Dynamic micro-optical coherence tomography enables structural and metabolic imaging of the mammalian cochlea

Abstract

Sensorineural hearing loss (SNHL) is caused by damage to the mechanosensory hair cells and auditory neurons of the cochlea. The development of imaging tools that can directly visualize or provide functional information about a patient's cochlear cells is critical to identify the pathobiological defect and determine the cells' receptiveness to emerging SNHL treatments. However, the cochlea's small size, embedded location within dense bone, and sensitivity to perturbation have historically precluded high-resolution clinical imaging. Previously, we developed micro-optical coherence tomography (μOCT) as a platform for otologic imaging in animal models and human cochleae. Here we report on advancing μOCT technology to obtain simultaneously acquired and co-localized images of cell viability/metabolic activity through dynamic μOCT (DμOCT) imaging of intracellular motion. DμOCT obtains cross-sectional images of ATP-dependent movement of intracellular organelles and cytoskeletal polymerization by acquiring sequential μOCT images and computing intensity fluctuation frequency metrics on a pixel-wise basis. Using a customized benchtop DμOCT system, we demonstrate the detailed resolution of anatomical and metabolic features of cells within the organ of Corti, via an apical cochleostomy, in freshly-excised adult mouse cochleae. Further, we show that DμOCT is capable of capturing rapid changes in cochlear cell metabolism following an ototoxic insult to induce cell death and actin stabilization. Notably, as few as 6 frames can be used to reconstruct cochlear DμOCT images with sufficient detail to discern individual cells and their metabolic state. Taken together, these results motivate future development of a DμOCT imaging probe for cellular and metabolic diagnosis of SNHL in humans.

Keywords: cochlea; hair cell; metabolic imaging; micro-optical coherence tomography; organ of Corti; sensorineural hearing loss.

Figure 1

Schematic of the DμOCT benchtop imaging system (A) and preparation of the inner ear specimen for imaging (B–D). (A) Simplified schematics of the DμOCT setup, with the organ of Corti within the mouse inner ear specimen displayed in the lower right-hand corner (not to scale). (B–D) Preparation and orientation of the adult mouse inner ear specimen for DμOCT imaging. The inner ear is positioned flat on the surface, resting on the inferior portion of the otic capsule and the anterior semicircular canal. Major anatomical landmarks including the RW and OW are identified prior to cochleostomy (B). An initial cochleostomy is created in the cochlear apex (white dotted circle) and widened to the demarcation line (yellow dotted circle) of the apical to mid-basal turn region (C). The inner ear is mounted on its anterior and posterior semicircular canals so the cochleostomy (yellow dotted circle) and the organ of Corti (orange dotted line) are facing upward for imaging, parallel to the bottom of the dish and surface of the medium (D). BS, non-polarizing beam splitter; DμOCT, dynamic micro-optical coherence tomography; FC, 90:10 fiber coupler; GM, 2-axis galvanometer mirror scanning system; L1, collimator; L2, 0.3 NA microscope objective; L3, 0.3 NA microscope objective; M, mirror; NA, numerical aperture; OW, oval window; RW, round window; TL, liquid tunable lens.

Figure 2

DμOCT cross-sectional image of the apical region of the adult mouse organ of Corti. DμOCT image of the adult mouse organ of Corti without (A) and with (B) anatomical features annotated. Static anatomical features (e.g., basilar membrane circled in white dashes) appear blue and Brownian motion-predominant areas, such as the tunnel of Corti, appear brown-red (triangle in A). Metabolically-active cell features appear green-yellow (nuclei, mitochondria, stereocilia). (C) Schematic of the organ of Corti, including the inner and outer hair cells and non-sensory cells (created with Biorender; www.biorender.com). DμOCT, dynamic micro-optical coherence tomography; Hz, hertz.

Figure 3

DμOCT image of adult mouse organ of Corti before (A) and after (B) formalin application. Images are an average of 5 consecutive DμOCT frames for improved clarity. Arrowheads point to examples of cell nuclei that shifted from active (green) to static (blue) post-formalin. Red arrows indicate the OHCs and IHCs. DμOCT, dynamic micro-optical coherence tomography; Hz, Hertz; IHC, inner hair cell; min, minutes; OHC, outer hair cell; TC, tunnel of Corti. * Indicates bubbles consistent with blebbing cells.

Figure 4

DμOCT images of adult mouse organ of Corti reconstructed from 150 contiguous frames over 1.5 s (A) and 6 contiguous frames spanning 375 ms (B). DμOCT, dynamic micro-optical coherence tomography; Hz, Hertz; ms, milliseconds; s, seconds; TC, tunnel of Corti.