Features

The Diamond Light Source

Dispensing
Dr Douglas Clarkson describes how a scientific tool in the UK could help extend our knowledge of ocular tissue structure

Figure 1: Aerial image of the Diamond Light Source (Image: Diamond Light Source Ltd 2023)

The Diamond Light Source is a unique scientific research tool in the United Kingdom known as a synchrotron that can produce intense beams of electromagnetic radiation to probe the characteristics of a wide range of materials.

The system serves a broad range of scientific investigations and has been used extensively in medical research. The synchrotron operates by accelerating electrons to speeds very close to that of light and bending the main beam at various stations using electromagnetic lenses.

Induced oscillations of the electron beam at these stages inject intense emissions of electromagnetic radiation into so called ‘beamlines’ where the ultra fine ‘filament’ of radiation produced is used to probe the properties of a wide range of materials.

A key advantage of the synchrotron is its ability to generate super intense beams, which significantly reduce the time taken to capture experimental data compared to alternative, conventional measuring techniques.

The Diamond Light Source is a not-for-profit limited company funded as a joint venture between UK Research & Innovation (UKRI) and the Wellcome Trust. A significant level of research utilises beam lines of intense x-ray radiation to probe diverse atomic and molecular structures.

This technique of x-ray diffraction was pioneered by Lawrence Bragg and William Henry Bragg around 1913 and was famously a method that provided clues to Crick and Watson regarding the structure of the DNA molecule.1

Another key function of the system is the ability to identify constituent atoms at ultra high spatial resolution, such as within individual cells. While the system involves a high degree of complexity, key building blocks of the system can be identified as outlined in figure 2.

Figure 2: Initially an electron gun produces a stream of low energy electrons, which is boosted by a linear accelerator to an energy of 100 MeV (Million electron volts). A booster synchrotron increases their final energy to 3GeV (giga electron volts) before being injected into the main storage ring which has a diameter of 560m and where the beam energy is maintained. The high energy electrons are guided through the various experimental beamline stations of which there are currently around 42. Vacuum pumps continually operate to maintain ultra high levels of vacuum in the system.
Diagram of Diamond Light Source: 1 injection system and linac; 2 Booster synchrotron; 3 Storage Ring; 4 Beamline; 5 Front End; 6 Optics Hutch;7 Experimental Hutch; 8 Control Cabin; 9 RF cavities; 10 Diamond House (Image: Diamond Light Source Ltd 2023)

 

A key part of the process of using the Diamond Light Source is appropriate preparation and preservation of tissue samples and where preservation of samples in transit is often undertaken at liquid nitrogen temperatures. The operation of each beam line is managed by a resident scientist who can advise on optimum use of sample stages, modes of detection and storage and analysis of data.

 

Eye Investigations

The various beamlines at the Diamond Light Source have been used to investigate the molecular structure of ocular tissues, principally of the cornea, sclera and lens. This has provided a unique perspective on various clinical conditions where deteriorating tissue function has been associated with changes in molecular self organisation.

In addition, the system has been used to investigate the atomic structure of retinal tissue using x-ray fluorescence as part of research into macular degeneration.

 

Corneal Cross Linking Therapies

While the use of riboflavin in association with UVA radiation to encourage corneal cross linking has gained in popularity, there has been uncertainty regarding the structural organisation of altered corneal tissue.

In the cross linking process, riboflavin is energised by the UVA radiation to create free radicals of oxygen, which encourage molecular bonds between individual molecules of collagen and increases the structural strength of the associated tissue.

A study by Hayes et al investigated the effect of cross linking a range of corneal samples with either hypo-osmolar or iso-osmolar riboflavin (dextran-based) solution where dextran acts as a deturgescent to maintain the state of relative dehydration of corneal tissue.3

Initial clinical practice has been with solutions containing dextran where a minimum corneal thickness of 400 microns has been used to decide on treatment suitability based on the potential adverse effect of dextran on the endothelium.

The use of hypo-osmolar (dextran-free) riboflavin has subsequently allowed thinner corneas to be treated in this way. In the study two keratoconus corneal button samples were utilised for cross linking together with 15 normal donor corneas, which were deemed to have low endothelial cell counts and consequently unsuitable for corneal transplants.

It was generally observed that fibril diameters and intrafibrillar spacings were increased with use of the hypo-osmolar riboflavin cross linking with UVA but were reduced with iso-molar riboflavin (with dextran) with UVA. The increased spacing with the dextran free solution (hypo-osmolar) is in part due to the additional hydration of tissue.

