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Serial electron microscopy techniques have proven to be a powerful tool in biology. Unfortunately, the data sets they generate lack robust and accurate automated segmentation algorithms. In this data descriptor publication, we introduce a serial focused ion beam scanning electron microscopy (FIB-SEM) dataset consisting of six outer hair cell (OHC) stereocilia bundles, and the supranuclear part of the hair cell bodies. Also presented are the manual segmentations of stereocilia bundles and the gold bead labeling of PKHD1L1, a coat protein of hair cell stereocilia important for hearing in mice. This depository includes all original data and several intermediate steps of the manual analysis, as well as the MATLAB algorithm used to generate a three-dimensional distribution map of gold labels. They serve as a reference dataset, and they enable reproduction of our analysis, evaluation and improvement of current methods of protein localization, and training of algorithms for accurate automated segmentation.

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In a recent study, we showed that one type of stereocilia surface specialization, the stereociliary coat, is formed at least in part by a newly identified stereociliary protein, Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1)17. This study was carried out using serial scanning electron microscopy of hair cell bundles immunolabeled with anti-PKHD1L1 and gold bead conjugated antibodies. We showed that PKHD1L1 is located at the tips of stereocilia, and quantified the gold bead distribution in different regions of the surfaces of stereocilia. PKHD1L1-deficient mice lack the surface coat at the upper but not lower regions of stereocilia, confirming that PKHD1L1 is a component of the surface coat, and they develop progressive hearing loss, showing that PKHD1L1 is required for normal hearing in mice.

In this study, only three rows of stereocilia were segmented, even when additional shorter stereocilia were observed. The shorter stereocilia were excluded from our analysis because their functional significance at that developmental stage is not clear, and they are not present in mature hair cells. Thus, for each dataset, a segmented bundle consisted of (1) the kinocilium, (2) the tallest row stereocilia, (3) the middle row stereocilia immediately next to the tallest row stereocilia, and (4) the short row of stereocilia, defined as shorter stereocilia immediately next to the ones from the middle row. Any stereocilia not in immediate proximity to the middle row stereocilia were excluded from the study. Sometimes the analyzed serial EM volume did not include the entire length of the stereocilium (for example, see Cell #344,45,46,47,48,49,50), in which case the stereocilia were still segmented, but excluded from the MATLAB analysis as described below.


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