Stem cells and the niche in which they reside feature a complex microenvironment with tightly regulated homeostasis, cell-cell interactions and dynamic regulation of metabolism. A significant number of organoid models has been described over the last decade, yet few methodologies can enable single cell level resolution analysis of the stem cell niche metabolic demands, in real-time and without perturbing integrity. Here, we studied the redox metabolism of Lgr5-GFP intestinal organoids by two emerging microscopy approaches based on luminescence lifetime measurement – fluorescence-based FLIM for NAD(P)H, and phosphorescence-based PLIM for real-time oxygenation. We found that exposure of stem (Lgr5-GFP) and differentiated (no GFP) cells to high and low glucose concentrations resulted in measurable shifts in oxygenation and redox status. NAD(P)H-FLIM and O₂-PLIM both indicated that at high ‘basal’ glucose conditions, Lgr5-GFP cells had lower activity of oxidative phosphorylation when compared with cells lacking Lgr5. However, when exposed to low (0.5 mM) glucose, stem cells utilized oxidative metabolism more dynamically than non-stem cells. The high heterogeneity of complex 3D architecture and energy production pathways of Lgr5-GFP organoids were also confirmed by the extracellular flux (XF) analysis. Our data reveals that combined analysis of NAD(P)H-FLIM and organoid oxygenation by PLIM represents promising approach for studying stem cell niche metabolism in a live readout.

干细胞及其所在的生态位具有复杂的微环境，具有严格调节的稳态、细胞间相互作用和代谢的动态调节。在过去的十年中，已经描述了大量的类器官模型，但很少有方法能够实时且不扰乱完整性地对干细胞生态位代谢需求进行单细胞水平分辨率分析。在这里，我们通过两种基于发光寿命测量的新兴显微镜方法研究了 Lgr5-GFP 肠道类器官的氧化还原代谢——基于荧光的 FLIM 用于 NAD(P)H，以及基于磷光的 PLIM 用于实时氧合。我们发现干细胞（Lgr5-GFP）和分化细胞（无 GFP）暴露于高和低葡萄糖浓度会导致氧合和氧化还原状态发生可测量的变化。NAD(P)H-FLIM和O ₂ -PLIM均表明在高“基础”葡萄糖条件下，与缺乏Lgr5的细胞相比，Lgr5-GFP细胞具有较低的氧化磷酸化活性。然而，当暴露于低（0.5 mM）葡萄糖时，干细胞比非干细胞更动态地利用氧化代谢。细胞外通量（XF）分析也证实了 Lgr5-GFP 类器官复杂 3D 结构和能量产生途径的高度异质性。我们的数据表明，NAD(P)H-FLIM 和 PLIM 类器官氧合的联合分析代表了在实时读数中研究干细胞生态位代谢的有前途的方法。