The study authors, however, suggest that there is greater uncertainty regarding the degree of absorption and stability of the hypo-osmolar film as identified by Wollensak et al.4 This observation possibly infers the use of elevated doses of UVA in a shorter time period than used for iso-molar treatments to maintain treatment effectiveness.

 

Structure of Biosynthetic Corneal Substitutes

As the number of biosynthetic corneal substitutes increases, there is growing interest in determining a wide range of physical parameters of such materials. The interest in such materials is driven by the acute shortage of donor corneal tissue.

Human cornea is identified as having an optimised molecular structure that confers unique properties with regard to optical transparency and mechanical strength, and where x-ray scattering techniques provide a unique method of identifying structural differences between such tissues.

The successful use of recombinant human collagen implants5,6 has indicated the potential of such material in treating a range of clinical conditions though the tensile strength of such material is significantly inferior to that of human corneal tissue. More recently acellular porcine-derived collagen has been identified as a more economical and practical collagen source.

Hayes et al describes a range of investigations undertaken on comparable laboratory fabricated collagen implant materials where a key investigation tool was the Diamond Light Source.7 Wide angle x-ray scattering (WAXS) was utilised to identify the predominant orientation of the collagen molecules in the various samples.

It was identified that the recombinant human collagen III (RHCIII) hydrogel samples were typically aligned along a common single axis direction while the human corneal samples showed alignment along two separate directions described as superior-inferior and nasal-temporal as indicated in figure 3.

Figure 3: Alignment pattern of collagen in RHCIII samples and human cornea7

 

 

This alternate collagen alignment in human corneas, which was also demonstrated using transmission electron microscopy (TEM), is responsible for their higher tensile strength. This factor is important for prevention of clinical conditions such as keratoconus. Small angle x-ray scattering was used to investigate the structure of human corneal samples and also of RHCIII hydrogel samples.

While a scattering pattern consistent with a D-periodicity (which is a way of describing a repeating banding pattern of collagen fibrils) of 65nm was identified with the human cornea (figure 4a) as expected, no pattern was observed for the RHCIII hydrogels since there was no structure of alternate collagen alignment within collagen fibrils to detect as indicated in figure 4b.

Figure 4: Small angle x-ray scatter (A) human cornea (B) RHCIII material7

 

Figure 5 indicates the origin of the D-period structure of collagen fibrils where in human cornea the individual monomers of typical length 300nm are separated in a specific layer by 40nm and with the adjoining layer offset by 25nm, which produces the observed periodicity centred on 65nm.

Figure 5: Origin of D-period in structure of collagen fibrils in human cornea

The WAXS technique provides, therefore, an important means to identify if such biosynthetic corneal substitutes reflect a moleculardeposition pattern that will ensure adequate structural strength. While such observations confirm the single directional alignment of RHCIII collagen, the challenge remains to replicate the alternate collagen alignment of human cornea.

Possible mechanisms to achieve alternate layer alignment could include creation of a multilayer ‘sandwich’ deposition of collagen with opposite alignments that are fused to a single continuous layer and also ‘printing’ alternate collagen layers with switched orientation.

Recently Lei et al has described a technique for introducing alternate alignments into artificially constructed collagen materials for artificial corneal substitutes.8 A similar process has previously been described by Chen et al.9

Role of Vitamin C in Collagen Structure

It is known that UVB radiation in the wavelength range 290nm to 320nm is a risk factor10 for the development of ocular disorders such as pterygium and climatic droplet keratopathy. It is likely that the presence of ascorbic acid (vitamin C) in the corneal epithelium acts to absorb UVB radiation and reduce damage to stromal tissue.

In addition, the lack of vitamin C in the cornea is thought to impair the formation of stable collagen fibrils and as a consequence adversely affect corneal transparency. These factors were investigated in animal studies by Hayes et al where four groups were established with combinations of normal/ low vitamin C dietary intake and presence/absence of UVB exposure.11

It was observed using small angle x-ray diffraction at the Diamond Light Source that collagen fibril separation was progressively reduced with low vitamin C diet and exposure to UVB, suggesting the impairment of collagen cross linking function. The clinical inference identified was that vitamin C may have a key role in protecting corneal stroma from the effects of excessive UVB exposure.

 

Variation of Corneal Structure with Depth

The distribution of collagen organisation in the cornea and sclera has been investigated by a wide range of researchers. An intriguing aspect of investigations12 using WAXS has identified subtle changes in collagen characteristics in moving from the central corneal region to the limbal border and also with depth.

