Three-dimensional Quantification of Intestinal Mucus Using Whole-mount Tissue Imaging

Summary

This study presents a 3D imaging method using whole-mount intestinal tissues and multi-photon microscopy to quantify secreted mucus, enabling precise volumetric analysis and visualization of mucus dynamics in response to stimuli like carbamoylcholine chloride.

Abstract

Mucus plays a critical role in maintaining intestinal homeostasis by facilitating digestion, forming a barrier against microbes, and regulating immune responses within the gut. Its secretion is modulated by various intrinsic and extrinsic factors, including microbial infections and inflammation. While traditional methods for mucus analysis rely on relative quantification, such as measuring mucus thickness in histological sections, these approaches provide limited insight into the actual volume and spatial distribution of secreted mucus within the intestinal lumen. Here, we present a detailed methodology for the absolute, three-dimensional quantification of mucus using whole-mount intestinal tissues and multi-photon microscopy. This protocol includes the preparation of ligated intestinal loops, treatment with carbamoylcholine chloride as a stimulus for goblet cell activation, tissue fixation, and staining for imaging. Using multi-photon microscopy, we acquire Z-stacked images of the luminal surface. These are processed in Imaris software to quantify the volume and spatial distribution of secreted mucus. We demonstrate that carbamoylcholine chloride robustly induces mucus secretion in both ileal and colonic tissues within 30 minutes. The secreted mucus appears in a sporadic and discontinuous manner throughout the lumen, underscoring the importance of volumetric analysis over traditional two-dimensional approaches. This protocol enables researchers to obtain absolute quantitative data and visualize mucus distribution in situ, offering a powerful tool for studying intestinal mucus dynamics under physiological and pathological conditions.

Introduction

The mucosal environment of the mammalian gastrointestinal tract is highly dynamic, supporting the colonization of a diverse microbial community. The intestinal epithelium consists of various specialized cells that collaborate to absorb nutrients, maintain barrier integrity, and communicate with the immune system¹. Among these, goblet cells store mucin granules and secrete mucus into the lumen under steady-state conditions. This mucus forms a critical physical barrier, preventing direct microbial contact with the epithelial layer, modulating tissue inflammation, and maintaining intestinal homeostasis^2,

Protocol

All animal experiments are approved by the Institutional Animal Care and Use Committee (IACUC) of Chang Gung University. The animal facility of Chang Gung University is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). No animal health concerns were observed in these studies.

1. Preparation of fixation base

1. Mix agarose powder with ddH₂O to a concentration of 4% (w/v) in a microwavable flask. Microwave for 2-3 min until it reaches a boil without tiny bubbles.
   NOTE: The volume depends on the number of samples; approximately 10 mL for a 6 cm dis....

Results

In this method, we quantified the volume of intestinal mucus from stacked images of whole-mount tissues acquired via multiphoton microscopy. Intestinal tissues were stained with fluorescent phalloidin and wheat germ agglutinin (WGA) to visualize cellular F-actin and mucus, respectively^15,16. In both ileal and colonic tissues treated with PBS, WGA⁺ mucus was mainly detected within goblet cells at the epithelial surface. Following carbamoylcholine chlori.......

Discussion

Here, we present a method for three-dimensional quantification of intestinal mucus using whole-mount tissue staining, multi-photon microscopy, and analysis with Imaris software. This procedure allows the observation of the discontinuous distribution of mucus within the intestinal lumen and provides an absolute measurement of mucus volume.

Discontinuous mucus distribution upon stimulation has also been observed in explant tissue culture systems⁸. In previous studies usin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2,2,2-tribromoethanol	Sigma-Aldrich	T48402
2-methyl-2-butanol	Sigma-Aldrich	240486
6 cm dish	Alpha Plus	16021-1
Agarose	Hispanagar	A02599
BSA	BioShop	ALB001
Carbamoylcholine chloride ≥98% (titration), crystalline	SIGMA	SI-C4382-1G
Copper wire (new)	TOP TECH WIRE & CABLE INDUSTRIAL	IW00125	Copper wire without insulation layer
Forceps	Shineteh	ST-M110
Gauze, sterile	GMTH	May-40
GraphPad Prism 8	GraphPad Software
Heating pad	CONFORT	SY-666
IMARIS 10.0.0	OXFORD INSTRUMENTS
Paraformaldehyde	BIOVOVAS	BL0415-1000
PBS	GENESTAR	BL0180-1000
Phalloidin Labeling Probes	Invitrogen	A12379
Scissors	Shineteh	02-1250.18
Triton X-100	Merck	1.08603.1000
Wheat Germ Agglutinin (WGA) Conjugates	Biotium	29027
Zeiss 7MP with a W Plan-Apochromat 20 x/1.0 DIC III water immersion objective (NA 1.0), Spectra-Physics Mai Tai HP ti sapphire femtosecond laser (without DeepSee module) with BP500 nm-550 nm and BP565 nm-610 nm detectors, and 750 nm laser wavelength			imaging platform used to acquire 3D stacks with a high-NA objective lens for deep tissue imaging; an excitation source for multi photon microscopy set to a wavelength of 750 nm