Determination of hair cell metabolic state in isolated cochlear preparations by two-photon microscopy

Currently there is no accepted method to measure the metabolic status of the organ of Corti. Since metabolism and mitochondrial dysfunction are expected to play a role in many different hearing disorders, here for the first time we employ two-photon metabolic imaging to assess the metabolic status of the cochlea. When excited with ultrashort pulses of 740-nm light, both inner and outer hair cells in isolated murine cochlear preparations exhibited intrinsic fluorescence. This fluorescence is characterized and shown to be consistent with a mixture of oxidized flavoproteins (Fp) and reduced nicotinamide adenine dinucleotide (NADH). The location of the fluorescence within hair cells is also consistent with the different mitochondrial distributions in these cell types. Treatments with cyanide and mitochondrial uncouplers show that hair cells are metabolically active. Both NADH and Fp in inner hair cells gradually become completely oxidized within 50 min from the time of death of the animal. Outer hair cells show similar trends but are found to have greater variability. We show that it is possible to use two-photon metabolic imaging to assess metabolism in the mouse organ of Corti.