Reactive molecular oxygen (O₂) plays important roles in bioenergetics and metabolism and is implicated in biochemical pathways underlying angiogenesis, fertilization, wound healing and regeneration. Here we describe how to use the scanning micro-optrode technique (SMOT) to measure extracellular fluxes of dissolved O₂. The self-referencing O₂-specific micro-optrode (also termed micro-optode and optical fiber microsensor) is a tapered optical fiber with an O₂-sensitive fluorophore coated onto the tip. The O₂ concentration is quantified by fluorescence quenching of the fluorophore emission upon excitation with blue–green light. The micro-optrode presents high spatial and temporal resolutions with improved signal-to-noise ratio (in the picomole range). In this protocol, we provide step-by-step instructions for micro-optrode calibration, validation, example applications and data analysis. We describe how to use the technique for cells (Xenopus oocyte), tissues (Xenopus epithelium and rat cornea), organs (Xenopus gills and mouse skin) and appendages (Xenopus tail), and provide recommendations on how to adapt the approach to different model systems. The basic, user-friendly system presented here can be readily installed to reliably and accurately measure physiological O₂ fluxes in a wide spectrum of biological models and physiological responses. The full protocol can be performed in ~4 h.

活性分子氧 (O ₂ ) 在生物能量学和新陈代谢中发挥重要作用，并参与血管生成、受精、伤口愈合和再生的生化途径。在这里，我们描述如何使用扫描微光极技术（SMOT）来测量溶解O ₂的细胞外通量。自参考O ₂特异性微光极（也称为微光极和光纤微传感器）是一种锥形光纤，其尖端涂覆有O ₂敏感荧光团。O ₂浓度通过蓝绿光激发时荧光团发射的荧光猝灭来定量。微光极具有高空间和时间分辨率以及改进的信噪比（在皮摩尔范围内）。在此协议中，我们提供了微光极校准、验证、示例应用和数据分析的分步说明。我们描述了如何将该技术用于细胞（爪蟾卵母细胞）、组织（爪蟾上皮和大鼠角膜）、器官（爪蟾鳃和小鼠皮肤）和附属物（爪蟾尾），并提供有关如何使该方法适应不同模型的建议系统。这里介绍的基本的、用户友好的系统可以很容易地安装，以可靠、准确地测量各种生物模型和生理反应中的生理 O _2通量。完整的协议可在约 4 小时内完成。