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

Abstract

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.

PSHG-TISS: A collection of polarization-resolved second harmonic generation microscopy images of fixed tissues

“…The SHG directional emission ratio (forward SHG to backward SHG ratio) was shown to be correlated with the fibril diameter and the collagen density 19 , 20 . Fractal analysis 18 , 21 was used to extract collagen-related morphological data from SHG images, while whole image Fast Fourier Transform 22 , 23 and wavelet transform 24 were able to quantify the texture and orientation changes of collagen. Inter-pixel relationship in SHG images were quantified by using texture analysis in which second-order image statistics are evaluated from the gray-level co-occurrence matrix (GLCM) 9 , 18 , 25 .…”

Quantitative analysis of second harmonic generated images of collagen fibers: a review

“…It is possible to determine fiber orientation using the FFT. It is the most used technique for this matter (Ayyalasomayajula et al 2019;Bueno et al 2013;Chiu 2010;Chow et al 2014;Forouhesh Tehrani et al 2021;Lau et al 2012;Lee et al 2019;Pijanka et al 2019;Robinson et al 2016;Sivaguru et al 2010;Wu et al 2011). This approach is also called FT-SHG imaging (Ambekar et al 2012a).…”

“…This approach is also called FT-SHG imaging (Ambekar et al 2012a). In Lee et al (2019), the authors used the FFT on the entire stack and then performed a segmentation on the transformed images to only keep the dominant fibers' direction. Once the segmentation achieved, it is possible to recover the angles' distribution that corresponds to each image.…”

“…The dominant orientation from a projection of all the SHG stack images can be extracted to recover a 2D image that contains all information from the entire stack (Hristu et al 2018). However, it is more common to use 2D images to estimate the orientation (Bueno et al 2013;Kabir et al 2013) and look at its evolution with respect to the stack depth (Lee et al 2019).…”