to previous work that demonstrated the success of hybrid polymer immunoisolation devices in vivo.

Elevated intraocular pressure (IOP) is a risk factor in glaucoma, a group of irreversible blinding diseases. Endogenous lipids may be involved in regulation of IOP homeostasis. We present comparative fold analysis of phospholipids and sphingolipids of aqueous humour and trabecular meshwork from human control vs primary open‐angle glaucoma and mouse control (normotensive) vs ocular hypertensive state. The fold analysis in control vs disease state was based on ratiometric mass spectrometric data for above classes of lipids. We standardized in vitro assays for rapid characterization of lipids undergoing significant diminishment in disease state. Evaluation of lipids using in vitro assays helped select a finite number of lipids that may potentially expand cellular interstitial space embedded in an artificial matrix or increase fluid flow across a layer of cells. These assays reduced a number of lipids for initial evaluation using a mouse model, DBA/2J with spontaneous IOP elevation. These lipids were then used in other mouse models for confirmation of IOP lowering potential of a few lipids that were found promising in previous assessments. Our results provide selected lipid molecules that can be pursued for further evaluation and studies that may provide insight into their function.

Purpose The aim of this study was to determine the roles of collagen XII in the regulation of stromal hierarchical organization, keratocyte organization, and corneal mechanics. Methods The temporal and spatial expression of collagen XII at postnatal days 4, 10, 30, 90, and 150 were evaluated in wild-type (WT) mice. The role of collagen XII in hierarchical organization was analyzed by measuring fibril diameter and density, as well as stromal lamellar structure, within ultrastructural micrographs obtained from WT and collagen XII-deficient mice (Col12a1–/–). Keratocyte morphology and networks were assessed using actin staining with phalloidin and in vivo confocal microscopy. The effects of collagen XII on corneal biomechanics were evaluated with atomic force microscopy. Results Collagen XII was localized homogeneously in the stroma from postnatal day 4 to day 150, and protein accumulation was shown to increase during this period using semiquantitative immunoblots. Higher fibril density (P < 0.001) and disruption of lamellar organization were found in the collagen XII null mice stroma when compared to WT mice. Keratocyte networks and organization were altered in the absence of collagen XII, as demonstrated using fluorescent microscopy after phalloidin staining and in vivo confocal microscopy. Corneal stiffness was increased in the absence of collagen XII. Young's modulus was 16.2 ± 5.6 kPa in WT and 32.8 ± 6.4 kPa in Col12a1–/– corneas. The difference between these two groups was significant (P < 0.001, t-test). Conclusions Collagen XII plays a major role in establishing and maintaining stromal structure and function. In the absence of collagen XII, the corneal stroma showed significant abnormalities, including decreased interfibrillar space, disrupted lamellar organization, abnormal keratocyte organization, and increased corneal stiffness.

PURPOSE This study presents a method to quantify micro-stiffness variations in experimental arteriovenous fistulae (AVF). METHODS AVF created by anastomosing the superficial epigastric vein to the femoral artery in Sprague-Dawley rats were allowed to remodel for 21 days before being harvested and preserved in culture medium. A custom atomic force microscope was used to measure microvascular stiffness (Young's modulus) in three areas of the AVF: the inflow artery, the juxta-anastomotic area, and the outflow vein. Morphometric measurements and collagen and elastin contents were also determined. RESULTS Atomic force microscopy indentation revealed an increased stiffness in the juxta-anastomotic area of the AVF compared to the outflow vein and inflow artery. The juxta-anastomotic area was also significantly stiffer than the contralateral vein. The lack of elasticity (higher Young's modulus) of the juxta-anastomotic region was associated with a thicker vascular wall that was rich in collagen but poor in elastin. CONCLUSIONS This study demonstrates for the first time the feasibility of using atomic force microscopy to measure local stiffness variations in experimental AVF. This technique could be instrumental in advancing our understanding of how micro-spatial organization of the AVF wall determines the overall biomechanical performance of this type of vascular access.

The purpose of this study was to evaluate elasticity and viscoelasticity in the anterior and deeper stromal regions of the cornea after cross linking with three different protocols using atomic force microscopy (AFM) through indentation. A total of 40 porcine corneas were used in this study and were divided into 4 groups (10 corneas per group): control (no treatment), Dresden (corneal epithelial debridement, riboflavin pretreatment for 30 min and a 3mw/cm(2) for 30 min UVA irradiation), accelerated (corneal epithelial debridement, riboflavin pretreatment for 30 min and a 30mw/cm(2) for 3 min UVA irradiation), and genipin (corneal epithelial debridement and submersion of anterior surface in a 1% genipin solution for 4 h). Elasticity and viscoelasticity were quantified using AFM through indentation for all corneas, for the anterior stroma and at a depth of 200 μm. For the control, Dresden, accelerated, and genipin groups, respectively, the average Young's modulus for the anterior stromal region was 0.60 ± 0.58 MPa, 1.58 ± 1.04 MPa, 0.86 ± 0.46 MPa, and 1.71 ± 0.51 MPa; the average for the 200 μm stromal depth was 0.08 ± 0.06 MPa, 0.08 ± 0.04 MPa, 0.08 ± 0.04 MPa, and 0.06 ± 0.01 MPa. Corneas crosslinked with the Dresden protocol and genipin were significantly stiffer than controls (p 0.05) in the anterior region only. For the control, Dresden, Accelerated, and genipin groups, respectively, the average calculated apparent viscosity for the anterior stroma was 88.2 ± 43.7 kPa-s, 8.3 ± 7.1 kPa-s, 8.1 ± 2.3 kPa-s, and 9.5 ± 3.8 kPa-s; the average for the 200 μm stromal depth was 35.0 ± 3.7 kPa-s, 49.6 ± 35.1 kPa-s, 42.4 ± 17.6 kPa-s, and 41.8 ± 37.6 kPa-s. All crosslinking protocols resulted in a decrease in viscosity in the anterior region only (p 0.05). The effects of cross-linking seem to be limited to the anterior corneal stroma and do not extend to the deeper stromal region. Additionally, the Dresden and genipin protocols seem to produce a stiffer anterior corneal stroma when compared to the accelerated protocol.

