Comparative analysis reveals Ce3D as optimal clearing method for in toto imaging of the mouse intestine.

Background: The intestinal wall has a complex topographical architecture. The multi-layered network of the enteric nervous system and its intercellular interactions are difficult to map using traditional section-based or whole-mount histology. With the advent of optical clearing techniques, it has become feasible to visualize intact tissue and organs in 3D. However, as yet, a gap still needs to be filled in that no in-depth analysis has been performed yet on the potential of different clearing techniques for the small intestine.

Aim: The goal of this study was to identify an optimal clearing protocol for in toto imaging of mouse intestinal tissue.

Methods: Five aqueous-based clearing protocols (SeeDB2, CUBIC, ScaleS, Ce3D, and UbasM) and four organic reagent-based clearing protocols (3DISCO, iDISCO+, uDISCO, and Visikol ) were assessed in segments of small intestine from CX3CR1 and wild-type mice. Following clearing, optical transparency, tissue morphology, green fluorescent protein (GFP) fluorescence retention, and compatibility with (immuno-)labeling were analyzed.

Key Results: All organic reagent-based clearing protocols-except for Visikol-rendered tissue highly transparent but led to substantial tissue shrinkage and deformation. Of the aqueous-based protocols, only Ce3D yielded full-thickness tissue transparency. In addition, Ce3D displayed excellent GFP retention and preservation of tissue morphology.

Conclusions: Ce3D emerged as a most efficient protocol for enabling rapid full-thickness 3D mapping of the mouse intestinal wall.