Optical clearing improves the imaging depth and signal‐to‐noise ratio for digital analysis and three‐dimensional projection of the human enteric nervous system

Background Due to the dispersed nature of neurites and fibers, the microtome‐based 2‐dimensional histology provides only a limited perspective of the enteric nervous system. To visualize the enteric plexus, we applied optical clearing to avoid scattering in the human ileum to facilitate photon penetration for 3‐dimensional (3‐D) microscopy of the neural tissue. Methods Human ileal specimens were derived by trimming the donor bowel due to its excess length during the clinical trial of small intestinal transplantation. The pan‐neuronal marker PGP9.5 was used as the immunostaining target to reveal the enteric plexuses. The labeled tissues were immersed in the optical‐clearing solution prior to deep‐tissue confocal microscopy. The serial sections were digitally analyzed and processed by reconstruction algorithms for 3‐D visualization. Key Results Optical clearing of the ileal specimen led to less fluorescence signal decay along the focal path in the tissue and a higher signal‐to‐noise ratio of the confocal micrographs in comparison with the untreated saline control. Taking advantage of the high signal‐to‐noise ratio images, we applied software‐based signal analysis to identify the presence of the nerve fibers and quantify the signal peaks. The image stacks derived from the serial anatomic micrographs created panoramic views of the gut wall innervations with their associated microstructures. Conclusions & Inferences We provide an optical approach to improve the imaging depth in 3‐D neurohistology of the human ileum. This methodology has significant promise in facilitating our understanding of the enteric nervous system in health and disease.