Semi-automated quantification of hair cells in the mature mouse utricle

PCP Auto Count: A Novel Fiji/ImageJ plug-in for automated quantification of planar cell polarity and cell counting

“…Previously published automated cell counting programs also often struggle to cope with overlapping cells, which can require advanced machine learning systems [38], labor intensive optical clearing methods [45], or user guided segmentation of cells prior to machine analysis [26]. In the case of inner ear tissues this issue can be particularly problematic where, for example, there are little to no options for the labeling of cell nuclei in the neurons of the auditory nerve which makes segmentation nearly impossible.…”

“…A similar problem persisted for decades with regard to sensory hair cells where myosin or other non-nuclear proteins were the best available markers. More recently, immunolabeling of the nuclear protein POU4F3 has been proposed in a semi-automated method for hair cell counting [26], but even with a nuclear marker, there appear to be difficulties with segmentation requiring manual separation of doublets and triplets of overlapping cells by the user. This problem may be partly solvable from the staining and imaging side of the approach where we have found that immunolabeling spectrin in hair cells provides better segmentation than staining of POU4F3 (data not shown).…”

“…However, consistently obtaining high quality and similar SNR micrographs, especially across different samples, is often challenging. Indeed, this is a limitation inherent in several previously published cell quantification programs as well [26,37,38]. While we were able to successfully run PCPA on images of suboptimal quality (both in terms of image dpi and antibody SNR ratio) by using the pre-processing steps recommended, we have also included several features in the PCPA outputs (i.e.…”

“…As the fusion of neighboring cells in micrographs is a common problem in automated image analysis [26][27][28], we developed an optional Doublet Splitting mode, where PCPA will separate two cells that touch or overlap by splitting the doublet mass in half, then treating each half as a discreet cell during subsequent steps (Fig B in S1). This optional function cannot recurse beyond separating two conjoined cells.…”

Maturation of type I and type II rat vestibular hair cells in vivo and in vitro

“…The postnatal maturation of the rat and mouse vestibular HCs and their fragility to experimental manipulation are significant factors that condition the establishment of in vitro models to study their biology and pathology. In one popular preparation, utricles are obtained from adult mice and are maintained as free floating specimens for a few (up to 7, usually 1 to 4) days (Cunningham et al, 2002;Cunningham, 2006;Sung et al, 2022). These explants have been frequently used to study ototoxicity and to evaluate otoprotective treatments in mature HCs, but culturing the sensory epithelium causes a cellular stress that probably masks the earliest responses to the toxicity being analyzed.…”