Irreversible optical clearing of sclera by dehydration and cross-linking

This study manipulates both clear cornea and opaque sclera by two dehydration processes for revealing the relationship between altered tissue structures and change in optical functions. In contrast to the high levels of light scattering in dehydrated tissues by critical point dry, a simple dehydration at 4-8 °C effectively and significantly improved their visible-light transmission, even in the sclera, with accompanying dense fiber packing. Further improvement in visible-light transmission, from 40-50% to 80-90%, has been achieved by flatting tissue surface with cover glasses during dehydration at low temperature. Such optical clearing of sclera by dehydration is reversible. However, chemical cross-linking effectively stabilizes their densely packed microscopic structures and visible-light transmission at over 50% irreversibly, even at wet conditions. Interestingly, the repetition of both low temperature dehydration/cross-linking treatments effectively reduced the required amounts of cross-linking reagents to keep a high transparency. Wet transparent cross-linked sclera can also show a characteristic strong tensile strength. Furthermore, rabbit corneal epithelium has regenerated on the transparent sclera with cross-linking in vitro.