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X-ray absorption near-edge structure (XANES) spectroscopy identifies differential sulfur speciation in corneal tissue

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Abstract

The chemical composition of tissues can influence their form and function. As a prime example, the lattice-like arrangement of collagen fibrils required for corneal transparency is controlled, in part, by sulfated proteoglycans, which, via core proteins, bind to the collagen at specific locations along the fibril axis. However, to date, no studies have been able to directly identify and characterize sulfur (S) in the cornea as a function of tissue location. In this study, X-ray absorption near-edge structure spectroscopy and micro-beam X-ray fluorescence (μ-XRF) chemical contrast imaging were employed to probe the nature of the mature (bovine) cornea as a function of position from the anterior sub-epithelial region into the deep stroma. Data indicate an inhomogeneity in the composition of S species in the first ≈50 μm of stromal depth. In μ-XRF chemical contrast imaging, S did not co-localize with phosphorous (P) in the deep stroma where sulfates are prominent. Rather, P is present only as isolated micrometric spots, presumably identifiable as keratocytes. This study lends novel insights into the elemental physiology of mature cornea, especially in relation to its S distribution; future studies could be applied to human tissues. Moreover, it defines an analytical protocol for the interrogation of S species in biological tissues with micrometric resolution.

Sulfur species distribution in corneal tissue. Spatial distribution of S (red) and P (green) extracted from µ-XRF maps of a bovine cornea cut. The incoming X-ray beam energy was tuned in order to enhance the absorption from sulfate (upper map) and thiol/monosulfide (lower map) groups, respectively

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Abbreviations

GCF:

Gaussian curve fitting

LCF:

Linear combination fitting

μ-XRF:

Micro-beam X-ray fluorescence

XANES:

X-Ray absorption near-edge structure

ESI-MS/MS:

Electrospray ionization tandem mass spectrometry

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Acknowledgments

The authors would like to express their gratitude to Dr. Rob Young and Ms. Frances Jones for help with specimen preparation, and also acknowledge the ESRF (http://www.esrf.eu) for providing access to synchrotron radiation facilities. This work is supported by a project grant from the UK Engineering and Physical Sciences Research Council (grant number EP/F034970 to AJQ). EK is the recipient of a Cardiff University President's Studentship.

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Authors and Affiliations

  1. X-Ray Microscopy Beamline ID21, European Synchrotron Radiation Facility, POB 220, Grenoble Cedex 9, France

    Giulia Veronesi & Marine Cotte

  2. Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff University, Maindy Road, Cardiff, CF24 4LU, Wales, UK

    Elena Koudouna & Andrew J. Quantock

  3. Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster, LA1 4YQ, UK

    Francis L. Martin

Authors
  1. Giulia Veronesi
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  2. Elena Koudouna
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  3. Marine Cotte
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  4. Francis L. Martin
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  5. Andrew J. Quantock
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Correspondence to Giulia Veronesi.

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Veronesi, G., Koudouna, E., Cotte, M. et al. X-ray absorption near-edge structure (XANES) spectroscopy identifies differential sulfur speciation in corneal tissue. Anal Bioanal Chem 405, 6613–6620 (2013). https://doi.org/10.1007/s00216-013-7120-x

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  • DOI: https://doi.org/10.1007/s00216-013-7120-x

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