Ph.D.Objectives: To determine the effect of hydration media on ex vivo cornealelasticity.Methods: Experiments wereconducted on 40porcineeyesretrievedfrom anabattoir (10 eyes each for phosphate-buffered saline (PBS), balanced saltsolution, Optisol,15%dextran). Theepithelium wasremoved,and thecorneawas excised with anintact scleral rim and placedin 20% dextran overnight torestore its physiological thickness. For each hydration media, corneas wereevenly divided into two groups: one with an intact scleral rim and the otherwithout. Corneas were mounted onto a custom chamber and immersed ina hydration medium for elasticity testing. Although in each medium, cornealelasticity measurements were performed for 2 hr: at 5-min intervals for thefirst 30 min and then 15-min intervals for the remaining 90 min. Elasticitytesting was performed using nanoindentation with spherical indenters, andYoung modulus was calculated using the Hertz model. Thickness measure-ments were taken before and after elasticity testing.Results: The percentage change in corneal thickness and elasticity wascalculated for each hydration media group. Balanced salt solution, PBS, andOptisol showed an increase in thickness and Young moduli for corneas withand without an intact scleral rim. Fifteen percent dextran exhibiteda dehydrating effect on corneal thickness and provided stable maintenanceof corneal elasticity for both groups.Conclusions: Hydration media affects the stability of corneal thickness andelasticity measurements over time. Fifteen percent dextran was most effectivein maintaining corneal hydration and elasticity, followed by Optisol.Key Words: Atomic force microscopy—Cornea—Mechanical properties—Hydration—Nanoindentantion.(Eye & Contact Lens 2014;0: 1–6)

The objective of this study is to further investigate the ultrastructural details of the surface of Bowman's membrane of the human cornea, using atomic force microscopy (AFM) images. One representative image acquired of Bowman's membrane of a human cornea was investigated. The three-dimensional (3-D) surface of the sample was imaged using AFM in contact mode, while the sample was completely submerged in optisol solution. Height and deflection images were acquired at multiple scan lengths using the MFP-3D AFM system software (Asylum Research, Santa Barbara, CA), based in IGOR Pro (WaveMetrics, Lake Oswego, OR). A novel approach, based on computational algorithms for fractal analysis of surfaces applied for AFM data, was utilized to analyze the surface structure. The surfaces revealed a fractal structure at the nanometer scale. The fractal dimension, D, provided quantitative values that characterize the scale properties of surface geometry. Detailed characterization of the surface topography was obtained using statistical parameters, in accordance with ISO 25178-2: 2012. Results obtained by fractal analysis confirm the relationship between the value of the fractal dimension and the statistical surface roughness parameters. The surface structure of Bowman's membrane of the human cornea is complex. The analyzed AFM images confirm a fractal nature of the surface, which is not taken into account by classical surface statistical parameters. Surface fractal dimension could be useful in ophthalmology to quantify corneal architectural changes associated with different disease states to further our understanding of disease evolution.

The purpose of this project was to assess anterior and posterior corneal stromal elasticity after corneal collagen cross linking (CXL) treatment in human cadaver eyes using Atomic Force Microscopy (AFM) through indentation. Twenty four human cadaver eyes (12 pairs) were included in this study and divided into 2 groups (6 pairs per group). In both groups, the left eye (OS) served as a control (no riboflavin or CXL treatment was performed) and the right eye (OD) underwent CXL treatment (30 min of riboflavin pretreatment followed by 30 min of exposure to 3 mW/cm 2 of ultraviolet light). In group 1, the anterior stroma was exposed by manual delamination of approximately 50 μm of the corneal stroma including Bowman's membrane. In group 2, the posterior stroma was exposed by delamination of the anterior 50% of the corneal stroma including Bowman's membrane. Delamination was performed after crosslinking treatment in the case of the treated eyes. In all eyes, the stromal elasticity was quantified using AFM through indentation. Young's modulus of elasticity for the anterior cornea (group 1) was 245.9 ± 209.1 kPa (range: 82.3–530.8 kPa) for the untreated control eyes, and 467.8 ± 373.2 kPa (range: 157.4–1126 kPa) for the CXL treated eyes. Young's modulus for the posterior cornea (group 2) was 100.2 ± 61.9 kPa (range: 28.1–162.6 kPa) for the untreated control eyes and 66.0 ± 31.8 kPa (range: 31.3–101.7 kPa) for the CXL treated eyes. Young's modulus of the anterior stroma significantly increased after CXL treatment ( p = 0.024), whereas the posterior stroma did not demonstrate a significant difference in Young's modulus after CXL treatment ( p = 0.170). The anterior stroma was stiffer than the posterior stroma for both the control and CXL treatment groups ( p = 0.077 and p = 0.023, respectively). Our findings demonstrate that stiffness of the anterior corneal stroma after CXL treatment seems to increase significantly, while the posterior stroma does not seem to be affected by CXL.

