Name: Live-Cell Imaging and Single-Cell Tracking to Monitor Clonal Dynamics and Fate Transitions in Human and Murine Intestinal Organoids (Video) | JoVE Journal
Uploaded: 2026-07-10T14:00:00.000+00:00
Duration: 7 min 39 s
Description: Explore live-cell imaging and single-cell tracking to monitor clonal dynamics and fate transitions in human and murine intestinal organoids. This protocol enables real-time observation of cell fate decisions at single-cell resolution.

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This research is funded by the Ministry of Education, Culture and Science of the Government of the Netherlands through the NWO Gravitation project Material Driven Regeneration (024.003.013 ZWK MDR) and by the European Research Council through the ERC Advanced Grant GutHormones (nr 101020405)

<ol>
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	<li>Clevers H. Shifting paradigms in tissue stem cell biology: insights from the intestine. Cell. 2026;189(3):706-24.</li>
	<li>Betjes MA, Kok RNU, Tans SJ, van Zon JS. Cell tracking with accurate error prediction. Nat Methods. 2025;22(11):2400-10.</li>
	<li>Sato T, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459(7244):262-5.</li>
	<li>Sato T, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett&#39;s epithelium. Gastroenterology. 2011;141(5):1762-72.</li>
	<li>He GW, et al. Optimized human intestinal organoid model reveals interleukin-22-dependency of Paneth cell formation. Cell Stem Cell. 2022;29(9):1333-45.e6.</li>
	<li>Artegiani B, et al. Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing. Nat Cell Biol. 2020;22(3):321-31.</li>
	<li>Celotti M, et al. Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools. STAR Protoc. 2024;5(3):103189.</li>
	<li>Krueger D, et al. Epithelial tension controls intestinal cell extrusion. Science. 2025;389(6764).</li>
	<li>Buissant des Amorie JR, et al. Intestinal tuft cell subtypes represent successive stages of maturation driven by crypt-villus signaling gradients. Nat Commun. 2025;16(1):6765.</li>
	<li>Huang L, et al. Tuft cells act as regenerative stem cells in the human intestine. Nature. 2024;634(8035):929-35.</li>
	<li>Wang D, et al. Interferon-responsive intestinal BEST4/CA7+ cells are targets of bacterial diarrheal toxins. Cell Stem Cell. 2025;32(4):598-612.e5.</li>
	<li>Jain A, et al. Morphodynamics of human early brain organoid development. Nature. 2025;644(8078):1010-9.</li>
	<li>Krotenberg Garcia A, van Rheenen J, Suijkerbuijk SJE. Generation of mixed murine organoids to model cellular interactions. STAR Protoc. 2021;2(4):100997.</li>
	<li>Krotenberg Garcia A, et al. Active elimination of intestinal cells drives oncogenic growth in organoids. Cell Rep. 2021;36(1):109307.</li>
	<li>Serra D, et al. Self-organization and symmetry breaking in intestinal organoid development. Nature. 2019;569(7754):66-72.</li>
	<li>de Medeiros G, et al. Multiscale light-sheet organoid imaging framework. Nat Commun. 2022;13(1):4864.</li>
	<li>Huelsz-Prince G, et al. Mother cells control daughter cell proliferation in intestinal organoids to minimize proliferation fluctuations. eLife. 2022;11.</li>
	<li>Zheng X, et al. Organoid cell fate dynamics in space and time. Sci Adv. 2023;9(33).</li>
	<li>Okkelman IA, Foley T, Papkovsky DB, Dmitriev RI. Live cell imaging of mouse intestinal organoids reveals heterogeneity in their oxygenation. Biomaterials. 2017;146:86-96.</li>
	<li>Gros A, et al. A quantitative pipeline for whole-mount deep imaging and analysis of multi-layered organoids across scales. eLife. 2026;14.</li>
	<li>Nagarajan S, Naganathan SR. Pushing the limits of automated cell tracking. Nat Methods. 2025;22(11):2236-8.</li>
	<li>Kok RNU, et al. OrganoidTracker: efficient cell tracking using machine learning and manual error correction. PLoS One. 2020;15(10).</li>
	<li>Martinez-Silgado A, et al. Differentiation and CRISPR-Cas9-mediated genetic engineering of human intestinal organoids. STAR Protoc. 2022;3(3):101639.</li>
	<li>van der Linden FH, Okkelman IA, Ioannou M, Dmitriev RI. A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium. Nat Commun. 2021;12(1):7159.</li>
	<li>Okkelman IA, Foley T, Papkovsky DB, Dmitriev RI. Extracellular Ca2+-sensing fluorescent protein biosensor based on a collagen-binding domain. ACS Appl Bio Mater. 2020;3(8):5310-21.</li>
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</ol>

H.C. is an inventor on patents related to organoid technology. All other authors declare no competing interests.

