Multimodal microscope for optical coherence microscopy, tomography, vibrometry, and two-photon microscopy in the living mouse cochlea.

Significance: Our understanding of mechanotransduction in mammalian inner ears remains incomplete, in part due to imaging limitations: current systems cannot simultaneously provide high-resolution images needed for subcellular analysis and the deep focus required for structural mechanics. Optical coherence tomography (OCT) enables structural and vibrational imaging through the bone of the intact cochlea in models such as mice, supporting studies of cochlear mechanics in animals with functional hearing. However, capturing both cellular ( < 10    μ m ) and structural ( > 200    μ m ) details requires rapid switching between optical configurations with numerical apertures ranging from 0.13 to 0.8. A spectral-domain OCT system combined with two-photon fluorescence microscopy (TPM) and interchangeable objectives could overcome this challenge, enabling high-precision vibration and fluorescence imaging across multiple scales in a single experiment. Aim: We aim to develop an integrated OCT and two-photon microscope optimized for imaging the morphology and function of the cochlea. Approach: We integrated a custom SD-OCT/TPM system into an upright microscope with a high-precision stage for animal positioning. The system uses two tunable liquid lenses to form a beam expander, enabling dynamic adjustment of the beam diameter at the back aperture of each objective. This optimized light throughput and maintained a high signal-to-noise ratio (SNR) across all objectives. In addition, we automated optical adjustments to facilitate seamless imaging with a wide range of objectives. Results: For each objective, we measured the SNR difference between a beam expanded to match the largest back aperture and a beam adjusted to match the back aperture of the objective. Except for the 4 × objective, the measured SNR improvements closely matched theoretical predictions. Using four selected objectives spanning the required numerical aperture (NA) range, we successfully imaged excised murine cochlea samples, obtaining relevant structural information across scales. In living murine models, we used TPM to locate fluorescent outer hair cells and make vibrometry measurements through the round window membrane. We found that hair cells, the basilar membrane, and the reticular lamina moved in phase in response to a 70 kHz stimulus at 90 dB SPL, consistent with expected cochlear mechanics. Conclusions: Automation and optimization of the optical system enabled seamless multiscale imaging of the murine cochlea, providing high-quality morphological, functional, and two-photon fluorescence images. The dynamic adjustment of the beam diameter within the microscope was essential for maintaining high SNR across a wide range of numerical apertures.