The purpose of this project is to measure the elasticity of the human and non-human primate lens capsule at the microscopic scale using Atomic Force Microscopy (AFM). Elasticity measurements were performed using AFM on the excised anterior lens capsule from 9 cynomolgus monkey (5.9–8.0 years), 8 hamadryas baboon (2.8–10.1 years), and 18 human lenses (33–79 years). Anterior capsule specimens were obtained by performing a 5mm continuous curvilinear capsulorhexis and collecting the resulting disk of capsular tissue. To remove the lens epithelial cells the specimen was soaked in 0.1% trypsin and 0.02% EDTA for five minutes, washed, and placed on a Petri dish and immersed in DMEM. Elasticity measurements of the capsule were performed with a laboratory-built AFM system custom designed for force measurements of ophthalmic tissues. The capsular specimens were probed with an AFM cantilever tip to produce force-indentation curves for each specimen. Young’s modulus was calculated from the force-indentation curves using the model of Sneddon for a conical indenter. Young’s modulus of elasticity was 20.1–131kPa for the human lens capsule, 9.19–117kPa for the cynomolgus lens capsule, and 13.1–62.4kPa for the baboon lens capsule. Young’s modulus increased significantly with age in humans (p=0.03). The age range of the monkey and baboon samples was not sufficient to justify an analysis of age dependence. The capsule elasticity of young humans (

The purpose of this study was to assess anterior and mid corneal stromal elasticity after high intensity (HI) corneal cross linking (CXL), with and without oxygen (O2) enrichment, and compare these results to conventional CXL. Experiments were performed on 25 pairs of human cadaver eyes, divided into four different groups. Group 1 included corneas that did not receive treatment and served as controls; Group 2 included corneas that received conventional CXL treatment (Dresden Protocol: corneal epithelial debridement, 30 min of riboflavin pretreatment followed by 30 min of exposure to 3 mW/cm2 of ultraviolet light); Group 3 included corneas that received HI CXL treatment (corneal epithelial debridement, 30 min of riboflavin pretreatment followed by 3 min of exposure to 30mW/cm2 of ultraviolet light); and Group 4 included corneas that received the same treatment as Group 3, except that they were enriched with oxygen (4 L per minute pure O2 gas stream) during ultraviolet irradiation. In each group, corneas were subdivided to assess anterior stromal elasticity and mid stromal elasticity. Corneal stromal elasticity was quantified using Atomic Force Microscopy (AFM) through micro-indentation. Young's modulus for the anterior corneal stroma was 14.5 ± 6.0 kPa, 80.7 ± 44.6 kPa, 36.6 ± 10.5 kPa, and 30.6 ± 9.2 kPa, for groups 1, 2, 3 and 4 respectively. Young's modulus for the mid corneal stroma was 5.8 ± 2.0 kPa, 20.7 ± 4.3 kPa, 12.1 ± 4.9 kPa, and 11.7 ± 3.7 kPa, for groups 1, 2, 3 and 4, respectively. In the anterior stromal region, conventional CXL demonstrated a significantly different result from the control, whereas the two HI CXL protocols were not significantly different from the control. There were no statistical differences between the two HI CXL protocols, although only the HI CXL protocol with O2 enrichment was significantly different from the conventional CXL group. In the mid stromal region, once again only conventional CXL demonstrated a significantly different result from the control. There were no statistical differences between the two HI CXL protocols, and both HI CXL protocols were significantly different from the conventional CXL group. Oxygen enriched HI CXL seems to offer similar changes in corneal elasticity when compared to HI CXL without the presence O2. Conventional CXL increases corneal stiffness more than HI CXL both with and without O2 enrichment.

Purpose To evaluate the effect of age, size, position, and species on the strength of minicapsulorhexes. Setting Surgical Suite and Laser Laboratory, Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA. Methods Capsulorhexes 0.7 to 2.3 mm in diameter were made centrally or peripherally in 35 eye-bank eyes and 32 rabbit eyes. A custom-made instrument stretched the capsulorhexes until rupture. Load and stretch at rupture were recorded. Results Maximum load and stretch were 26.3 mN ± 20.3 (SD) and 50% ± 18% for central and 50.8 ± 20.5 mN and 69% ± 17% for peripheral capsulorhexes in eye-bank eyes and 19.8 ± 15.2 mN and 38% ± 13% for central and 13.5 ± 9.5 mN and 30% ± 7% for peripheral capsulorhexes in rabbit eyes. Peripheral capsulorhexes were stronger and more elastic than central capsulorhexes in eye-bank eyes, and maximum load and stretch increased statistically with the capsulorhexis diameter. Conclusions Peripheral minicapsulorhexes were more resistant to rupture than central capsulorhexes in eye-bank eyes, probably because of increased lens capsule thickness at the periphery. An increase in capsulorhexis diameter increased the resistance to rupture.