This protocol provides a step-by-step workflow for live-cell confocal imaging and single-cell tracking in human and murine intestinal organoids, built around two complementary experimental systems: mosaic organoids that resolve individual cell behavior within a dense epithelium, and fate reporter organoids expressing MUC2-mNeonGreen and DEFA5-dsRed for monitoring secretory cell type transitions in real time.
Compared to fixed-timepoint methods such as immunofluorescence or single-cell RNA sequencing, live imaging captures the dynamics of transitions rather than snapshots. Compared to Cre-based lineage tracing, it provides the temporal resolution to identify individual fate decisions. The mosaic approach adds internally controlled comparisons within the same tissue context, avoiding confounding organoid-to-organoid variability.
Several steps in the protocol are critical for success. For a mosaic with salt-and-pepper distribution dissociation of organoids to single cells must be thorough but gentle: incomplete dissociation produces large clonal patches, while excessive dissociation reduces viability (Section 4.3). The 24 h aggregation step on a flat surface before re-embedding in BME is necessary for proper mixing of the two populations; omitting it results in poorly integrated mosaics (Section 4.6&#8211;4.8). Phototoxicity management during imaging requires careful calibration, starting with conservative laser settings and increasing only if the tracking software produces excessive false negatives (Section 6.8).
The main limitations relate to the inherent constraints of organoid systems and live imaging. Organoids lack stromal, vascular, and immune components that may influence cell behavior in vivo. For confocal microscopy, imaging depth is limited to approximately 100 &#181;m from the coverslip, beyond which light scattering degrades signal quality and tracking accuracy. Multiphoton and light-sheet microscopy can substantially extend this depth range at the cost of additional instrumentation and protocol adaptation. The semi-automated tracking approach, while substantially more efficient than manual annotation, still requires considerable correction time, particularly in dense tissues (Section 7.4).
Both strategies are readily extensible. By varying the reporter co-expressed with the nuclear marker, different aspects of single-cell behavior can be examined, from cortical Myosin-II for mechanical dynamics to junctional E-cadherin for adhesion remodeling, calcium biosensors such as tq-Ca-FLITS and ColBD-Twitch, or metabolic biosensors such as SoNar for cellular NAD+/NADH redox state, enabling simultaneous readout of cell identity, lineage, and physiological state within the same organoid9,25,26,27. Adjusting the mixing ratio tunes the analysis: sparse labeled cells enable single-cell attribution of membrane signals, while sparse unlabeled cells reveal neighborhood responses. The fate reporter strategy is extendable to any endogenous locus amenable to CRISPR-HOT or HDR-based tagging. Combining both approaches within the same organoid would allow simultaneous tracking of lineage commitment and single-cell dynamics.
More broadly, the imaging and analysis pipeline is applicable to organoids derived from other epithelial tissues, including gastric, colonic, hepatic, and pancreatic models. Induced pluripotent stem cell (iPSC)-derived organoids are a promising extension, as H2B-based nuclear labeling and mosaic generation by cell mixing are directly transferable, and the pipeline may enable patient-specific studies of cell fate and competitive dynamics. Synthetic embryo models such as gastruloids and stem cell-derived embryoids are also amenable to single-cell tracking, though their more complex geometry and higher nuclear density may require retraining of the a semi-automated organoid cell-tracking software tool neural network model and adaptation of imaging parameters. Brain organoids present additional challenges due to their large size and optical density, which limit confocal imaging depth. Furthermore, brain organoids require weeks to months of differentiation before meaningful imaging experiments can be performed, substantially increasing experimental timelines compared to intestinal organoids. Some brain organoid protocols also rely on suspension culture rather than BME embedding, which is incompatible with the sample preparation approach described here and would require adaptation to matrix-embedded or surface-attached formats before imaging. Light-sheet microscopy, which offers reduced phototoxicity and greater imaging depth, may be better suited for these systems13. Together, these extensions highlight the broad potential of the pipeline across diverse 3D model systems beyond the epithelial organoids described here.

