ABSTRACT: The hair bundle is the mechanosensory organelle of hair cells that detects mechanical stimuli caused by sounds, head motions, and fluid flows. Each hair bundle is an assembly of cellular-protrusions called stereocilia, which differ in height to form a staircase. Stereocilia have different heights, widths, and separations in different species, sensory organs, positions within an organ, hair-cell types, and even within a single hair bundle. The dimensions of the stereociliary assembly dictate how the hair bundle responds to stimuli. These hair-bundle properties have been measured previously only to a limited degree. In particular, mammalian data are either incomplete, lack control for age or position within an organ, or have artifacts owing to fixation or dehydration. Here, we provide a complete set of measurements for postnatal day (P) 11 C57BL/6J mouse apical inner hair cells (IHCs) obtained from living tissue, tissue mildly-fixed for fluorescent imaging, or tissue strongly fixed and dehydrated for scanning electronic microscopy (SEM). We found that hair bundles mildly-fixed for fluorescence had the same dimensions as living hair bundles, whereas SEM-prepared hair bundles shrank uniformly in stereociliary heights, widths, and separations. By determining the shrinkage factors, we imputed live dimensions from SEM that were too small to observe optically. Accordingly, we created the first complete blueprint of a living IHC hair bundle. We show that SEM-prepared measurements strongly affect calculations of a bundle's mechanical properties - overestimating stereociliary deflection stiffness and underestimating the fluid coupling between stereocilia. The methods of measurement, the data, and the consequences we describe illustrate the high levels of accuracy and precision required to understand hair-bundle mechanotransduction.