Samples from peripheral cornea were investigated using the European Synchrotron in Grenoble and the central cornea samples at the Diamond Light Source using WAXS. The limbal region marks a change in the curvature of the eye and this is associated with a contour of increased tensile strength associated with increased degrees of alignment of collagen fibrils as described by Aghamohammadzadeh et al.13

The structural integrity of the cornea and sclera as a whole is determined by the combined interactions of its connected tissues. By scanning tissue samples taken at different depths as five sections each of 100 microns thickness, it was possible to demonstrate that this increased alignment takes place in the more posterior layers of the sclera and takes up a tangential alignment parallel to the corneal/scleral boundary.

There is particular interest in this level of corneal structure since it directly affects the refractive contour of the cornea. In addition, disruption of corneal layers at the limbus by, for example, laser refractive surgery has the potential to induce post procedure astigmatism due to the altered structural strength of eye in this region. A wide range of other investigative techniques, are also used in conjunction with x-ray scattering to characterise such structures.

 

Investigation of Graft-Host Collagen Status in Penetrating Keratoplasty

The reduced structural strength of the corneal stroma after radical procedures such as penetrating keratoplasty has for some time been suspected14 as an underlying cause of eventual graft failure. Boote et al describe the use of x-ray scattering to quantify stromal collagen ultrastructure from three eyes of two patients who had previously undergone penetrating keratoplasty procedures.15

A common observation of wound margins was that the alignment of collagen fibrils as evidenced by x-ray diffraction tended to align along the wound margin and not span across it, which is likely to have reduced the strength of the graft-host interface even after the tissue appears fully healed.

This was identified as a probable underlying cause of eventual graft failure. This prompts speculation regarding practice and processes that could improve the integrity of the graft-host interface, either at initial surgery or as post surgical management.

 

Collagen Structures in Cases of High Myopia

The increasing prevalence of myopia has encouraged research into the molecular constructs in the posterior sclera around the optic nerve head (ONH). A study by Markov et al used x-ray diffraction at the Diamond Light Source to compare the tissue samples from seven non-myopic globes and two highly myopic globes.16

In the non-myopic tissue samples, there was observed a highly aligned development of circular ‘rings’ of aligned collagen around the ONH while in the highly myopic specimens there was interruption observed of the circumferential collagen orientation, with the implication that the ONH could be less well protected against mechanical stress forces.

The researchers indicate, however, that while such re-modelling of tissue is observed in cases of high myopia, it is not clear if this is as a result of eye lengthening or due to greater stress forces arising from increased intraocular pressures or eye movements in enlarged eyes.

 

X-Ray fluorescence Studies of Retinal Tissue

X-ray fluorescence provides a mechanism to investigate the atomic composition of material samples and has been used, for example, to investigate the composition of retinal drusen samples17 as a possible indicator of the progression of age-related macular degeneration.

In x-ray fluorescence, x-rays eject electrons from inner energy levels, which results in characteristic x-ray radiation of lower energies as atoms return to their ground state energy levels.

Fingerprints of up to 10 separate elements can be simultaneously identified in this way. Use was made of beamline I18 at Diamond for microfocus spectroscopy with a beam size of 2.0µm x 2.5µm and an energy range from 2.05keV to 20.5keV. A comparable beamline, I22 is indicated in figure 6.

Figure 6: A section of beamline I22 – indicating high degree of complexity of function (Image: Diamond Light Source Ltd 2023)

The identification of calcified nodules at a specific phase of age-related macular degeneration suggests that an imbalance of Ca2+ and Mg2+ regulation may be a factor in disease progression, through the field of study is challenging based on the complex metabolic pathways identified.

Additional scanning technologies of this specific beamline include µX-ray fluorescence imaging, X-ray Excited Optical Luminescence (XEOL) and X-ray Fluorescence Tomography. A detailed description of the capabilities of the I18 beamline has been provided by Mosselmans et al.18

This particular research work highlights the broad range of collaborating groups involved and also the range of highly developed multimodal research tools/technologies available for such investigations.

 

Summary

The Diamond Light Source provides a unique research tool for investigation of tissue structures and molecular composition in the eye. Such research indicates that a wide range of clinical conditions may have their origin in adverse changes in molecular structure and organisation.

Perhaps this provides some clues regarding mechanisms for preventing deterioration of and restoring the natural structures of ocular tissues. 

  • Dr Douglas Clarkson is a Research Fellow at UHCW NHS Trust in Coventry with a wide interest in optical technologies applied to diagnostic specialties and modes of treatment which include lasers and non-coherent light sources.