The intestinal epithelium is one of the most rapidly self-renewing tissues in the body, with most cells replaced within three to five days. This continuous turnover is sustained by Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5)-positive stem cells residing at the base of intestinal crypts, which give rise to transit-amplifying progenitors that differentiate into the specialized cell types of the intestinal lining as they migrate toward the villus tip1. The major differentiated lineages include absorptive enterocytes, mucus-secreting goblet cells, hormone-producing enteroendocrine cells, and antimicrobial Paneth cells2. How individual cells commit to specific fates, how collective cell behavior emerges from individual cell dynamics, and how tissue-level coordination is achieved remain fundamental questions in intestinal biology. Answering these questions requires experimental approaches that capture cellular behavior at single-cell resolution, in real time, and over timescales relevant to differentiation and migration3.
Intestinal organoids, three-dimensional (3D) self-organizing structures grown from single stem cells or tissue fragments, recapitulate key aspects of epithelial architecture, self-organization, and cell-type diversity4,5,6. Since their development, organoids have become an indispensable tool for studying intestinal biology, disease modeling, and drug responses. Both murine and human intestinal organoids can be maintained long-term in culture, genetically modified using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based approaches, and combined into mosaic structures by mixing dissociated single cells of different genotypes or reporter backgrounds prior to re-embedding in basement membrane extract7,8,9. Mosaic organoids combine cells of distinct genetic or fluorescent backgrounds within a single structure, so that the populations experience identical niche signals and mechanical context and can be compared with built-in internal controls. This genetic tractability makes them particularly suitable for live-imaging studies that require precise labeling of specific cell populations6,10,11,12. Reporters that label cells sporadically, such as cell-type-specific fate markers, produce isolated fluorescent cells that can be traced and analyzed individually through single-cell tracking methods. However, a persistent challenge arises when membrane-localized and cytoskeletal proteins, such as cell junctions or cortical Myosin-II, are labeled uniformly across the epithelium. Their signals cannot be assigned to individual cells because neighboring cells share their boundaries at the resolution of confocal microscopy. Mosaic organoids overcome this limitation: when a cell expressing a fluorescent reporter is surrounded by unlabeled neighbors, its membrane and cytoskeletal signals can be unambiguously attributed to that single cell, revealing how it remodels its cytoskeleton during migration, division, or extrusion9,13. Adjusting the labeling ratio inverts this view, allowing the neighborhood response around a unlabeled cell to be captured instead. Histone H2B nuclear reporters such as H2B-mCherry or H2B-iRFP serve as tools to identify the mosaic pattern and locate informative cell constellations for analysis. Beyond single-cell resolution, mosaic organoids also enable direct fitness comparisons between genotypes: because both populations share the same niche, differences in cell lifetime or extrusion rate provide an internally controlled readout of competitive fitness9,14,15.
Live-cell imaging of organoids, however, presents specific technical challenges. Their 3D geometry requires confocal or light-sheet microscopy for optical sectioning16,17,18,19. Capturing slow processes such as differentiation and migration demands imaging durations of 24 h or more, with careful optimization of laser power, exposure time, and imaging intervals to limit phototoxicity16,18. The embedding matrix and culture medium must support organoid health while remaining compatible with the microscope optics. Furthermore, extracting quantitative single-cell information from dense 3D time-lapse datasets requires dedicated computational tools to detect and link cells across hundreds of time points. Beyond confocal microscopy and light-sheet microscopy, multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) offer additional capabilities such as reduced phototoxicity and label-free metabolic readouts20,21, though confocal microscopy remains the most widely accessible and versatile platform for multi-color live imaging of fluorescently labeled organoids.
Several approaches have been developed for tracking cells in organoids, ranging from manual annotation to semi-automated and fully automated deep learning-based pipelines22. OrganoidTracker is a semi-automated software tool that uses neural networks to detect nuclei in 3D confocal stacks and links detections across time points to reconstruct cell trajectories and lineage trees3,23. It has been validated for tracking hundreds of cells over multiple days in intestinal organoids and provides a flexible framework for quantifying cell division, migration, differentiation, and extrusion events. Its support for multi-channel imaging makes it directly compatible with both mosaic organoids carrying spectrally distinct nuclear reporters and organoids expressing cell-type-specific fate markers.
In this protocol, we describe a modular workflow that integrates confocal live-cell imaging and single-cell tracking with two complementary reporter strategies in human and murine intestinal organoids. The first strategy uses mosaic murine organoids generated by mixing two cell populations carrying distinct H2B&#160;nuclear reporters (H2B-mCherry and H2B-iRFP) at defined ratios (Figure 1A). One population additionally carries a co-expressed reporter of interest, such as Myosin-II-mNeonGreen, whose membrane-localized signal can be unambiguously attributed to individual cells when they are surrounded by neighbors carrying only the second nuclear label. This enables direct observation of how a single cell remodels its cytoskeleton during migration, division, or extrusion within an otherwise intact tissue. The second strategy uses cell-type-specific fate reporters in human intestinal organoids, Mucin 2 (MUC2) to label goblet cells and Defensin Alpha 5 (DEFA5) to label Paneth cells, enabling real-time monitoring of secretory cell type transitions (Figure 1B). Together, these two systems illustrate how a shared imaging and tracking pipeline can be adapted to diverse biological questions simply by swapping the reporter strategy (Figure 1C,D). The sections that follow provide all steps needed to implement this workflow, from organoid culture through mosaic generation, image acquisition, and quantitative single-cell analysis.