A low-cost, high-resolution, fibre optic system for non-contact thickness measurement of ocular tissues in cadaver eyes was developed. The optical system uses direct modulation at 100 kHz of a 670 nm laser diode delivered to a single-mode fibre coupler. The output of the fibre coupler is focused onto the tissue using a high numerical aperture aspheric lens mounted on a motorized translation stage. Light reflected from the sample is collected by the fibre coupler and sent to a photoreceiver. Peaks in the power signal are detected when the focal point of the aspheric lens coincides with the tissue boundaries. The thickness is proportional to the distance between successive peaks. The optical system has a sensitivity of 52 dB, which corresponds to a detectable change in the refractive index of 0.005 at a boundary-assuming specular reflection. With a focusing numerical aperture of 0.68, the resolution is 4.0 µm and the precision is ± 0.2 µm. The optical system was used to measure the corneal layers and lens capsule thickness of cadaver monkeys eyes. The system was able to measure the thickness of the epithelium, stroma and Descemet's membrane of the cornea, as well as the lens capsule and lens epithelial cell layer.

Creating high-quality corneal cuts in clinically acceptable treatment times has been a critical aspect of femtosecond laser corneal refractive surgery since its introduction in the late 1990s [1]. Considerable effort has gone into establishing laser-scanning designs, laser-scanning parameters, scanning patterns, and surgical techniques to produce the high-quality flaps, keratoplasties, and other femtosecond laser incisions that are in widespread use today. The surface quality of femtosecond laser cuts has steadily improved over the earliest instruments in which energies of several microjoules and spot spacings on the order of 10 μm were used [1]. Faster and more accurate scanning systems, smaller laser focal spots, and lower energies have been the driving technological force enabling the trends of higher-quality cut surfaces and minimal opaque bubble layer (OBL) effects. As flap generation performed with the femtosecond laser keratome was perfected, lamellar resection quality approached [2] and then equaled the quality of resections produced by mechanical blades [3].

PURPOSE: To assess the cut quality of the anterior and posterior surfaces of intrastromal refractive lenticules removed during small incision lenticule extraction (SMILE). METHODS: The VisuMax femtosecond laser (500 kHz; Carl Zeiss Meditec, Dublin, CA) was used to perform SMILE on 8 eyes of 5 individuals to correct only myopia (no cylinder). The cut energy index was 26 (equivalent to an energy of 130 nJ) with a 2.5 × 2.5 μ m spot/track separation. The lenticule diameter was 6.5 mm with a minimum edge thickness of 15 μ m and the cap diameter was 7.3 mm with an intended thickness of 120 μ m. After laser treatment, the lenticule was loosened with a spatula and removed with forceps. The extracted lenticules were placed in 2% formalin and sent for imaging with an environmental scanning electron microscope. Images of the anterior and posterior surfaces of the lenticules were obtained at multiple magnifications (100×, 250×, and 500×). Surface quality was evaluated by an investigator who specializes in electron microscopy using three criteria: overall surface regularity, percent of surface irregularity, and position of irregular area. RESULTS: Both the anterior and posterior surfaces of the extracted lenticules were smooth and absent of surface irregularities. The cut edges also appeared uniform. Jagged edges were seen in several images, but were clearly caused by the forceps during extraction. CONCLUSIONS: Using the VisuMax laser to perform SMILE produces smooth cuts absent of surface irregularities. [ J Refract Surg. 2014;30(6):388–393.]

Corneal biomechanics is an essential parameter for developing diagnostic and treatment methods of corneal-related diseases. It is widely accepted that corneal mechanical strength stems from the stroma's collagenous composition. However, more comprehensive insight into the mechanical properties within the stroma is needed to improve current corneal diagnostic and treatment techniques. The purpose of this study was to perform elasticity characterization of anterior and posterior stromal regions of human corneas using atomic force microscopy (AFM). Nine pairs of human whole globes were placed in 20% Dextran solution, cornea side down, to restore the corneal thickness to physiological levels (400–600 μm). The epithelium and Bowman's membrane were removed from all eyes. Anterior stromal AFM elasticity testing was then performed on left (OS) eyes. Additional stroma was removed from right (OD) eyes to allow posterior stromal measurements at a depth of 50% of the original thickness. All experiments were performed with corneas submerged in 15% Dextran to maintain corneal hydration. The results of the study showed that the Young's modulus of elasticity of the anterior stroma (average: 281 ± 214 kPa; range: 59–764 kPa) was significantly higher than that of the posterior stroma (average: 89.5 ± 46.1 kPa; range: 29–179 kPa) (p = 0.014). In addition, a linear relationship was found between the posterior stromal elasticity and anterior stromal elasticity (p = 0.0428). On average, the elasticity of the posterior stroma is 39.3% of the anterior stroma. In summary, there appears to be an elasticity gradient within the corneal stroma, which should be considered in the design and development of corneal diagnostic and treatment methods to enhance efficacy.

