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Updated: Aug 5, 2026
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Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids
Published on: June 5, 2020
Live-Cell Imaging and Single-Cell Tracking to Monitor Clonal Dynamics and Fate Transitions in Human and Murine
Xuan Zheng1, Daniel Krueger1, Johan H van Es1
1Hubrecht Institute for Developmental Biology and Stem Cell Research; Oncode Institute; Royal Netherlands Academy of Arts and Sciences (KNAW); University Medical Center Utrecht.
Journal of Visualized Experiments : Jove
|July 27, 2026
Summary
This study introduces a new live-cell imaging protocol for tracking single cells in intestinal organoids over 72 hours. The method uses mosaic organoids and fate reporters to reveal real-time cell dynamics and lineages in epithelial self-renewal.
Area of Science:
- Epithelial biology
- Stem cell research
- Developmental biology
Background:
- The intestinal epithelium renews rapidly via stem cell division, differentiation, and migration.
- Understanding cell fate commitment and clonal dynamics requires real-time, single-cell resolution methods.
- Intestinal organoids offer a controlled model for studying epithelial self-organization and cell diversity.
Purpose of the Study:
- To develop and present a protocol for long-term (up to 72 hours) confocal live-cell imaging and single-cell tracking in human and murine intestinal organoids.
- To enable the study of cell fate commitment, clonal dynamics, and epithelial self-renewal processes.
- To provide a framework adaptable to other epithelial organoid systems.
Main Methods:
- Generation of mosaic organoids by combining differentially labeled cell populations for single-cell resolution of reporters.
- Utilization of cell-type-specific fate reporters (MUC2, DEFA5) to monitor real-time secretory cell transitions.
- Long-term confocal live-cell imaging, sample preparation minimizing phototoxicity, and semi-automated single-cell tracking using OrganoidTracker.
Main Results:
- Successful long-term live-cell imaging and single-cell tracking in intestinal organoids.
- Real-time monitoring of cell fate transitions and reconstruction of cell trajectories and lineages.
- Demonstration of mosaic organoid utility for resolving reporter signals in dense epithelia.
Conclusions:
- The presented protocol enables detailed, real-time analysis of cellular behavior and lineage in intestinal organoids.
- This framework is adaptable to various epithelial organoid models, advancing studies in homeostasis and disease.
- The method provides crucial insights into the mechanisms governing epithelial self-renewal and cell fate decisions.