<table><tbody><tr><td>A83-01</td><td>Tocris&amp;nbsp;</td><td>2939</td><td></td></tr><tr><td>Accutase Cell Detachment Solution</td><td>Innovative Cell Technologies</td><td>AT104-500</td><td></td></tr><tr><td>Advanced Dulbecco&#039;s Modified Eagle Medium/F12 (Advanced DMEM/F12)</td><td>Invitrogen</td><td>&amp;nbsp;12634-010</td><td></td></tr><tr><td>B27 supplement&amp;nbsp;</td><td>Thermo Fisher</td><td>17504044</td><td></td></tr><tr><td>Blasticidin</td><td>InvivoGen</td><td>ant-bl-05</td><td></td></tr><tr><td>Cell strainer (40um)&amp;nbsp;</td><td>Greiner Bio-one</td><td>542140</td><td></td></tr><tr><td>CHIR99021</td><td>Stemgent</td><td>04-0004-base</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>SP8</td><td>with climate control for temperature and CO2</td></tr><tr><td>Cultrex Basement Membrane Extract (BME)</td><td>R&amp;amp;D Systems, Bio-Techne</td><td>3533-001-02</td><td></td></tr><tr><td>Fiji</td><td>open source</td><td></td><td>open source https://imagej.net/software/fiji/</td></tr><tr><td>Glass bottom plates</td><td>CellVis</td><td>P24-1.5H-N</td><td></td></tr><tr><td>Glutamax&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>35050-061</td><td></td></tr><tr><td>HEPES</td><td>Gibco&amp;nbsp;</td><td>15630-080</td><td></td></tr><tr><td>N2 supplement&amp;nbsp;</td><td>Thermo Fisher</td><td>17502048</td><td></td></tr><tr><td>N-acetylcysteine</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A9165</td><td></td></tr><tr><td>NEPA electroporation system</td><td>NEPAGENE</td><td>NEPA21</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich&amp;nbsp;</td><td>N0636</td><td></td></tr><tr><td>NucSpot Live</td><td></td><td></td><td>far-red live-cell nuclear dye</td></tr><tr><td>Noggin conditioned medium (U-Protein Express)</td><td>IPA Therapeutics</td><td>N002</td><td></td></tr><tr><td>OrganoidTracker</td><td>open source</td><td></td><td>open source https://organoidtracker.org/</td></tr><tr><td>p2T-CAG-mT2TP</td><td>homemade</td><td></td><td>protocol to generate the plasmid DOI: 10.1126/science.adr8753</td></tr><tr><td>p2T-H2B-iRFP-IRES-PuroR</td><td>homemade</td><td></td><td>protocol to generate the plasmid DOI: 10.1126/science.adr8753</td></tr><tr><td>p2T-H2B-mCherry-IRES-BlastR&amp;nbsp;</td><td>homemade</td><td></td><td>protocol to generate the plasmid DOI: 10.1126/science.adr8753</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco&amp;nbsp;</td><td>11548876</td><td></td></tr><tr><td>Primocin</td><td>InvivoGen</td><td>ant-pm-05</td><td></td></tr><tr><td>Prostaglandin E2</td><td>Tocris&amp;nbsp;</td><td>2296</td><td></td></tr><tr><td>puromycin</td><td>InvivoGen</td><td>ant-pr</td><td></td></tr><tr><td>Recombinant human EGF</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>Recombinant Human IL-22</td><td>Peprotech</td><td>200-22</td><td></td></tr><tr><td>Recombinant human R-spondin 1</td><td>Thermo Fisher</td><td>120-38-5UG</td><td></td></tr><tr><td>Recombinant murine EGF</td><td>Thermo Fisher&amp;nbsp;</td><td>315-09-500UG</td><td></td></tr><tr><td>Recombinant murine Noggin</td><td>Thermo Fisher</td><td>250-38-100UG</td><td></td></tr><tr><td>R-spondin conditioned medium (U-Protein Express)</td><td>IPA Therapeutics</td><td>R001</td><td></td></tr><tr><td>SiR-DNA</td><td></td><td></td><td>far-red live-cell DNA dye</td></tr><tr><td>SB202190 (p38 inhibitor)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S7076</td><td></td></tr><tr><td>Suspension plate for organoid culture</td><td>Greiner</td><td>665102</td><td></td></tr><tr><td>Trypan blue&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>15250061</td><td></td></tr><tr><td>TrypLE</td><td>Gibco&amp;nbsp;</td><td>12605-010</td><td></td></tr><tr><td>Wnt surrogate</td><td>U-Protein Express</td><td>&amp;nbsp;custom order</td><td></td></tr><tr><td>Wnt3A conditioned medium</td><td>homemade</td><td></td><td>The production of Wnt3A conditioned medium can be found here:&amp;nbsp; https://doi.org/10.1002/cpim.106</td></tr><tr><td>Y-27632 dihydrochloride</td><td>Abmole Bioscience</td><td>M1817</td><td></td></tr></tbody></table>

live-cell imaging and single-cell tracking to monitor clonal dynamics and fate transitions in human and murine intestinal organoids

Live-Cell Imaging and Single-Cell Tracking to Monitor Clonal Dynamics and Fate Transitions in Human and Murine Intestinal Organoids

