We can see clearly now

The cornea is the eye's window – it allows us to see. Its transparency has baffled scientists, but physicists are at last revealing its complex structure, as Simon Hadlington reports

The eye is often cited as one of evolution's most awesome creations – a window on the world in which specialised cells are stimulated by light to produce a volley of electrical impulses that are sent to the brain to be processed, resulting in what we call "sight".

However, the eye would not function if its own window – the cornea – was not transparent. Yet despite decades of speculation and theory, the absence of hard experimental data has meant that the exact nature of the transparency of the cornea has remained uncertain.

But now, physicists in the UK are on the verge of cracking the problem. By using some of the world's most powerful X-rays, the researchers are revealing the intricate structure of the cornea, providing the definitive information that the theoreticians have lacked until now. Importantly, the research is also shedding new light on diseases of the eye, where the structure of the cornea breaks down, resulting in loss of vision. The work is yielding new information for eye surgeons about how best to operate on the cornea to enable healing to take place as quickly as possible and without scarring, and to explain why laser surgery to correct defects in people's vision sometimes leads to a haze on the surface of the cornea. It is also giving scientists clues about what would be required successfully to create a biologically-based artificial cornea for transplanting into patients whose own cornea has failed through disease or injury.

The cornea is remarkable because it must combine two fundamental properties that are difficult to reconcile in a biological tissue: toughness and transparency. It needs to be transparent to allow light to enter, and it needs to be tough because it is exposed on the surface of the body and must be able to withstand the inevitable batterings. Most animal tissues, however, are inherently opaque.

At the Department of Optometry and Vision Sciences at Cardiff University, Professor Keith Meek and his colleagues Professors Stuart Hodson and Bruce Caterson and Dr Andrew Quantock have recently been awarded a £1.1m grant from the Medical Research Council to provide definitive experimental evidence that accounts for the cornea's transparency.

"The cornea is classified as a connective tissue, and is made largely of the protein collagen, which forms fibres that are not transparent," says Professor Meek. "Collagen itself is the main scaffold protein of the body – the principal component of skin, bones and ligaments."

So the question is: how can light be transmitted through a structure that is made of an opaque substance? The answer lies in the phenomenon of interference. If light waves strike a very small aperture, they spread outwards as they emerge from the other side. If two such apertures are adjacent, the emerging light waves can "interfere" with each other, either destructively, in which case they cancel each other out, or constructively, whereby they reinforce one another. A diffraction grating is an arrangement of many adjacent apertures; depending on their geometry, a variety of interference patterns can be obtained. Certain arrangements of the grating will allow virtually all the incident light to be transmitted. In effect, the cornea is an exquisite diffraction grating.

Keith Meek explains: "The cornea is continuous with the white of the eye, the sclera – it is made of essentially the same material. However, under the electron microscope, the collagen fibres in the sclera are thick and irregularly arranged in relation to each other. In the cornea, the fibres are very narrow, are spaced evenly and parallel to each other."

There are between 200 and 300 layers of these parallel, narrow, collagen fibres in the cornea, each layer oriented at a slightly different angle to the one below it. Distributed between these layers are specialised flat cells, which replenish the components of the cornea, slowly under normal circumstances but very quickly if the cornea becomes damaged.

The Cardiff researchers have used powerful X-rays generated at the Synchrotron Radiation Source at Daresbury in Cheshire to investigate the arrangement of the collagen fibres in the cornea. By passing the X-rays through the structure and analysing the pattern that they produce, it is possible to measure very precisely the size, pattern and orientation of the fibres.

Each fibre is about 30 nanometres thick (a nanometre is a millionth of a millimetre), and the distance between the centres of neighbouring fibres is around 60 nanometres. Between the fibres are jelly-like molecules called proteoglycans, of which there are several types, and which are thought to act as spacers, retaining the correct distance between the collagen fibres.

"The transparency of the cornea is to do with the spacing between the collagen fibres," says Professor Meek. "When light enters the cornea, it strikes the collagen fibres and effectively bounces off in random directions. The spacing and arrangement of the fibres is such that any light waves that bounce off and meet other light waves destructively interfere and cancel each other out. However, those light waves in the straight-through direction effectively reinforce each other – they constructively interfere. In this way, around 96 per cent of the light entering the eye passes through this diffraction grating."

The regular spacing between the collagen fibres is therefore crucial to the cornea's transparency. When this precise arrangement of the fibres breaks down, the light waves start to scatter within the structure, causing opacity. In some diseases, it appears that the proteoglycan spacers fail to do their job properly, causing the pattern of the fibres to become disturbed.

Professor Caterson, a biochemist, is developing a series of antibodies that will be able to attach specifically to each of the different types of proteoglycan to reveal their precise location within the structures. By mapping their distribution in healthy and diseased corneas, it should be possible to get an idea of their exact function within the tissue.

Dr Quantock, meanwhile, has been awarded a grant by the Engineering and Physical Sciences Research Council to track how the arrangement of the collagen fibres correlates with the transparency of the cornea as it develops in the growing embryo of a chick. "By taking the cornea at different points in its development, it is possible to measure its transparency against the arrangement of the collagen fibres," he says.

"What we have found is that as the cornea develops, the collagen fibres get closer: early in the development, the spacing is wide and the fibres are relatively disorganised. At this stage, the cornea lets only around 40 per cent of light pass through it. However, within three or four days, the structure has become much more uniform and 90 per cent of light can get in."

The work with the X-rays is also providing vital information about the shape of the cornea. "The cornea's shape is crucial to its function," says Professor Meek. "The cornea is the most important refracting component of the eye because most of the eye's focusing power comes from the cornea, not the lens." This is why it is possible to correct some vision defects by using a laser to burn off the top layers of the cornea to flatten it slightly.

The X-ray diffraction studies have shown the arrangement of the bundles of fibres of collagen not only in the cornea itself, but also in the tissue surrounding it. "We have shown that there is a collar of protein running around the edge of the cornea that we believe is crucial for retaining its shape and function," says Professor Meek. "Because we can see which ways the fibres are running, we can give this information to surgeons, who might prefer to make an incision along the fibres rather than across them."

The structural information is also leading to a better understanding of why laser- correction of vision defects can lead to a haze on the surface of the eye. "It had previously been thought that the cloudiness after some laser treatments was because the new collagen laid down as scar tissue was not as precisely defined as it needs to be, with larger fibres put down at the incorrect spacing," says Professor Meek. "However, our modelling studies have shown that this is in fact unlikely to be the case. It seems more likely to have something to do with the specialised cells that are distributed throughout the cornea to replace the collagen and proteoglycans – but we do not yet know for sure."