Probing Multiscale Collagenous Tissue by Nonlinear Microscopy

Imaging of thick biological samples has been very challenging because of severe light scattering. The transparent cornea with the unique organization of stromal collagen makes it a good candidate for deep imaging and is responsible for mechanical strength and optical clarity of the eye. However, limitation on traditional histology method provides incomplete spatial information and details on the structural organization of corneal tissue is still not sound. Second harmonic generation (SHG) microscopy is a noninvansive and nonstained technique to characterize the macromolecular organization of collagen in biological tissues. Through the combination of SHG microcopy and optimized Fourier-transform analysis, adult and embryonic chick corneas are investigated. Our results show that the anterior stroma demonstrates a fanlike distribution of rotated fibrous lamellae. In comparison with the anterior structure, the posterior stroma maintains a nonrotating pattern while increasing the depth of corneal tissue. In particular, the rotational pattern in anterior stroma exhibits a potential role of corneal maturation. Moreover, SHG microscopy in combination with the Fourier-transform-based analysis exhibits a useful tool in determination of collagen alignment in biological tissues and discrimination of diseases.