The intestinal epithelium undergoes rapid self-renewal through stem cell division, cell differentiation, and migration. Understanding how individual cells commit to specific fates and how clonal dynamics emerge within this tissue requires methods that capture cellular behavior in real time and at single-cell resolution. Intestinal organoids recapitulate key features of epithelial self-organization and cell-type diversity, making them a powerful system for studying these processes in a controlled setting. Here we present a protocol for long-term confocal live-cell imaging (up to 72 h) and single-cell tracking in human and murine intestinal organoids, built around two complementary reporter strategies. First, we describe the generation of mosaic organoids by combining differentially labeled cell populations, enabling single-cell resolution of membrane-localized and cytoskeletal reporters that cannot otherwise be attributed to individual cells in a dense epithelium. Second, we use cell-type-specific fate reporters (MUC2 for goblet cells and DEFA5 for Paneth cells) to monitor secretory cell type transitions in real time. The protocol covers organoid culture, mosaic organoid formation, sample preparation with strategies to minimize phototoxicity, image acquisition over several days, and semi-automated single-cell tracking using OrganoidTracker, which reconstructs cell trajectories and lineages over time. While demonstrated in intestinal organoids, this framework is readily adaptable to other epithelial organoid systems, including gastric, pancreatic, and colonic models, broadening its utility for studies of epithelial biology, homeostasis, and disease.

