Visualization of Stem Cell Niche by Fluorescence Lifetime Imaging Microscopy
Abstract
Fluorescence lifetime imaging microscopy (FLIM), enabling live quantitative multiparametric analyses, is an emerging bioimaging approach in tissue engineering and regenerative medicine. When combined with stem cell-derived intestinal organoid models, FLIM allows for tracing stem cells and monitoring of their proliferation, metabolic fluxes, and oxygenation. It is compatible with the use of live Matrigel-grown intestinal organoids produced from primary adult stem cells, crypts, and transgenic Lgr5-GFP mice. In this chapter we summarize available experimental protocols, imaging platforms (one- and two-photon excited FLIM, phosphorescence lifetime imaging microscopy (PLIM)) and provide the anticipated data for FLIM imaging of the live intestinal organoids, focusing on labeling of cell proliferation, its colocalization with the stem cell niche, measured local oxygenation, autofluorescence, and some other parameters. The protocol is illustrated with examples of multiparameter imaging, employing spectral and "time domain"-based separation of dyes, probes, and assays.
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Fluorescence lifetime imaging microscopy has emerged as a refined method to assess proliferation in 3D models with improved specificity and reduced signal interference.
“fluorescence lifetime imaging microscopy (FLIM) has also emerged as a refined method to assess proliferation in 3D models with improved specificity and reduced signal interference [270,271].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [270]
Cites
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mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake
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Shah, S., & D’Souza, G. G. M. (2025) · Cells · doi:10.3390/cells14100732 · Cited by 66 · see citing works
“More recently, fluorescence lifetime imaging microscopy (FLIM) has also emerged as a refined method to assess proliferation in 3D models with improved specificity and reduced signal interference [270,271].”
Okkelman, I. A., Zhou, H., Borisov, S. M., Debruyne, A. C., Lefebvre, A. E. Y. T., Leomil Zoccoler, M., Chen, L., Devriendt, B., & Dmitriev, R. I. (2025) · Light: Science & Applications · doi:10.1038/s41377-025-01949-0 · Cited by 28 · see citing works
Self-citation
- Authors
- Okkelman, I. A., Puschhof, J., Papkovsky, D. B., & Dmitriev, R. I.
- Affiliations
- University College Cork (IE)
- Royal Netherlands Academy of Arts and Sciences (NL)
- Corresponding author
- Dmitriev, Ruslan I.
- Venue
- Methods in Molecular Biology
- Publisher
- Springer US
- Published
- 2020
- Volume
- 2171
- Pages
- 65-97
- ISSN
- 1064-3745 (ISSN-L)
- 1940-6029
- OpenAlex
- W3044474256
- Cited by
- 15
- References
- 4
- Citations by year
- 2026: 1 · 2025: 2 · 2023: 3 · 2022: 4 · 2021: 2 · 2020: 2 · 2019: 1
- FWCI
- 6.05
- Type
- book chapter
- Language
- en
- Open access
- closed
- Locations
- Methods in molecular biology (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Optical Imaging and Spectroscopy Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Keywords
- stem cell niche, intestinal organoids, fluorescence lifetime imaging, cell proliferation, oxygenation, autofluorescence, crypt, time-domain separation
- MeSH terms
- Animals, Microscopy, Fluorescence, Organoids, Software, Tissue Engineering, Cell Proliferation, Mice, Stem Cell Niche, Optical Imaging
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