Preservation of three-dimensional spatial structure in the gut microbiome

Area of Science:

• Microbiology
• Histology
• Bioimaging

Background:

• Accurate analysis of gut microbiota and luminal contents requires preserving the gut's native 3D structure.
• Conventional embedding media like OCT, paraffin, and polyester waxes can cause spatial redistribution of gut contents.
• Developing robust sample preparation methods is crucial for high-resolution gut imaging.

Purpose of the Study:

• To evaluate and optimize mouse gut preparation methods for preserving micron-scale spatial structure.
• To identify embedding media that prevent redistribution of luminal contents during sample processing.
• To establish a protocol compatible with various fluorescence imaging and labeling techniques.

Main Methods:

• Evaluation of common embedding media (OCT, paraffin, polyester waxes) for their effect on gut structure.
• Testing a hydrophilic methacrylate resin (Technovit H8100) for its ability to preserve 3D organization.
• Optimization of a mouse intestinal preparation protocol using Technovit H8100.
• Compatibility testing with microbial fluorescence in situ hybridization (FISH), immunostaining, WGA, and DAPI staining.
• Assessment of mucus visualization with paraformaldehyde (PFA) and Carnoy's fixative.

Main Results:

• Common embedding media (OCT, paraffin, waxes) caused redistribution of gut luminal contents.
• Technovit H8100 effectively preserved the three-dimensional organization of the mouse gut.
• The optimized protocol supported multiple labeling techniques, including FISH, immunostaining, WGA, and DAPI.
• Mucus visualization was achievable with both PFA and Carnoy's fixative.
• Simultaneous visualization of microbial spatial patterns and host-derived landmarks was enabled.

Conclusions:

• Technovit H8100 is a superior embedding medium for preserving mouse gut 3D structure and luminal contents.
• The developed protocol facilitates detailed spatial analysis of the gut microbiome and its environment.
• This method enables comprehensive imaging of gut biogeography at the micron scale.