Following the protocol described in Section 4, mosaic organoids containing H2B-mCherry- and H2B-iRFP-labeled cell populations form within 48 h of seeding. Successful mosaic formation is characterized by either a salt-and-pepper distribution or defined boundaries between the two populations, depending on the degree of dissociation used. A salt-and-pepper distribution refers to a random, single-cell intermixing of the two populations, in which cells of each population are interspersed at the single-cell level, with no large clonal domains. Defined boundaries, by contrast, result in coherent patches of each cell population within the same organoid. The degree of cell dispersal is controlled by the duration of the dissociation step and the vigor of pipetting: thorough single-cell dissociation produces salt-and-pepper mosaics, while gentle fragmentation preserves small clonal clusters (Figure 3A).
The mosaic labeling enables single-cell analysis of processes that cannot be resolved in uniformly labeled tissues. When an individual cell co-expressing a reporter of interest (e.g., a fluorescent Myosin-II reporter) is surrounded by unlabeled neighbors, its cytoskeletal dynamics during migration, division, or extrusion can be unambiguously attributed to that cell (Figure 3B). Nuclear H2B reporters facilitate the identification of informative cell constellations within the mosaic, and per-cell measurements, such as lifetime, division rate, and reporter intensity dynamics, can be extracted from the tracking data (Figure 3C). When two genotypes are mixed, these per-cell metrics can be compared across populations to assess competitive fitness differences.
In human intestinal organoids carrying the MUC2-mNeonGreen and DEFA5-dsRed fate reporters, secretory cell fate commitment is evident as a gradual increase in reporter fluorescence over days. Organoids cultured in Maturation Medium were transferred to glass-bottom plates on day 7 and imaged by confocal live-cell microscopy (Figure 4A). Time-lapse imaging reveals two distinct reporter dynamics: goblet cells activate and maintain high MUC2-mNeonGreen expression throughout the imaging period, while Paneth cells display initial MUC2-mNeonGreen activation followed by progressive signal decrease and subsequent DEFA5-dsRed activation6 (Figure 4A). Single-cell quantification confirms this sequential transition at the level of individual cells (Figure 4B), suggesting that Paneth cell commitment proceeds through a transient MUC2-expressing intermediate state rather than directly from an unlabeled progenitor. Averaged intensity curves across multiple tracked cells confirm that this pattern is consistent at the population level, with Paneth cell precursors displaying a characteristic inverse relationship between MUC2-mNeonGreen and DEFA5-dsRed signals (Figure 4C). Together, these data demonstrate that the dual reporter system resolves distinct secretory fate trajectories at single-cell resolution.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/72058/72058fig1.jpg" /> 
Figure 1: The workflow that integrates confocal live-cell imaging and single-cell tracking with two complementary reporter strategies in human and murine intestinal organoids. (A) Schematic overview of the mosaic organoid generation workflow. Murine intestinal organoids expressing H2B-mCherry (magenta) or H2B-iRFP (green) are dissociated into single cells, mixed at defined ratios, and seeded onto a BME-coated plate for 24 h aggregate formation before re-embedding in BME. (B) Schematic overview of human intestinal organoids differentiation. Human organoids expressing MUC2-mNeonGreen (goblet cells, green) and DEFA5-dsRed (Paneth cells, magenta) endogenous knock-in reporters are differentiated using a two-step protocol. Organoids are first cultured in Patterning Medium for 14 days, followed by Maturation Medium for 7&#8211;10 days. During differentiation, organoids transition from a cystic, spherical morphology to an extensively budded structure that harbors major intestinal cell types, including MUC2-mNeonGreen-positive goblet cells and DEFA5-dsRed-positive Paneth cells. (C) 3D time-lapse confocal imaging of a murine mosaic intestinal organoid expressing nuclear reporters. (D) Single-cell tracking and lineage reconstruction using a semi-automated organoid cell-tracking software tool. Detected nuclei are linked across time points to reconstruct cell trajectories and lineage trees, enabling quantification of cell division, migration, and extrusion events. <a href="https://www.jove.com/files/ftp_upload/72058/72058fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/72058/72058fig2.jpg" /> 
Figure 2: Comparison of healthy and phototoxic human intestinal organoids during live confocal imaging. (A) Representative brightfield and fluorescence images of a healthy human intestinal organoid. Healthy organoids display clear cystic or budding morphology with no cell debris in the surrounding medium. (B) Representative brightfield and fluorescence images of a human intestinal organoid showing phototoxic damage. Signs of phototoxicity include accumulation of cell debris in the surrounding medium, nuclear condensation, and arrested cell division. H2B-mCherry fluorescence images are shown as maximum intensity projections. Scale bars, 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/72058/72058fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/72058/72058fig3.jpg" /> 
Figure 3: Mosaic organoid generation and single-cell resolution of cytoskeletal dynamics in murine intestinal organoids. (A) Schematic of mosaic organoid generation by dissociation, mixing at defined ratios, and re-aggregation. Representative confocal images of mosaic organoids generated at cell ratios of 1:9, 1:5, and 1:2 (H2B-mCherry, green; H2B-iRFP, magenta) using single-cell dissociation (top row) or small-fragment dissociation (bottom row). Single-cell dissociation produces a salt-and-pepper mixing pattern, while small-fragment dissociation results in larger clonal patches. Scale bars, 50 &#181;m. (B) Confocal images of mosaic organoids in which a dense (left) or sparse (right) population expresses Myosin-II-mNeonGreen (green), while the other population expresses only H2B-iRFP (magenta). Dashed boxes indicate the regions magnified in (C). Scale bars, 20 &#181;m. (C) Time-lapse series showing cell extrusion at 8 min intervals, aligned to the moment of cell removal (t = 0 min). Top row (arrowheads): extrusion of a unlabeled cell surrounded by Myosin-II-mNeonGreen-expressing neighbors, revealing the neighborhood response. Bottom row (asterisks): extrusion of an individual Myosin-II-mNeonGreen-expressing cell surrounded by unlabeled neighbors, enabling unambiguous attribution of cortical myosin dynamics to the extruding cell. Scale bars, 10 &#181;m. Right: normalized Myosin-II-mNeonGreen fluorescence intensity over time for extruding cells, aligned to the moment of cell removal (t = 0 min). Myosin-II accumulates progressively in the minutes preceding extrusion, peaking at the time of removal. Shaded area indicates standard deviation. <a href="https://www.jove.com/files/ftp_upload/72058/72058fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/72058/72058fig4v2.jpg" /> 
