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The lens, a major optical component of the eye, has a gradient refractive index, which is required to provide sufficient refractive power and image quality. The refractive index variations across the lens are dependent on the distributions and concentrations of the varying protein classes. In this study, we present the first measurements of the refractive index in the in situ eye lens from five species using a specially constructed X-ray Talbot grating interferometer. The measurements have been conducted in two planes: the one containing the optic axis (the sagittal plane) and the plane orthogonal to this (the equatorial plane). The results show previously undetected discontinuities and fluctuations in the refractive index profile that vary in different species. These may be linked to growth processes and may be the first optical evidence of discrete developmental stages.

The eye lens grows throughout life by cell accrual on its surface and can change shape to adjust the focussing power of the eye. Varying concentrations of proteins in successive cell layers create a refractive index gradient. The continued growth of the lens and age-related changes in proteins render it less able to alter shape with loss of capacity by the end of the sixth decade of life. Growth and protein ageing alter the refractive index but as accurate measurement of this parameter is difficult, the nature of such alterations remains uncertain. The most accurate method to date for measuring refractive index in intact lenses has been developed at the SPring-8 synchrotron. The technique, based on Talbot interferometry, has an X-ray source and was used to measure refractive index in sixty-six human lenses, aged from 16 to 91 years. Height and width were measured for forty-five lenses. Refractive index contours show decentration in some older lenses but individual variations mask age-related trends. Refractive index profiles along the optic axis have relatively flat central sections with distinct micro-fluctuations and a steep gradient in the cortex but do not exhibit an age-related trend. The refractive index profiles in the equatorial aspect show statistical significance with age, particularly for lenses below the age of sixty that had capacity to alter shape in vivo. The maximum refractive index in the lens centre decreases slightly with age with considerable scatter in the data and there are age-related variations in sagittal thickness and equatorial height.

De-epithelialised and de-endothelialised bovine corneal stromas with a hydration of 3.2 equilibrated at 154 mM NaCl and buffered at pH 7.4 had their optical density (400-750 nm) measured. Stromas equilibrated against 10, 20, 30, 50 or 100 mM NaCl made isotonic to 154 mM NaCl by supplementing with sorbitol were progressively more transparent as NaCl increased. Hypertonic equilibration against 300, 600 or 1000 mM NaCl resulted in a progressive loss of transparency compared with 154 mM NaCl. Light scattering as a function of wavelength fitted a l _3 function well for 10, 30, 50, 100 and 154 mM NaCl preparations between 450 and 650 nm, but not at higher wavelengths. However, hypertonic 300, 600 and 1000 mM NaCl preparations showed a l _2 dependence in the 450-750 nm range. Experiments with 154 mM NaCl and either 0 or 300 mM sorbitol suggested that the changes in light scattering in hypertonic preparations are unlikely to be caused by osmotic alterations to the stromal keratocytes. Psychophysical studies of the optical transmission function of preparations indicated that corneal stromas dialysed against 154 mM NaCl had usable optical properties, but preparations dialysed against 10 mM NaCl were effectively unable to transmit an image. The results are related to the known increase of fixed negative charge in the corneal matrix when chloride ions are adsorbed onto the matrix. It is suggested that the ordering force between corneal collagen fibrils, generated in part by anion binding, may be crucial to the physiological functioning of the visual system.

Keywords: eye lens refractive index zones of discontinuity light scatter implant design a b s t r a c t The structural basis of zones of discontinuity in the living human eye lens has not been elucidated, and there is no conclusive explanation for what relevance they may have to the structure and function of the lens. Newly developed synchrotron radiation based X-ray Talbot interferometry has enabled the detection of subtle fluctuations in the human eye lens which, when used in mathematical modelling to simulate reflected and scattered light, can recreate the image of the lens seen in the living human eye. The results of this study show that the zones of discontinuity may be caused by subtle fluctuations in the refractive index gradient as well as from random scattering in the central regions. As the refractive index contours are created by cell layers with progressively varying protein concentrations, the zones are linked to growth and will contain information about ageing and development. The index gradient is important for image quality and fluctuations in this gradient may add to quality optimisation and serve as models for designs of new generation implant lenses.

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Chondroitin sulfate (CS) is an important component of the extracellular matrix in multiple biological tissues. In cornea, the CS glycosaminoglycan (GAG) exists in hybrid form, whereby some of the repeating disaccharides are dermatan sulfate (DS). These CS/DS GAGs in cornea, through their presence on the proteoglycans, decorin and biglycan, help control collagen fibrillogenesis and organization. CS also acts as a regulatory ligand for a spectrum of signaling molecules, including morphogens, cytokines, chemokines, and enzymes during corneal growth and development. There is a growing body of evidence that precise expression of CS or CS/DS with specific sulfation motifs helps define the local extracellular compartment that contributes to maintenance of the stem cell phenotype. Indeed, recent evidence shows that CS sulfation motifs recognized by antibodies 4C3, 7D4, and 3B3 identify stem cell populations and their niches, along with activated progenitor cells and transitional areas of tissue development in the fetal human elbow. Various sulfation motifs identified by some CS antibodies are also specifically located in the limbal region at the edge of the mature cornea, which is widely accepted to represent the corneal epithelial stem cell niche. Emerging data also implicate developmental changes in the distribution of CS during corneal morphogenesis. This article will reflect upon the potential roles of CS and CS/DS in maintenance of the stem cell niche in cornea, and will contemplate the possible involvement of CS in the generation of eye-like tissues from human iPS (induced pluripotent stem) cells.