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Visualization of Stem Cell Niche by Fluorescence Lifetime Imaging Microscopy

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Book cover Intestinal Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2171))

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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Acknowledgments

This work was supported by the Science Foundation Ireland grant 12/RC/2276 (D.B.P., I.A.O.) and by the Agilent University Research Program (ACT-UR) No. 4225 (R.I.D.). We thank Dr. H. Glauner, Dr. L. Alvarez and team at the Leica training Centre (Mannheim, Germany) for support with demonstration of Leica SP8 Falcon systems.

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Authors and Affiliations

  1. School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland

    Irina A. Okkelman, Dmitri B. Papkovsky & Ruslan I. Dmitriev

  2. Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Utrecht, The Netherlands

    Jens Puschhof

  3. Oncode Institute, Utrecht, The Netherlands

    Jens Puschhof

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  1. Irina A. Okkelman
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  2. Jens Puschhof
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  3. Dmitri B. Papkovsky
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  4. Ruslan I. Dmitriev
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Correspondence to Ruslan I. Dmitriev .

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Editors and Affiliations

  1. Division of Cancer and Stem Cells, School of Medicine, Biodiscovery Institute, Centre for Cancer Sciences, University of Nottingham, Nottingham, UK

    Paloma Ordóñez-Morán

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Okkelman, I.A., Puschhof, J., Papkovsky, D.B., Dmitriev, R.I. (2020). Visualization of Stem Cell Niche by Fluorescence Lifetime Imaging Microscopy. In: Ordóñez-Morán, P. (eds) Intestinal Stem Cells. Methods in Molecular Biology, vol 2171. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0747-3_5

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  • DOI: https://doi.org/10.1007/978-1-0716-0747-3_5

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  • Print ISBN: 978-1-0716-0746-6

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