Figure 4: Real-time monitoring of secretory cell fate transitions in human intestinal organoids using MUC2-mNeonGreen and DEFA5-dsRed fate reporters. <a href="https://www.jove.com/files/ftp_upload/72058/72058fig4largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
(A) Representative time-lapse confocal images of a human intestinal organoid cultured in Maturation Medium and imaged from day 7 of maturation. MUC2-mNeonGreen (green) marks goblet cells and DEFA5-dsRed (magenta) marks Paneth cells. Scale bar, 20 &#181;m. (B) Single-cell quantification of MUC2-mNeonGreen (green) and DEFA5-dsRed (magenta) fluorescence intensity over time for a representative Paneth cell, showing initial MUC2-mNeonGreen activation followed by progressive signal decrease and subsequent DEFA5-dsRed onset. Fluorescence intensity is shown in arbitrary units (a.u.). (C) Averaged fluorescence intensity curves for goblet cells (n = 7 cells) and Paneth cell precursors (n = 8 cells) showing MUC2-mNeonGreen (green) and DEFA5-dsRed (magenta) signal dynamics over time6. For Paneth cell precursors, time is aligned to the first appearance of DEFA5-dsRed signal (t = 0 h). Goblet cells maintain consistently high MUC2-mNeonGreen intensity over time, while Paneth cell precursors display a characteristic inverse relationship between MUC2-mNeonGreen and DEFA5-dsRed signals. Shaded areas indicate standard deviation.
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	<tbody>
		<tr>
			<td>Component&#160;</td>
			<td>Stock concentration</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Advanced Dulbecco&#39;s Modified Eagle Medium/F12 (Advanced DMEM/F12)</td>
			<td></td>
			<td>Base medium</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1M</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Glutamax&#160;</td>
			<td>200mM</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>N2 supplement&#160;</td>
			<td>100x</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>B27 supplement&#160;</td>
			<td>50x</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>500mM</td>
			<td>1mM</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>10000U/ml, 10000ug/ml</td>
			<td>100U/ml, 100ug/ml</td>
		</tr>
		<tr>
			<td>Recombinant murine EGF</td>
			<td></td>
			<td>50ng/ml</td>
		</tr>
		<tr>
			<td>Recombinant murine Noggin</td>
			<td></td>
			<td>100ng/ml</td>
		</tr>
		<tr>
			<td>Recombinant human R-spondin 1</td>
			<td></td>
			<td>500ng/ml</td>
		</tr>
	</tbody>
</table>
Table 1: Growth medium for murine intestinal organoids.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Advanced Dulbecco&#39;s Modified Eagle Medium/F12 (Advanced DMEM/F12)</td>
			<td></td>
			<td>Base medium</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1M</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Glutamax&#160;</td>
			<td>200mM</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>B27 supplement&#160;</td>
			<td>50x</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>10000U/ml, 10000ug/ml</td>
			<td>100U/ml, 100ug/ml</td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>50mg/ml</td>
			<td>100ug/ml</td>
		</tr>
		<tr>
			<td>R-spondin conditioned medium (U-Protein Express)</td>
			<td></td>
			<td>2%</td>
		</tr>
		<tr>
			<td>Noggin conditioned medium (U-Protein Express)</td>
			<td></td>
			<td>2%</td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>500mM</td>
			<td>1mM</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>1M</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Human recombinant EGF</td>
			<td>500ug/ml</td>
			<td>50ng/ml</td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>5mM</td>
			<td>500nM</td>
		</tr>
		<tr>
			<td>SB202190 (p38 inhibitor)</td>
			<td>30mM</td>
			<td>3uM</td>
		</tr>
		<tr>
			<td>Prostaglandin E2</td>
			<td>10mM</td>
			<td>1uM</td>
		</tr>
		<tr>
			<td>Wnt surrogate</td>
			<td></td>
			<td>1000x</td>
		</tr>
	</tbody>
</table>
Table 2: Expansion medium for human intestinal organoids.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component&#160;</td>
			<td>Stock concentration</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Advanced Dulbecco&#39;s Modified Eagle Medium/F12 (Advanced DMEM/F12)</td>
			<td></td>
			<td>Base medium</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1M</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Glutamax&#160;</td>
			<td>200mM</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>B27 supplement&#160;</td>
			<td>50x</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>10000U/ml, 10000ug/ml</td>
			<td>100U/ml, 100ug/ml</td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>50mg/ml</td>
			<td>100ug/ml</td>
		</tr>
		<tr>
			<td>R-spondin conditioned medium (U-Protein Express)</td>
			<td></td>
			<td>2%</td>
		</tr>
		<tr>
			<td>Noggin conditioned medium (U-Protein Express)</td>
			<td></td>
			<td>2%</td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>500mM</td>
			<td>1mM</td>
		</tr>
		<tr>
			<td>Human recombinant EGF</td>
			<td>500ug/ml</td>
			<td>50ng/ml</td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>5mM</td>
			<td>500nM</td>
		</tr>
		<tr>
			<td>Wnt surrogate</td>
			<td></td>
			<td>1000x</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>3mM</td>
			<td>3uM</td>
		</tr>
		<tr>
			<td>Recombinant Human IL-22</td>
			<td>10 ug/ml</td>
			<td>2ng/ml</td>
		</tr>
	</tbody>
</table>
Table 3: Patterning medium for human intestinal organoids.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component&#160;</td>
			<td>Stock concentration</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Advanced Dulbecco&#39;s Modified Eagle Medium/F12 (Advanced DMEM/F12)</td>
			<td></td>
			<td>Base medium</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1M</td>
			<td>10mM</td>
		</tr>
		<tr>
			<td>Glutamax&#160;</td>
			<td>200mM</td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>B27 supplement&#160;</td>
			<td>50x</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>10000U/ml, 10000U/ml</td>
			<td>100U/ml, 100U/ml</td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>50mg/ml</td>
			<td>100ug/ml</td>
		</tr>
		<tr>
			<td>R-spondin conditioned medium (U-Protein Express)</td>
			<td></td>
			<td>2%</td>
		</tr>
		<tr>
			<td>Noggin conditioned medium (U-Protein Express)</td>
			<td></td>
			<td>2%</td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>500mM</td>
			<td>1mM</td>
		</tr>
		<tr>
			<td>Human recombinant EGF</td>
			<td>500ug/ml</td>
			<td>50ng/ml</td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>5mM</td>
			<td>500nM</td>
		</tr>
		<tr>
			<td>Wnt surrogate</td>
			<td></td>
			<td>30000x</td>
		</tr>
		<tr>
			<td>Recombinant Human IL-22</td>
			<td>10 ug/ml</td>
			<td>2ng/ml</td>
		</tr>
	</tbody>
</table>
Table 4: Maturation medium for human intestinal organoids.

