Second harmonic generation imaging reveals asymmetry in the rotational helicity of collagen lamellae in chicken corneas.

High tensile strength and optical clarity are unique properties of the cornea. These features are dictated by the three-dimensional architecture of corneal lamellae. Therefore, understanding the microscopic details of the cornea's structural organization may contribute to the development of artificial cornea for the treatment of corneal diseases. In this study, the combination of forward second harmonic generation (SHG) microcopy and fast Fourier-transform based image analysis was used to characterize the depth-dependent superstructure of chicken corneal stroma. Our results show that from the surface, adjacent lamellae of anterior chicken cornea lamella rotate in a counterclockwise direction, and the same rotational helicity is observed in left and right corneas. Furthermore, the overall average rotational pitch of lamellae is 0.92 ± 0.11 degree/µm which persists for 176 ± 14 µm in the anterior stroma. As depth further increased, the rate of lamellar rotation decreases. Upon reaching posterior stroma, lamellar orientation remains constant. Throughout the stroma, collagen lamellae in chicken rotate a total of 169 ± 21 degrees. The lack of lamellar rotation in posterior stroma suggests that packing efficiency cannot be used to explain the helicity of depth-dependent rotation of anterior stroma. In addition, although the right cornea has a higher rotational pitch (0.95 ± 11 vs 0.90 ± 10 degrees/µm) and thinner anterior stroma (173 ± 13 vs 179 ± 14 µm) than the left cornea, the two effects cancel each other out and result in similar total angular rotation of anterior stroma (161 ± 23 and 165 degrees ± 21). Finally, our observation of a total angular rotation of 169 ± 21 degrees shows that within experimental error, chicken cornea lamellae rotate around 180 degrees or half of a complete turn. Additional studies are needed to arrive at an explanation of chicken superstructure in three dimensions.