FM-AFM experiments were performed using a Bruker Bioscope Catalyst AFM system (Bruker Inc., Santa Barbara, CA, USA) mounted on an inverted Axiovert 200M microscope system (Carl Zeiss, Göttingen, Germany) equipped with a confocal LSM 510 Meta (Carl Zeiss) and a 40× (0.6 numerical aperture; Plan-Apochromat) objective lens (Carl Zeiss). The AFM biological system was placed on a vibration isolation table (Kinetic Systems, Boston, MA, USA). A heating stage (Bruker) was used to maintain the physiological temperature (37°C) of explants during measurements. Modified triangular, gold-coated, silicon nitride AFM cantilevers with a 10-μm borosilicate glass attached microsphere were obtained from Novascan (Novascan, Ames, IA, USA). The cantilevers were precalibrated by the company before microsphere attachment with a calibrated spring constant of ~0.12 N m−1. We confirmed calibrations by using the thermal fluctuation tune method (29) built in the AFM system. AFM microcantilevers’ calibrated spring constants were 0.1 to 0.17 N m−1.

Once the cochlear explant was placed in the AFM X-Y sample stage, the cantilever was positioned in liquid far above the sample surface and allowed to thermally equilibrate. For noncontact acoustic FM-AFM experiments, tapping mode AFM was engaged. Immediately, the cantilever tune mode was launched to choose the driving frequency. An initial frequency sweep was performed to locate fπ/2. Because we used piezo-driven excitation in liquids, a forest of peaks is observed (41). We chose the largest peak found with a well-defined phase change, with a typical resonant frequency of 30 to 45 kHz. Next, the cantilever was approached and gently placed in contact with the hair cell bundle. Then, the cantilever tune mode was launched and initially set to position the microsphere of the cantilever 1 μm above the hair bundle, and the phase lag between the piezo and the cantilever was set to π/2. In addition, the drive oscillatory amplitude of the piezo was adjusted to ensure that the cantilever oscillation amplitude at fπ/2 was 5 nm or below. Frequency sweeps were recorded with a 10-kHz frequency range around fπ/2 for multiple distances between the bead and the hair bundle from 1 μm or 500 nm down to 50 nm. Last, the vibrating cantilever was moved 4 μm away for the surface, and a final frequency sweep was recorded to acquire f∞. Note that this methodology derives from (25).

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