We describe confocal imaging and tracking pipelines applied to two reporter strategies: mosaic organoids for resolving individual cell behavior and fate reporter organoids for monitoring cell type transitions in human and murine intestinal organoids.

The intestinal epithelium undergoes rapid self-renewal through stem cell division, cell differentiation, and migration. Understanding how individual cells ...

Our research focuses on studying how individual cells make their fates decisions and coordinate their behaviors within the self-renewing intestinal epithelium. Light microscopy cannot resolve a ubiquitous marker, for example, the membrane compartment of a cell or cytoskeleton, and attribute it actually to one cell or its neighbors. But mosaic organoids can actually help with that limitation.To begin, obtain the required reagents and validated murine and human fluorescent reporter intestinal organoid lines. Passage both organoid lines into Wnt-conditioned medium three days before mosaic organoid generation to promote stem cell expansion and maximize cell viability during dissociation. Prepare 5% BME in DMEM. Coat each well of a 48-well plate with 20 microliters of the prepared BME to provide a thin adhesive layer for cell attachment, then incubate at 37 degrees Celsius for one hour. Next, dissolve the BME by adding ice-cold base medium. Collect organoids from each line separately by centrifugation at 300 g for five minutes at 4 degrees Celsius. Dissociate organoids into single cells by incubating them in an enzymatic cell detachment solution for 5 to seven minutes in a water bath at 37 degrees Celsius. Using a P1000 pipette, repeatedly pipette the suspension to aid mechanical dissociation. Monitor the dissociation under a bright field microscope. Stop the reaction by adding excess ice-cold base medium when predominantly single cells or small organoid fragments of 2 to 10 cells are visible. Then pellet the cells by centrifugation at 300 g for five minutes at 4 degrees Celsius. Assess cell viability and count the viable cells using trypan blue exclusion. Combine the two cell populations at the desired ratio to obtain a total of 300,000 cells in 500 microliters of Wnt-conditioned medium, supplemented with 10 micromolar Y-27632 dihydrochloride for murine mosaic organoids or human expansion medium for human mosaic organoid cultures. Mix thoroughly by gentle pipetting. Then, remove the coating medium from the 48-well plates and seed the cell suspension into the coated wells. Centrifuge the plate at 200 g for five minutes at room temperature, and incubate at 37 degrees Celsius. Prepare a P200 pipette tip by bending it. After 24 hours, mechanically detach the cell layer by scraping with the bent pipette tip, then flush thoroughly with ice-cold base medium. Collect the detached aggregates by centrifugation at 300 g for three minutes at 4 degrees Celsius, without additional mechanical shearing. Resuspend the aggregates in BME and plate into imaging-compatible sample carriers. Culture the plated aggregates in ENR medium for murine and human expansion medium for human mosaic organoids.Use glass-bottom imaging chambers to ensure optical compatibility with high-numerical-aperture confocal objectives. Plate the organoid-BME suspension as a thin dome near the center of the glass bottom. Incubate the plate at 4 degrees Celsius for 5 to 10 minutes to allow the organoids to sink toward the glass bottom by gravity. Transfer the plate to 37 degrees Celsius and allow the BME to solidify for 20 to 30 minutes. Add 500 microliters of pre-warmed growth medium to each well. Allow the organoids to recover and establish their morphology for at least 24 hours before beginning live imaging. Acquire long-term time-lapse images and perform single-cell tracking and lineage reconstruction for downstream analysis.Successful mosaic formation was characterized by either a salt-and-pepper distribution or defined boundaries between the two populations, depending on the degree of dissociation used. Thorough single-cell dissociation produced salt-and-pepper mosaics, while gentle fragmentation preserve small clonal clusters. When unlabeled neighbors surround an individual cell, co-expressing a reporter of interest, its cytoskeletal dynamics during migration, division, or extrusion can be unambiguously attributed to that cell. Nuclear H2B reporters facilitated the identification of informative cell constellations within the mosaic and per-cell measurements such as lifetime division rate and reporter intensity dynamics were extracted from the tracking data. Goblet cells activated and maintained high MUC2-mNeonGreen expression throughout the imaging period, while Paneth cells displayed initial MUC2-mNeonGreen activation followed by progressive signal decrease and subsequent DEFA5-dsRed activation. Single-cell quantification confirmed this sequential transition at the level of individual cells. Averaged intensity curves across multiple tracked cells confirmed that this pattern was consistent at the population level, with Paneth cell precursors displaying a characteristic inverse relationship between MUC2-mNeonGreen and DEFA5-dsRed signals.This protocol allows researchers to study how an individual cells remodels its cytoskeleton, for example during division, during migration, or for example, during extrusion in an intact tissue. The important consideration is choosing the labeling ratio, as sparse reporter cells isolate single-cell signals, while sparse, unlabeled cells reveal the neighborhood responses. Future studies can combine fate reporters with mosaic labeling in one organoid to link fate commitments towards single-cell cytoskeleton dynamics.

live-cell-imaging-single-cell-tracking-to-monitor-clonal-dynamics

Research

JoVE Journal

Biology

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used.  This technique can be applied to any cell type.

Single Cell Fate Mapping in Zebrafish

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Explore live-cell imaging and single-cell tracking to monitor clonal dynamics and fate transitions in human and murine intestinal organoids. This protocol enables real-time observation of cell fate decisions at single-cell resolution.

Live-Cell Imaging and Single-Cell Tracking to Monitor Clonal Dynamics and Fate Transitions in Human and Murine Intestinal Organoids (Video) | JoVE Journal

Live-Cell Imaging and Single-Cell Tracking to Monitor Clonal Dynamics and Fate Transitions in Human and Murine Intestinal Organoids

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