Raman Fiber Photometry for Understanding Mitochondrial Superoxide Burst and Extracellular Calcium Ion Influx upon Acute Hypoxia in the Brain of Freely Moving Animals

Abstract Developing a novel tool capable of real‐time monitoring and simultaneous quantitation of multiple molecules in mitochondria across the whole brain of freely moving animals is the key bottleneck for understanding the physiological and pathological roles that mitochondria play in the brain events. Here we built a Raman fiber photometry, and created a highly selective non‐metallic Raman probe based on the triple‐recognition strategies of chemical reaction, charge transfer, and characteristic fingerprint peaks, for tracking and simultaneous quantitation of mitochondrial O 2 .− , Ca 2+ and pH at the same location in six brain regions of free‐moving animal upon hypoxia. It was found that mitochondrial O 2 .− , Ca 2+ and pH changed from superficial to deep brain regions upon hypoxia. It was discovered that hypoxia‐induced mitochondrial O 2 .− burst was regulated by ASIC1a, leading to mitochondrial Ca 2+ overload and acidification. Furthermore, we found the overload of mitochondrial Ca 2+ was mostly attributed to the influx of extracellular Ca 2+ .