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^2,3. Dysregulation of mucus secretion compromises barrier function and has been associated with inflammation and tumorigenesis^4,5,6,7.

Intestinal mucus secretion can be triggered by intrinsic factors, such as the neurotransmitter acetylcholine, and by microbial components from commensals^8,9. Given the complexity of the gut environment, many factors influencing goblet cell mucus secretion remain unexplored. Therefore, a precise method for quantifying luminal mucus is essential for investigating how different stimuli affect secretion dynamics. Traditional approaches mainly rely on relative quantification: for example, measuring mucus thickness using fluorescent or charcoal particles in ex vivo intestinal explant systems^10,11, or assessing the distance between luminal microbes and the epithelium in stained tissue sections^12,13. While these methods are convenient for relative comparisons, they may not capture the full distribution or three-dimensional structure of secreted mucus, particularly since secreted mucus does not always uniformly cover the epithelium.

In this study, we present a method for three-dimensional visualization and absolute quantification of luminal mucus using whole-mount intestinal tissues. This approach requires fresh tissue fixation with an appropriate fixative to preserve structural integrity and morphology, followed by fluorescent staining and imaging using a confocal or multiphoton microscope. By administering test agents directly into ligated intestinal loops of deeply anesthetized mice, followed by immediate fixation, the method preserves native mucus architecture and allows analysis of secretion kinetics. Compared to sectioned tissues, where the imaging depth is primarily limited by tissue thickness, the imaging depth of whole-mount tissues (without delipidation) reaches approximately 200-300 µm, constrained by light scattering and tissue opacity. Using this technique, we show that carbamoylcholine chloride (CCh), an acetylcholine analog whose receptors are expressed on goblet cells¹⁴, robustly induces rapid mucus secretion. Notably, the secreted mucus forms both a continuous layer along the epithelium and discontinuous structures within the lumen, features that are difficult to detect in conventional tissue sections. This protocol provides a robust platform for quantifying the three-dimensional distribution of intestinal mucus and for comparing the mucus-inducing potential of various stimuli.

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 dish.
2. Let the agarose solution cool down to ~60 °C in ~3 min. Pour the agarose into a 6 cm dish carefully until the liquid height reaches half of the dish (approximately 10 mL for a 6 cm dish).
3. Place the dish with lid in a closed area for 20-30 min, until the agarose has completely solidified.

2. Preparation of fixation material

1. Prepare new copper wires and cut them into 5 mm lengths. Each piece of tissue requires 6-8 pieces of wire.

3. Preparation of CCh solution

1. Dissolve crystalline CCh in sterile PBS buffer to a final concentration of 10 µM (200 µL for one intestinal loop).

4. Preparation of injectable anesthetics

1. To prepare the stock solution for anesthesia, dissolve 2,2,2-tribromoethanol in 2-methyl-2-butanol.
2. Use sterile physiologic saline to dilute the stock solution, so that the final working solution contains 30 mg/mL 2,2,2-tribromoethanol and 3% (v/v) 2-methyl-2-butanol.
3. Vortex thoroughly for ~1 min until no pellet remains in solution (maximum rate: 8).
4. Store the stock solution in the dark at 4 ˚C to avoid degradation; dispose of the stock 1 month after preparation.
5. Before anesthesia, draw the stock solution into sterile PBS and mix well to ensure even dispersion, achieving a final dose of 250 mg/kg per mouse.
   CAUTION: 2,2,2-Tribromoethanol has neurotoxicity, and 2-methyl-2-butanol is volatile and has irritating odor. Proper protective equipment should be used. For disposal, collect the waste in a sealed container and follow the institution's hazardous chemical waste disposal procedures.

5. Intestinal inoculation via loop injection

1. Anesthetize a 6-8-week-old mouse with intraperitoneal injection of 200 µL of 2,2,2-tribromoethanol (250 mg/kg per mouse), and place the mouse on a heating pad to maintain its body temperature. Confirm the depth of anesthesia via a toe-pinch.
2. After disinfecting the skin surface with alcohol, make an incision in the mouse's left lower abdomen using operating scissors and expose the cecum under anesthesia.
3. Identify the ileum or proximal colon, place the intestinal loop on a sterile gauze, and use arterial clamps to secure both ends, creating a 3-cm-long closed loop.
4. Rinse the organ on the gauze with sterile PBS.
5. Inject sterile PBS or CCh reagent in a volume no more than 200 µL into the ligated intestinal loop under anesthesia.
   NOTE: Rinse regularly the exposed intestine with physiologic saline throughout the treatment period to keep it moist. Ensure regular monitoring of anesthetic depth (toe pinch) and monitoring of vital signs.

6. Tissue harvest, fixation, and staining

1. After 30 min of treatment, euthanize the mouse by cervical dislocation, and harvest the intestinal loop.
2. Use forceps to hold the intestinal loop and gently rinse it in sterile PBS.
3. Gently cut open the intestinal loop longitudinally and rinse the tissue in sterile PBS.
4. Flatten the tissue and use copper wire segments to anchor it onto the agarose in a 6 cm dish.
5. Fix the tissue with 10 mL of 4% paraformaldehyde solution for 12-24 h in the dish.
6. Wash the tissue with PBS at least 3x to remove residual paraformaldehyde.
7. Immerse the tissue in 10 mL of blocking buffer (PBS supplemented with 0.4% Triton X-100 and 3% bovine serum albumin [BSA]) at 4 ˚C for 12-24 h in the dish.
8. Stain the tissue with Wheat Germ Agglutinin (WGA, 1:200) and Phalloidin (1:500) in blocking buffer, shaking at 120 rpm at 4 ˚C for 12-24 h in the dish.
   NOTE: To minimize dye consumption, the tissue can be anchored on solidified agarose in a 3 cm dish immersed in 2 mL of dye buffer for staining.
   CAUTION: Paraformaldehyde poses an inhalation hazard. Proper protective equipment should be used during handling, such as operation in a fume hood. For disposal, collect the waste in a sealed container and follow the institution's hazardous chemical waste disposal procedures.

7. Image capture by multiphoton microscopy

1. Wash out the remaining staining buffer at least 3x with PBS.
2. Anchor the tissue on agarose with copper wires in a 6 cm dish in 10 mL of PBS.
3. Acquire 100 optical sections (covering 99 µm in depth) of intestinal tissue and associated secreted mucus using a multiphoton microscope equipped with a high NA objective lens for deep tissue imaging. Use an excitation source for multiphoton microscopy set to a wavelength of 750 nm. Collect emission signals using BP500-550 nm and BP565-610 nm bandpass filters. Set the scanning speed to 8.
   NOTE: For multiphoton microscopy, the maximum scanning speed is 9, with higher values indicating faster acquisition. The speed setting depends on the capacity of the microscope. When using the imaging platform used to acquire 3D stacks, we recommend a scanning speed of 8 to balance acquisition time and image quality.

8. Image analysis and mucus quantification (Figure 1)

1. To measure the volume of each mucus particle (in the WGA channel), open the image stack in Imaris by dragging and dropping the file onto the Arena page. Click the Surface icon to begin the surface creation workflow. Follow the standard steps to segment and quantify particle volumes within the z-stack. Specific parameters and processing steps are detailed in Figure 1.
2. Exclude mucus particles smaller than 1,000 µm³, which are mostly goblet cells (Figure 2A,B).
3. Sum up the volume of secreted mucus for each group.

9. Statistical analysis

1. Analyze and compare total volume for each group. Enter the value of mucus volume in Column and analyze using a non-parametric Mann-Whitney test.

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