Purpose To quantify the cut quality of lamellar dissections made with the femtosecond laser using atomic force microscopy (AFM). Setting Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA. Design Experimental study. Methods Experiments were performed on 3 pairs of human cadaver eyes. The cornea was thinned to physiologic levels by placing the globe, cornea side down, in 25% dextran for 24 hours. The eyes were reinflated to normal pressures by injecting a balanced salt solution into the vitreous cavity. The eyes were placed in a holder, the epithelium was removed, and the eyes were cut with a Visumax femtosecond laser. The energy level was 180 nJ for the right eye and 340 nJ for the left eye of each pair. The cut depths were 200 μm, 300 μm, and 400 μm, with the cut depth maintained for both eyes of each pair. A 12.0 mm trephination was then performed. The anterior portion of the lamellar surface was placed in a balanced salt solution and imaged with AFM. As a control, the posterior surface was placed in 2% formalin and imaged with environmental scanning electron microscopy (SEM). Four quantitative parameters (root-mean-square deviation, average deviation, skewness, kurtosis) were calculated from the AFM images. Results From AFM, the 300 μm low-energy cuts were the smoothest. Similar results were seen qualitatively in the environmental SEM images. Conclusion Atomic force microscopy provided quantitative information on the quality of lamellar dissections made using a femtosecond laser, which is useful in optimizing patient outcomes in refractive and lamellar keratoplasty surgeries. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

Purpose. To characterize the age dependence of shape, refractive power, and refractive index of isolated lenses from nonhuman primates. Methods. Measurements were performed on ex vivo lenses from cynomolgus monkeys (cyno: n = 120; age, 2.7-14.3 years), rhesus monkeys (n = 61; age, 0.7-13.3 years), and hamadryas baboons (baboon: n = 16; age, 1.7-27.3 years). Lens thickness, diameter, and surface curvatures were measured with an optical comparator. Lens refractive power was measured with a custom optical system based on the Scheiner principle. The refractive contributions of the gradient, the surfaces, and the equivalent refractive index were calculated with optical ray-tracing software. The age dependence of the optical and biometric parameters was assessed. Results. Over the measured age range isolated lens thickness decreased (baboon: -0.04, cyno: -0.05, and rhesus: -0.06 mm/y) and equatorial diameter increased (logarithmically for the baboon and rhesus, and linearly for cyno: 0.07 mm/y). The isolated lens surfaces flattened and the corresponding refractive power from the surfaces decreased with age (-0.33, -0.48, and -0.68 D/y). The isolated lens equivalent refractive index decreased (only significant for the baboon, -0.001 D/y), and as a result the total isolated lens refractive power decreased with age (baboon: -1.26, cyno: -0.97, and rhesus: -1.76 D/y). Conclusions. The age-dependent trends in the optical and biometric properties, growth, and aging, of nonhuman primate lenses are similar to those of the pre-presbyopic human lens. As the lens ages, the decrease in refractive contributions from the gradient refractive index causes a rapid age-dependent decrease in maximally accommodated lens refractive power.

In recent years, the atomic force microscope (AFM) has become an important tool in ophthalmic research. It has gained popularity largely because AFM is not restricted by the diffraction limits of light microscopy and can be applied to resolve images with molecular resolution. AFM is a minimally invasive technique and can be used to visualize molecular structures under near-physiological conditions. In addition, the AFM can be employed as a force apparatus to characterize the viscoelastic properties of biomaterials on the micron level and at the level of individual proteins. In this article, we summarize recent AFM studies of ocular tissues, while highlighting the great potential of AFM technology in ophthalmic research. Previous research demonstrates the versatility of the AFM as high resolution imaging technique and as a sensitive force apparatus for probing the mechanical properties of ocular tissues. The structural and mechanical properties of ocular tissues are of major importance to the understanding of the optomechanical functions of the human eye. In addition, AFM has played an important role in the development and characterization of ocular biomaterials, such as contact lenses and intraocular lenses. Studying ocular tissues using Atomic Force Microscopy has enabled several advances in ophthalmic research.

Accommodation is a process that relies on the change in curvature of the crystalline lens to focus on objects at different distances.1-5 According to the Helmholtz theory of accommodation,3 these changes in curvature are induced as the zonules apply or release a load on the lens equator.6-9 When the ciliary muscle is relaxed, the zonules are taut, imparting a load on the lens capsule, causing it to flatten. This flatter curvature lowers the optical power of the eye, allowing the observer to focus on distant objects. When the ciliary muscle is contracted, the tension on the zonules is diminished, reducing the load on the lens capsule, allowing the lens to assume a more curved form. This increase in curvature increases the optical power of the eye, allowing the observer to focus on nearby objects. Since the zonules are responsible for transferring the force from the ciliary body to the lens, they serve as a critical component of the accommodative apparatus. Rohen10 described the zonular apparatus of both humans and monkeys as consisting of two functionally different sets of zonular fibers, the main fibers and the tension fibers, where the main fibers, which Rohen also refers to as anterior zonules, consist of all fibers connecting to the anterior, posterior, and equatorial region of the lens. Other anatomic investigations have led to a description of the structural arrangement of Rohen's main fibers as consisting of two primary groups with zonules inserting into the anterior and posterior lens surface and a secondary group with zonules inserting into the equatorial lens surface.8,11,12 In the remainder of this article, we will refer to these three groups as the anterior, posterior, and equatorial zonules, as illustrated in Figure 1. Using scanning electron microscopy to investigate the architecture of the accommodative apparatus, Streeten and Pulaski13 showed that there is a connection between the posterior zonules and the hyaloid membrane, and recently, using environmental scanning electron microscopy (eSEM), Bernal et al.14 verified this connection. The posterior zonular fibers are connected directly to the hyaloid membrane, as they run through and along the hyaloid membrane before they connect to the posterior capsule.13,14 In contrast, the anterior zonules are directly connected from the anterior capsule to the ciliary body. Figure 1 Schematic showing the lens (L), ciliary body (CB), anterior zonules (AZ), equatorial zonules (EZ), posterior zonules (PZ), and hyaloid membrane (HM). Since the anterior and posterior zonules do not attach to the lens in exactly the same manner, the respective effect of each might also be different. The asymmetry in the anchorage points and orientation of the anterior and posterior zonular fibers suggest a different mode of action of each. The goal of the present study is to determine the effects of the difference in anterior and posterior zonule anchorage points and orientation by quantifying the role of anterior zonular transection on the lens diameter and power of nonhuman primate eyes during simulated accommodation in a lens stretcher.

