Experimental setup

All methods were carried out in accordance with relevant guidelines and regulations. The experimental protocols were approved by an institutional committee (Technische Universität Dresden, Ethikkommision an der TU Dresden, Fetscherstr. 74, 01307 Dresden). Informed consent was obtained from all subjects and/or their legal guardian(s). The TBs included in this study were either fresh and kept refrigerated in isotonic NaCl-solution or freshly frozen upon harvesting and subsequently defrosted immediately before the measurement. The following is a general description of preparation and setup pertaining to the most commonly used way of determining the METF in TB specimens^5,13. We will be labeling this approach as method “A”. Laboratory specific details or deviations from the general description are given below (see “Laboratory specifics”).

Following basic preparation (removal of connective tissue and muscles), access to the middle ear is accomplished via mastoidectomy and subsequent posterior tympanotomy. The cartilage and skin parts of the external ear canal are removed, leaving only the bony part of the external ear canal. The tympanic segment of the facial nerve is removed partially or completely in order to allow line-of-sight to the SFP for the LDV measurements. Care is taken to leave the whole ossicular chain, the attached ligaments and muscles (Musculus stapedius and Musculus tensor tympani), the TM, and the inner ear intact. In the process, the middle ear is inspected by the surgeon for any abnormalities. Pathological mobility of the OC and any other issues pertaining to sound transmission (such as a damaged cochlea or TM) are noted if detected. Note that the methods for inspection may vary, commonly this includes visual and tactile inspection. Reflective foil or beads are then placed onto the SFP. LDV measurements are acquired at a single point near the center of the SFP. For accurate measurements, it is important to remove excess water droplets from the SFP to ensure a reliable LDV signal. At the same time, the TB should be kept moist for the duration of the experiment so as to avoid changes in sound transmission due to drying of tissue, which typically begin to occur after about 60 min of drying at room temperature¹⁴. For METF measurements of the intact OC, this is not an issue because these are done immediately upon commencement of the experiment and usually do not require more than an hour.

An earphone for sound excitation is inserted into the ear canal and tightly sealed with an earplug to avoid uncontrolled loss of sound pressure during measurements. Excitement sound pressure levels of about 94–100 dB SPL are used to minimize nonlinear transmission effects. The ear canal is sometimes replaced partially or completely by an artificial volume. The probe microphone is inserted so that the tip of the probe sits a few millimeters in front of the TM. There are different ways of achieving this placement (see laboratory specifics below), but when done correctly they can be assumed to be equivalent in outcome. This is because the METF is per definition normalized to input pressure, and the measured sound pressure in TB experiments is insensitive to exact microphone position¹⁴. The METF is calculated by analogy to Eq. (¹) with the respective discrete signals Yk=ak·VkLDV and Xk=bk·VkProbe, where Vk denotes the magnitude of the fast Fourier transform (FFT) of the voltage received, ak and bk are calibration factors and the index k symbolizes discrete frequency. Note that due to anatomical restrictions, the laser beam is usually pointed at the SFP at an incidence angle Θ>0 to the normal axis of the SFP plane. This means that the measurement potentially underestimates the actual motion magnitude, because dmeasured=dactual·cos(θ), where d is the magnitude of stapes displacement. Therefore Θ itself or a range for the angle is usually recorded along with the experimental data.