3D imaging and pathological analysis of microglia in LPS-treated mice with light-sheet fluorescence microscopy

Although two-dimensional (2D) histology and immunohistochemistry techniques have long been established and successfully applied to obtain structural information from tissues, recent advances in tissue clearing and expansion approaches combined with light sheet microscopy have led to the realization of three-dimensional (3D) nondestructive pathology, which may revolutionize our knowledge of the morphology of an organ or the whole body in its true state. Employing these 3D technologies, we obtained imaging data of microglia in whole hippocampus of mice. We established a simple procedure to analyze the 3D structures of microglia using the commercial software Amira. Major 3D structural parameters, including the volume of the whole cell, volume of the cell body, Feret's maximum/minimum diameter, number of total branches, fractal dimension, convex hull, and number of intersections,were measured. We found that lipopolysaccharide administration markedly changed multiple 3D morphological parameters of microglia. 3D analysis of microglial structures provides a more comprehensive evaluation than 2D analysis, which can be applied in future neuroscience research.