Purpose To determine whether changes in elastic properties of the lens capsule ex vivo with age contribute to the forces necessary for accommodation. Methods Postmortem human (n = 22; age average: 41 +/- 17 years; range: 6-7) and cynomolgus monkey (n = 19; age average: 7.7 +/- 1.8 years, range: 4.2-10) tissues including the lens, capsule, zonules, ciliary body, and sclera were mounted in an optomechanical lens-stretching system. Starting at 0 load, the sclera was symmetrically stretched to 2 mm in 0.25-mm steps at a speed of 0.1 mm x s(-1). The load and lens diameter were measured at each step. The lens contents were removed through a mini-capsulorhexis. The stretching cycles were repeated on the empty capsular bag. The forces necessary to stretch the natural lens and empty bag were quantified as a function of age and compared. Results The force needed to stretch the empty lens capsule was independent of age (human, 2.6-34.9 g/mm [25.2-342.7 mN/mm]; monkey, 8.2-21.3 g/mm [80.3-208.6 mN/mm]). The ratio of the force necessary to stretch the empty lens capsule to the force necessary to stretch the natural lens decreased with age in the human and monkey lenses (P = 0.003, P = 0.72, respectively). Conclusions The mechanical properties of the empty lens capsule assessed ex vivo in a lens stretcher remain constant with age, suggesting that the changes in elasticity of the lens capsule do not play a significant role in presbyopia. In young eyes, the lens capsule determines the force necessary to stretch the whole lens. The age-related increase in force needed to stretch the lens is due to changes in the lens contents.

Purpose To characterize the age dependence of isolated human crystalline lens power and quantify the contributions of the lens surfaces and refractive index gradient. Methods Experiments were performed on 100 eyes of 73 donors (average 2.8 +/- 1.6 days postmortem) with an age range of 6 to 94 years. Lens power was measured with a modified commercial lensmeter or with an optical system based on the Scheiner principle. The radius of curvature and asphericity of the isolated lens surfaces were measured by shadow photography. For each lens, the contributions of the surfaces and the refractive index gradient to the measured lens power were calculated by using optical ray-tracing software. The age dependency of these refractive powers was assessed. Results The total refractive power and surface refractive power both showed a biphasic age dependency. The total power decreased at a rate of -0.41 D/y between ages 6 and 58.1, and increased at a rate of 0.33D/y between ages 58.1 and 82. The surface contribution decreased at a rate of -0.13 D/y between ages 6 and 55.2 and increased at a rate of 0.04 D/y between ages 55.2 and 94. The relative contribution of the surfaces increased by 0.17% per year. The equivalent refractive index also showed a biphasic age dependency with a decrease at a rate of -3.9 x 10(-4) per year from ages 6 to 60.4 followed by a plateau. Conclusions The lens power decreases with age, due mainly to a decrease in the contribution of the gradient. The use of a constant equivalent refractive index value to calculate lens power with the lens maker formula will underestimate the power of young lenses and overestimate the power of older lenses.

Several intraocular lens designs and surgical approaches have been proposed to restore dynamic accommodation in the presbyopic eye.1–5 One of the fundamental hurdles in the development of such approaches is the absence of a definitive explanation for the progressive decrease in the accommodative ability with age, which leads to presbyopia. Most likely, presbyopia results from a combination of factors affecting several of the anatomic structures involved in accommodation.6–9 Most of the current theories of presbyopia attribute the loss of accommodation directly or indirectly to age-related changes in the lens itself.7,10–14 Alternatively, Strenk et al.6 recently suggested that presbyopia may also be due to an inward movement of the ciliary muscle that occurs with age and reduces the tension on the zonules when the ciliary muscle is relaxed. Croft et al.15 proposed that a reduction in the forward movement of the ciliary body could contribute to the decrease in lens accommodation with age. The relation between the changes in zonular tension and the resultant changes in lens shape and dioptric power is a key characteristic of these current theories of presbyopia and of the accommodation mechanism in general. The relation between the lens biometric and optical responses has been relatively well characterized in vivo.16–23 In contrast, the relation between the forces of accommodation and the resultant biometric and optical responses remains to be quantified. Unfortunately, there is currently no suitable technique for direct measurements of the accommodation force in absolute, physical units in an in vivo setting. Direct quantitative measurements of the force–power relation have been obtained on human tissue postmortem by simulating the force exerted on the zonules during disaccommodation. The first studies of this kind appear to be those of Pau,24 who simulated the force of accommodation on the lens by attaching weights to the ciliary body. Fisher25 later designed a device that mimicked the pull on the zonules by spinning isolated lenses to produce a centrifugal force and a second, more sophisticated, system26 that simulated accommodation by radial stretching and recorded the changes in lens focal length, curvature, and thickness. Fisher estimated the stretching force indirectly, by comparing the thickness measured in the stretching device with the thickness produced when the same lens was submitted to a spinning experiment. Around the same time, Sunderland and O’Neill27 developed a system that simulated accommodation by applying circumferential forces to isolated feline lenses, using an approach similar to that of Pau.24 More recently, Van Alphen and Graebel28 relied on uniaxial loading experiments to estimate the relative force required to stretch the lens, zonules, ciliary body, and choroid. Pierscionek29,30 and Glasser and Campbell31 have developed experimental setups that stretch lenses to simulate accommodation. Their studies focused on quantifying biometric changes and did not include force measurements. Several other lens-stretching or -spinning systems have been designed recently32,33 (Michail H et al. IOVS 2004;45:ARVO E-Abstract 1726) to study the optomechanical response of the lens. The purpose of the present study was to quantify the power- and diameter-force characteristics of primate lenses by using a lens-stretching system (EVAS; Ex Vivo Accommodation System, designed and constructed in-house at the Ophthalmic Biophysics Center) that allows direct measurements of the force required to stretch the crystalline lens to its unaccommodated state while simultaneously measuring the associated change in lens power and equatorial diameter. Results obtained with rhesus monkey, cynomolgus monkey, and human crystalline lenses are presented.

