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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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 (O 2 ) 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/cm 2 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/cm 2 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 O 2 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 O 2 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 O 2 . Conventional CXL increases corneal stiffness more than HI CXL both with and without O 2 enrichment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2012 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

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 5 mm 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 5 min, 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 20.1-131 kPa for the human lens capsule, 9.19-117 kPa for the cynomolgus lens capsule, and 13.1-62.4 kPa 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 (<45 years) was not statistically different from that of the monkey and baboon. In humans, there is an increase in lens capsule stiffness at the microscale that could be responsible for an increase in lens capsule bulk stiffness., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: To demonstrate the feasibility of measuring the elasticity of intact crystalline lenses using atomic force microscopy (AFM)., Methods: AFM elasticity measurements were performed on intact lenses from 18 fresh cynomolgus monkey cadaver eyes (4-10 years old, <1 day postmortem) that had been left attached to their zonule-ciliary body-sclera framework. The eyes were prepared by bonding a plastic ring on the sclera after removal of the conjunctival, adipose, and muscle tissues. The posterior pole was sectioned, with the excess vitreous removed, and the eye's anterior section was placed on a Teflon slide to protect the posterior pole of the lens. The cornea and iris were then sectioned. The lens-zonule-ciliary body-sclera section was then placed in a Petri dish filled with balanced salt solution in an AFM system designed for force measurements. Next, the central pole of the anterior surface of the intact lens was probed with the AFM cantilever tip. The recorded AFM cantilever deflection-indentation curves were used to derive force-indentation curves for the lens after factoring out the deflection of the cantilever on a hard surface. Young's modulus of the lens was calculated from the force-indentation relation using the Hertz model., Results: Young's modulus was 1,720+/-880 Pa (range: 409-3,210 Pa) in the 18 cynomolgus monkey lenses., Conclusions: AFM can be used to provide measurements of the elasticity of the whole lens including the capsule. Values obtained using AFM on cynomolgus monkey lenses are similar to published values obtained using dynamic mechanical analysis on young human lenses.