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

Abstract

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.

Intracellular Coenzymes as Natural Biomarkers for Metabolic Activities and Mitochondrial Anomalies

“…Two-photon fluorescence microscopy of intrinsic NADH has been used to monitor energy metabolism in macrophages, pancreatic islet cells, skeletal muscle cells [142–144], brain slices [42,139], cardiomyocytes [29], human breast normal and cancer cells [53], cochlea [145] and murine skeletal muscle in vivo [146]. In addition, the redox and metabolic states of a cell can be determined using ratiometric measure of FAD and NADH autofluorescence [29,98].…”

Mitochondrial Redox Imaging for Cancer Diagnostic and Therapeutic Studies

“…48-52 However, the snapfreezing method, which is important to preserve the in vivo metabolic status, was often not used for tissue sample preparation in ex vivo studies. 48,49,51,52 Without the snap-freezing procedure, redox states ex vivo may deviate significantly from the true redox states in vivo after sample preparation. Therefore, we choose only to review the ex vivo studies with the proper snap-freezing procedure and in vivo imaging studies in the following section.…”

Multiphoton microscopic imaging of adipose tissue based on second‐harmonic generation and two‐photon excited fluorescence

“…TPEF microscopy presents a noninvasive and high-resolution laser scanning enabling deep optical tissue imaging while maintaining tissue viability (Zipfel et al 2003a;Squirrell et al 1999;Centonze and White 1998;Wilder et al 2004). Moreover, this technique enables visualization of cellular and subcellular structures based on the intrinsic fluorophores such as NAD(P)H, FAD or porphyrins contained in tissues, without any exogenous fluorescent agents (Zoumi et al 2002;Zipfel et al 2003b), and thus can provide an estimate of a metabolism-related redox ratio (Tiede et al 2007;Zhuo et al 2007a). Additionally, SHG microscopy was also used for investigating collagen fibers and the modification in tumor growth (Brown et al 2003).…”