Noël M. Ziebarth

Optical Power of the Isolated Human Crystalline Lens

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.

Surface quality of human corneal lenticules after SMILE assessed using environmental scanning electron microscopy.

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 μm spot/track separation. The lenticule diameter was 6.5 mm with a minimum edge thickness of 15 μm and the cap diameter was 7.3 mm with an intended thickness of 120 μm. 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.

Multiscale Investigation of the Depth-Dependent Mechanical Anisotropy of the Human Corneal Stroma

PURPOSE To investigate the depth-dependent mechanical anisotropy of the human corneal stroma at the tissue (stroma) and molecular (collagen) level by using atomic force microscopy (AFM). METHODS Eleven human donor corneas were dissected at different stromal depths by using a microkeratome. Mechanical measurements were performed in 15% dextran on the surface of the exposed stroma of each sample by using a custom-built AFM in force spectroscopy mode using both microspherical (38-μm diameter) and nanoconical (10-nm radius of curvature) indenters at 2-μm/s and 15-μm/s indentation rates. Youngs modulus was determined by fitting force curve data using the Hertz and Hertz-Sneddon models for a spherical and a conical indenter, respectively. The depth-dependent anisotropy of stromal elasticity was correlated with images of the corneal stroma acquired by two-photon microscopy. RESULTS The force curves were obtained at stromal depths ranging from 59 to 218 μm. At the tissue level, Youngs modulus (ES) showed a steep decrease at approximately 140-μm stromal depth (from 0.8 MPa to 0.3 MPa; P = 0.03) and then was stable in the posterior stroma. At the molecular level, Youngs modulus (EC) was significantly greater than at the tissue level; EC decreased nonlinearly with increasing stromal depth from 3.9 to 2.6 MPa (P = 0.04). The variation of microstructure through the thickness correlated highly with a nonconstant profile of the mechanical properties in the stroma. CONCLUSIONS The corneal stroma exhibits unique anisotropic elastic behavior at the tissue and molecular levels. This knowledge may benefit modeling of corneal behavior and help in the development of biomimetic materials.

Crystalline lens MTF measurement during simulated accommodation

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.

Impact of Hydration Media on Ex Vivo Corneal Elasticity Measurements

Objectives: To determine the effect of hydration media on ex vivo corneal elasticity. Methods: Experiments were conducted on 40 porcine eyes retrieved from an abattoir (10 eyes each for phosphate-buffered saline (PBS), balanced salt solution, Optisol, 15% dextran). The epithelium was removed, and the cornea was excised with an intact scleral rim and placed in 20% dextran overnight to restore its physiological thickness. For each hydration media, corneas were evenly divided into two groups: one with an intact scleral rim and the other without. Corneas were mounted onto a custom chamber and immersed in a hydration medium for elasticity testing. Although in each medium, corneal elasticity measurements were performed for 2 hr: at 5-min intervals for the first 30 min and then 15-min intervals for the remaining 90 min. Elasticity testing was performed using nanoindentation with spherical indenters, and Young modulus was calculated using the Hertz model. Thickness measurements were taken before and after elasticity testing. Results: The percentage change in corneal thickness and elasticity was calculated for each hydration media group. Balanced salt solution, PBS, and Optisol showed an increase in thickness and Young moduli for corneas with and without an intact scleral rim. Fifteen percent dextran exhibited a dehydrating effect on corneal thickness and provided stable maintenance of corneal elasticity for both groups. Conclusions: Hydration media affects the stability of corneal thickness and elasticity measurements over time. Fifteen percent dextran was most effective in maintaining corneal hydration and elasticity, followed by Optisol.

The effect of nicotine on the mechanical properties of mesenchymal stem cells

PURPOSE: To measure the elasticity of the nucleus and cytoplasm of human mesenchymal stem cells (MSCs) as well as changes brought about by exposure to nicotine in vitro. METHODS: MSCs were synchronized to the G(0) stage of the cell cycle through serum deprivation techniques. The cells were then treated with medium containing nicotine (0.1 µM, 0.5 µM, and 1 µM). Atomic force microscopy was then used to measure the Youngs modulus of both the nucleus and cytoplasm of these cells. RESULTS: For both unsynchronized and synchronized cells, the nucleus was softer than the cytoplasm, although this difference was not found to be statistically significant. The nucleus of cells treated with nicotine was significantly stiffer than the control for all concentrations. The cytoplasm was significantly stiffer in nicotine-treated cells than in control cells for the 0.5 µM and 1.0 µM concentrations only. CONCLUSIONS: The results of this study could suggest that nicotine affects the biophysical properties of human MSCs in a dose-dependent manner, which may render the cells less responsive to mechanoinduction and other physical stimuli.

Effect of Anterior Zonule Transection on the Change in Lens Diameter and Power in Cynomolgus Monkeys during Simulated Accommodation

PURPOSE To quantify the role of anterior zonular tension on the optomechanical lens response during simulation of accommodation in primates. METHODS Postmortem cynomolgus monkey eyes (n = 14; age range, 3.0-11.5 years) were dissected leaving intact the lens, zonules, ciliary body, hyaloid membrane, anterior vitreous, and a scleral rim. The lens was mounted in a lens-stretching system and stretched radially in step-wise fashion. The load, and the lens diameter and power were measured at each step and the diameter- and power-load relationships were quantified. The anterior zonular fibers were then transected, and the experiment was repeated. The equatorial lens diameter and lens optical power before and after zonular transection were compared. RESULTS Stretching increased the lens diameter by 0.25 +/- 0.09 mm (median +/- interquartile range) before and 0.25 +/- 0.19 mm after zonular transection. Stretching decreased the lens power by 13.0 +/- 6.5 D before and 10.6 +/- 8.0 D after zonular transection. The load required to change the diameter of the lens by 1 mm decreased from 18.8 +/- 10.7 g before to 15.0 +/- 7.8 g after zonular transection. The absolute change in power per gram of loading decreased from 2.5 +/- 1.1 before to 2.0 +/- 1.2 D after zonular transection. CONCLUSIONS The cynomolgus monkey lens retains a significant fraction of its accommodative ability after transection of the anterior zonules in simulated accommodation experiments.

Fractal Analysis of AFM Images of the Surface of Bowman’s Membrane of the Human Cornea

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.

Quality of corneal lamellar cuts quantified using atomic force microscopy

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.

Refractive Power and Biometric Properties of the Nonhuman Primate Isolated Crystalline Lens

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.