The purpose of this study was to assess the condition of human lenses (obtained from an eye bank) and of fresh monkey lenses, and to determine the effects of maintaining these lenses in various liquid preservation media.Freshly excised human and monkey lenses were maintained for 5 h in one of four solutions (Balanced Saline Solution [BSS], Ringer's Solution, Dulbecco's Modified Eagle Medium with Ham's F-12 [DMEM/F-12/F-12], and Tissue Culture Medium 199 [TC-199]) using a custom-designed, temperature-regulated testing cell. A modified optical comparator and digital camera were used to photograph magnified lens profiles and measure lens diameter and thickness. Lens volume was then calculated assuming rotational symmetry about the optical axis.Seven of the 33 human lenses exhibited extensive swelling and separation of the capsule from the lens cell mass prior to the incubation. During incubation, for 12/22 of the remaining human and 27/27 of the monkey lenses, thickness increased by 1.0-1.8%, diameter decreased by 0.7-1.6% and the volume was essentially unchanged. Substantial swelling and capsular separation were observed in 10 of the 22 human lenses, 7/10 for those maintained in salt solutions, and 3/12 for those in tissue culture media. Lens volumes increased by an average of 6.8%, due to an 8.7% increase in the thickness, while the diameter decreased by 0.9%. These changes appeared to be independent of postmortem time and donor age.Culture media are more effective than simple salt solutions in maintaining lens physical integrity during short-term incubations. Substantial uptake of water, accompanied by separation of the capsule from the lens cell mass, occurs at various stages during storage and experimental manipulations in60% of human lenses obtained from the eye bank. Data obtained with such lenses will not be representative of the true ex vivo state. It is recommended that lenses be assessed to determine if swelling has taken place before acceptance of data.

PURPOSE. To measure interspecies thickness differences in the central anterior and posterior capsules of postmortem crystalline lenses, by a technique that maintains the anatomic integrity of the lens. METHODS. Central capsule thickness was measured with a custom-built, noncontact optical system, using a focus detection technique. Anterior and posterior lens capsule thickness measurements were performed on 22 human, 29 monkey, and 34 New Zealand White rabbit intact postmortem lenses in situ. Eyes were prepared for optical measurements by bonding a PMMA ring to the sclera in the region of the ciliary body after the conjunctiva, adipose, and muscle tissues were removed. The posterior pole was removed by making a circumferential incision through the sclera approximately 7 mm posterior to the limbus. Excess vitreous was removed to expose the posterior capsule surface, and the eye assembly was placed on a Teflon slide. The cornea and iris were sectioned to expose the anterior capsule surface. After the experiments, the lenses were excised, placed in 10% buffered formalin, and prepared for histology. Lens capsule thickness was measured from the histologic slides and compared to the optical results. RESULTS. Central anterior lens capsule thickness was 8.2 5.5 (human), 7.5 4.4 (monkey), and 10.7 4.2 (rabbit) m optically and 12.4 2.5 (human), 10.7 3.7 (monkey), and 10.4 2.0 (rabbit) m histologically. Central posterior capsule thickness was 6.3 2.2 (human), 5.9 1.7 (monkey), and 7.8 2.3 (rabbit) m optically and 4.1 1.5 (human), 3.5 1.6 (monkey), and 4.7 2.5 (rabbit) m histologically. CONCLUSIONS. The central anterior and posterior lens capsule thicknesses do not appear to vary considerably among human, rabbit, and monkey eyes. There were significant differences between optical in situ measurements and histology, which indicates that histologic preparation may affect lens capsule thickness. (Invest Ophthalmol Vis Sci. 2005;46:1690‐1697)

