Visualization of Stem Cell Niche by Fluorescence Lifetime Imaging Microscopy

Area of Science:

• Biomedical Engineering
• Regenerative Medicine
• Bioimaging

Background:

• Fluorescence lifetime imaging microscopy (FLIM) is an advanced bioimaging technique.
• It enables live, quantitative, multiparametric analyses crucial for tissue engineering and regenerative medicine.
• Stem cell-derived intestinal organoids are valuable models for studying gut biology.

Purpose of the Study:

• To detail experimental protocols for applying FLIM to live intestinal organoids.
• To demonstrate FLIM's capability in tracing stem cells and monitoring their proliferation, metabolic activity, and oxygenation.
• To provide anticipated data for FLIM imaging of intestinal organoids.

Main Methods:

• Utilized one- and two-photon excited FLIM and phosphorescence lifetime imaging microscopy (PLIM).
• Employed live Matrigel-grown intestinal organoids from primary adult stem cells, crypts, and Lgr5-GFP mice.
• Applied spectral and time-domain separation of dyes, probes, and assays for multiparameter imaging.

Main Results:

• Demonstrated FLIM's compatibility with live intestinal organoids.
• Showcased the ability to label cell proliferation and its colocalization with the stem cell niche.
• Presented measurements of local oxygenation and autofluorescence within organoids.

Conclusions:

• FLIM is a powerful tool for live, quantitative multiparametric analysis in intestinal organoid research.
• This approach facilitates stem cell tracing and functional monitoring in regenerative medicine.
• The summarized protocols and data provide a foundation for future FLIM applications in tissue engineering.