The hair cell bodies have been partially sectioned away, with their stereocilia bundles still embedded within the resin block and pointing down, as shown in f. White arrow, IHC region black arrow, OHC region. (e) Following transmission electron microscopy imaging of ultrathin sections to confirm proper tissue orientation and location within the resin block, the block was mounted for FIB-SEM, and observed with an insertable backscatter detector to identify hair cell bodies. (d) Tissue block trimming and sectioning to approach the hair cell stereocilia bundles using ultramicrotomy. Yellow circles were added to schematically represent gold beads. (b) Immunogold labeling and EM staining with heavy metals. (a) Organ of Corti dissection, tissue fixation. Llustration of the workflow outlining the major steps of the current study. 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. 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. 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.
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. Unfortunately, the data sets they generate lack robust and accurate automated segmentation algorithms.
Serial electron microscopy techniques have proven to be a powerful tool in biology.