Purpose: To design and test an optical system to measure the optical quality of post mortem lenses during simulated accommodation. Methods: An optical bench top system was designed to measure the point spread function and calculate the modulation transfer function (MTF) of monkey and human ex-vivo crystalline lenses. The system consists of a super luminescent diode emitting at 850nm, collimated into a 3mm beam which is focused by the ex-vivo lens under test. The intensity distribution at the focus (point spread function) is re-imaged and magnified onto a beam profiler CCD camera. The optical quality in terms of spatial frequency response (modulation transfer function) is calculated by Fourier transform of the point spread function. The system was used on ex-vivo lenses with attached zonules, ciliary body and sclera. The sclera was glued to 8 separate PMMA segments and stretched radial by 5mm on an accommodation simulating lens stretching device. The point spread function was measured for each lens in the relaxed and stretched state for 5 human (ages 38-86 years) and 5 cynomolgus monkey (ages 53 - 67 months) fresh post mortem crystalline lenses. Results: Stretching induced measurable changes in the MTF. The cutoff frequency increased from 54.4±13.6 lp/mm unstretched to 59.5±21.4 lp/mm stretched in the post-presbyopic human and from 51.9±24.7 lp/mm unstretched to 57.7±18.5 lp/mm stretched cynomolgus monkey lenses. Conclusion: The results demonstrate the feasibility of measuring the optical quality of ex-vivo human and cynomolgus monkey lenses during simulated accommodation. Additional experiments are underway to quantify changes in optical quality induced by stretching.

Purpose: To non-invasively measure the thickness of the anterior and posterior lens capsule, and to determine if it significantly changes during accommodation. Methods: Anterior and posterior capsule thickness was measured on post-mortem lenses using a non-contact optical system using a focus-detection technique. The optical system uses a 670nm laser beam delivered to a single-mode fiber coupler. The output of the fiber coupler is focused on the tissue surface using an aspheric lens (NA=0.68) mounted on a translation stage with a motorized actuator. Light reflected from the sample surface is collected by the fiber coupler and sent to a photoreceiver connected to a computer-controlled data acquisition system. Optical intensity peaks are detected when the aspheric lens is focused on the capsule boundaries. The capsule thickness is equal to the distance traveled between two peaks multiplied by the capsule refractive index. Anterior and posterior lens capsule thickness measurements were performed on 18 cynomolgus (age average: 6±1 years, range: 4-7 years) eyes, 1 rhesus (age: 2 years) eye, and 12 human (age average: 65±16, range: 47-92) eyes during simulated accommodation. The mounted sample was placed under the focusing objective of the optical system so that the light was incident on the center pole. Measurements were taken of the anterior lens capsule in the unstretched and the stretched 5mm states. The lens was flipped, and the same procedure was performed for the posterior lens capsule. Results: The precision of the optical system was determined to be ±0.5um. The resolution is 4um and the sensitivity is 52dB. The human anterior lens capsule thickness was 6.0±1.2um unstretched and 4.9±0.9um stretched (p=0.008). The human posterior lens capsule was 5.7±1.2um unstretched and 5.7±1.4um stretched (p=0.974). The monkey anterior lens capsule thickness was 5.9±1.9um unstretched and 4.8±1.0um stretched (p=0.002). The monkey posterior lens capsule was 5.9±2.0um unstretched and 5.1±1.3um stretched (p=0.128). Conclusions: The results indicate that the primate anterior lens capsule thickness changes during accommodation.

To report a case of interface deposits after Descemet stripping automated endothelial keratoplasty (DSAEK) imaged by means of an electron microscope.An 88-year-old man was referred with a history of corneal edema resulting from pseudophakic bullous keratopathy. A DSAEK was performed on his left eye without complications; however, the follow-up examination revealed a well-attached graft with persistent interface opacities at the donor-recipient interface. The DSAEK was repeated in this eye 1 year after the first surgery because of these corneal opacities that interfered with his vision.In the immediate postoperative period, the patient had adequate visual acuity with intact graft placement and a clear interface. Pathology and electron microscope analysis were performed on the removed endothelial graft, which revealed diffuse particles on the stromal surface of the endothelial graft.A repeated DSAEK procedure sufficiently removed this patient's corneal opacities and improved the visual acuity. The opacity is believed to have occurred because of residual viscoelastic material, which was used to maintain anterior chamber volume during surgery. This solution must be thoroughly removed to avoid similar complications.

Purpose: To design a non-contact optical system to measure lens capsule thickness in cadaver eyes. Methods: The optical system uses a 670nm laser beam delivered to a single-mode fiber coupler. The output of the fiber coupler is focused onto the tissue using an aspheric lens (NA=0.68) mounted on a motorized translation stage. Light reflected from the sample is collected by the fiber coupler and sent to a silicon photodiode connected to a power meter. Peaks in the power signal are detected when the focal point of the aspheric lens coincides with the capsule boundaries. The capsule thickness is proportional to the distance between successive peaks. Anterior and posterior lens capsule thickness measurements were performed on 13 human, 10 monkey, and 34 New Zealand white rabbit lenses. The cadaver eyes were prepared for optical measurements by bonding a PMMA ring on the sclera. The posterior pole was sectioned, excess vitreous was removed, and the eye was placed on a Teflon slide. The cornea and iris were then sectioned. After the experiments, the lenses were excised, placed in 10% buffered formalin, and prepared for histology. Results: Central anterior lens capsule thickness was 9.4±2.9μm (human), 11.2±6.6μm (monkey), and 10.3±3.6μm (rabbit) optically and 14.9±1.6μm (human), 17.7±4.9μm (monkey), and 12.6±2.3μm (rabbit) histologically. The values for the central posterior capsule were 9.4±2.9μm (human), 6.6±2.5μm (monkey), and 7.9±2.3μm (rabbit) optically and 4.6±1.4μm (human), 4.5±1.2μm (monkey), and 5.7±1.7μm (rabbit) histologically. Conclusions: This study demonstrates that a non-contact optical system can successfully measure lens capsule thickness in cadaver eyes.