TY - CHAP
T1 - Visualization of stem cell niche by fluorescence lifetime imaging microscopy
AU - Okkelman, Irina A.
AU - Puschhof, Jens
AU - Papkovsky, Dmitri B.
AU - Dmitriev, Ruslan I.
N1 - Publisher Copyright:
© Springer Science+Business Media, LLC, part of Springer Nature 2020.
PY - 2020
Y1 - 2020
N2 - 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.
AB - 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.
KW - Cell proliferation
KW - FLIM
KW - Intestinal organoids
KW - Live cell imaging
KW - PLIM
KW - Real-time oxygenation
KW - Stem cell niche
UR - https://www.scopus.com/pages/publications/85088526341
U2 - 10.1007/978-1-0716-0747-3_5
DO - 10.1007/978-1-0716-0747-3_5
M3 - Chapter
C2 - 32705636
AN - SCOPUS:85088526341
T3 - Methods in Molecular Biology
SP - 65
EP - 97
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -