Label-free cleared tissue microscopy and machine learning for 3D histopathology of biomaterial implants

ABSTRACT Tissue clearing of whole intact organs has enhanced imaging by enabling the exploration of tissue structure at a subcellular level in three-dimensional space. Although clearing and imaging of the whole organ have been used to study tissue biology, the microenvironment in which cells evolve to adapt to biomaterial implants or allografts in the body is poorly understood. Obtaining high-resolution information from complex cell-biomaterial interactions with volumetric landscapes represents a key challenge in the fields of biomaterials and regenerative medicine. To provide a new approach to examine how tissue responds to biomaterial implants, we apply cleared tissue light-sheet microscopy and three-dimensional reconstruction to utilize the wealth of autofluorescence information for visualizing and contrasting anatomical structures. This study demonstrates the adaptability of the clearing and imaging technique to provide sub-cellular resolution (0.6μm isotropic) 3D maps of various tissue types, using samples from fully intact peritoneal organs to volumetric muscle loss injury specimens, with and without biomaterials implants. We further apply computational-driven image classification to analyze the autofluorescence spectrum at multiple emission wavelengths to categorize tissue types in the tissue-biomaterial microenvironment.