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Updated: May 23, 2026
Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine
Published on: June 24, 2019
Visualization of mucosal homeostasis via single- and multiphoton intravital fluorescence microscopy
Cassie Xu1, Yuelei Shen, Dan R Littman
1Department of Microbiology and Immunology, Indiana University School of Medicine, South Bend, IN, USA.
Abstract:
FIVM has provided many insights into the regulation of immunity. We report the validation of an approach for visualizing murine small bowel via single- and multiphoton FIVM. Tissue damage is limited to ∼200 μm, immediately adjacent to the incision, as confirmed by intravital PI staining. Treatment with 10 KDa dextran-FITC and 70 KDa dextran-TR confirms that perfusion is intact. Selective filtration of 10 KDa but not 70 KDa dextran from the blood indicated that kidney function is also intact. Interestingly, lamina propria vasculature is semipermeable to 10 KDa dextran. Next, reporter mice expressing egfp from the CX3CR1 locus, egfp from the FoxP3 locus, or RFP from the IL-17F locus were used to track DC subsets, FoxP3(+) Tregs, or Th17f cells, respectively. Resident cx3cr1(+/egfp) cells were sessile but actively probed the surrounding microenvironment. Both T cell populations patrol the lamina propria, but the Th17f cells migrate more rapidly than Tregs. Together, these data demonstrate intact vascular perfusion, while intravitally visualizing the mucosal surface of the small bowel. Lastly, the cx3cr1(+) DCs and T cells display activity similar to that found in steady-state, secondary lymphoid organs.
Insights
This study validates intravital multiphoton microscopy for visualizing the murine small bowel, confirming intact vascular perfusion and immune cell activity in the lamina propria.
Area of Science:
- Immunology
- Gastroenterology
- Microscopy
Background:
- Intravital microscopy offers insights into immune regulation.
- Visualizing the small bowel's immune microenvironment is crucial.
Purpose of the Study:
- To validate single- and multiphoton intravital fluorescence microscopy (FIVM) for visualizing the murine small bowel.
- To assess vascular perfusion and immune cell dynamics in the small bowel mucosa.
Main Methods:
- Single- and multiphoton FIVM of murine small bowel.
- Intravital propidium iodide (PI) staining to assess tissue damage.
- Dextran-FITC and dextran-TR perfusion and filtration assays.
- Utilizing reporter mice (CX3CR1-GFP, FoxP3-GFP, IL-17F-RFP) to track immune cells.
Main Results:
- Minimal tissue damage (∼200 μm) adjacent to incision.
- Intact vascular perfusion and kidney function confirmed by dextran filtration.
- Lamina propria vasculature demonstrated semipermeability to 10 KDa dextran.
- CX3CR1+ DCs were sessile but probed microenvironment; Th17f cells migrated faster than Tregs.
- Immune cell activity mirrored that in secondary lymphoid organs.
Conclusions:
- Validated FIVM for visualizing murine small bowel immune microenvironment.
- Demonstrated intact vascular perfusion and kidney function.
- Characterized distinct migratory behaviors of DC subsets, Tregs, and Th17f cells in the lamina propria.