 

References

  1. Watson, James D, and Francis HC Crick. “Molecular Structure of Nucleic Acids.” Nature 171 (1953): 737–8.
  2. Meek KM, Quantock AJ. The use of X-ray scattering techniques to determine corneal ultrastructure. Prog Retin Eye Res. 2001 Jan;20(1):95-137
  3. Hayes S, Boote C, Kamma-Lorger CS, Rajan MS, Harris J, Dooley E et al. Riboflavin/UVA collagen cross-linking-induced changes in normal and keratoconus corneal stroma. PLoS One. 2011;6(8):e22405.
  4. Wollensak G, Aurich H, Wirbelauer C, Sel S. Significance of the riboflavin film in corneal collagen crosslinking. J Cataract Refract Surg. 2010 Jan;36(1):114-20
  5. Fagerholm P, Lagali NS, Merrett K, Jackson WB, Munger R, Liu Y et al. A biosynthetic alternative to human donor tissue for inducing corneal regeneration: 24-month follow-up of a phase 1 clinical study. Sci Transl Med. 2010 Aug 25;2(46):46ra61.
  6. Fagerholm P, Lagali NS, Ong JA, Merrett K, Jackson WB, Polarek JW et al. Stable corneal regeneration four years after implantation of a cell-free recombinant human collagen scaffold. Biomaterials. 2014 Mar;35(8):2420-7
  7. Hayes S, Lewis P, Islam MM, Doutch J, Sorensen T, White T et al. The structural and optical properties of type III human collagen biosynthetic corneal substitutes. Acta Biomater. 2015 Oct;25:121-130
  8. Lei M, Zhang S, Zhou H, Wan H, Lu Y, Lin S et al. Electrical Signal Initiates Kinetic Assembly of Collagen to Construct Optically Transparent and Geometry Customized Artificial Cornea Substitutes. ACS Nano. 2022 Jul 26;16(7):10632-10646
  9. Chen Z, Liu X, You J, Song Y, Tomaskovic-Crook E, Sutton G et al. Biomimetic corneal stroma using electro-compacted collagen. Acta Biomater. 2020 Sep 1;113:360-371
  10. Delic NC, Lyons JG, Di Girolamo N, Halliday GM. Damaging Effects of Ultraviolet Radiation on the Cornea. Photochem Photobiol. 2017 Jul;93(4):920-929. doi: 10.1111/php.12686. Epub 2017 Feb 28. PMID: 27935054.
  11. Hayes S, Cafaro TA, Boguslawska PJ, Kamma-Lorger CS, Boote C, Harris J et al. The effect of vitamin C deficiency and chronic ultraviolet-B exposure on corneal ultrastructure: a preliminary investigation. Mol Vis. 2011;17:3107-15
  12. Kamma-Lorger CS, Boote C, Hayes S, Moger J, Burghammer M, Knupp C et al. Collagen and mature elastic fibre organisation as a function of depth in the human cornea and limbus. J Struct Biol. 2010 Mar;169(3):424-30
  13. Aghamohammadzadeh H, Newton RH, Meek KM. X-ray scattering used to map the preferred collagen orientation in the human cornea and limbus. Structure. 2004 Feb;12(2):249-56
  14. Hayes S, Young R, Boote C, Hawksworth N, Huang Y, et al. (2010) A structural investigation of corneal graft failure in suspected recurrent keratoconus. Eye 24: 728–734.
  15. Boote C, Dooley EP, Gardner SJ, Kamma-Lorger CS, Hayes S, Nielsen K et al. Quantification of collagen ultrastructure after penetrating keratoplasty - implications for corneal biomechanics. PLoS One. 2013 Jul 5;8(7):e68166.
  16. Markov PP, Eliasy A, Pijanka JK, Htoon HM, Paterson NG, Sorensen T et al. Bulk changes in posterior scleral collagen microstructure in human high myopia. Mol Vis. 2018 Dec 30;24:818-833
  17. Tan ACS, Pilgrim MG, Fearn S, Bertazzo S, Tsolaki E, Morrell AP et al. Calcified nodules in retinal drusen are associated with disease progression in age-related macular degeneration. Sci Transl Med. 2018 Nov 7;10(466):eaat4544.
  18. Mosselmans JFW, Quinn PD , Dent AJ. Cavill SA, Moreno SD et al. I18 – the microfocus spectroscopy beamline at the Diamond Light Source, J. Sync. Rad. 2009, 16: 818-824