Abstract
The unique organization of the corneal stromal collagen is responsible for mechanical strength and optical clarity of the
eye. However, factors and reasons on formation of the corneal stroma is still not fully understood. Second-harmonic
generation (SHG) is a nonlinear second order optical process occurring in noncentrosymmetric systems with a large
hyperpolarizability. Through the combination of the second harmonic generation (SHG) microcopy and optimized
Fourier-transform analysis, mature chicken corneas are investigated to probe the depth-dependent collagen organization
of the corneal stroma. Our results show that the anterior stroma behaves like a fan-like distribution of successively and
counterclockwisely rotated fibrous lamellae for paired corneas from the same chicken. However, the posterior stroma
maintains a non-rotating pattern while increasing in depth. Surprisingly, the thickness of the anterior stroma remains
almost constant throughout the temporal-nasal direction, but the posterior stroma does not behave the same. Through
quantitative analysis, the natural transition of the anterior and posterior stroma is also determined. These findings
enhance our understanding of the collagen-rich tissue in the chicken cornea model. Moreover, the Fourier-transformbased
modality, in combination with SHG microscopy, serves as a promising tool to determine collagen alignment in
embryonic development, tissue engineering and corneal diseases.
