Second Harmonic Generation (SHG) microscopy has emerged as a powerful label-free tool for studying collagen microstructure in biological tissues, with increasing interest in the backward-detected signal due to its potential for translation to in vivo imaging contexts. The origin of backward-SHG signal is not fully understood and, as such, interpretation of images remains challenging, as it represents complex optical interactions. This has led to misinterpretations of the collagen structure of biological tissues. Using a custom optical chamber allowing bidirectional SHG imaging of the cornea under controlled pressure, we assessed the spatial relationship between features in backward images and the underlying structural features of collagen in the paired forward images. Collagen waviness (crimp) was identified to be a key structural feature responsible for periodic bands of fluctuating signal intensity in the backward SHG images. Additionally, we demonstrate the potential of SHG imaging for investigating the biomechanical mechanisms relating to collagen and build-upon previous theoretical and experimental frameworks explaining forward and backward signal generation and current limitations in their interpretation.
Boutkhil et al. (Wed,) studied this question.