Whole-tissue quantification of the spatial distribution of mitotic cells along the epididymal tubule.

<p><b>(A–C)</b> Overview of the procedure for digital image processing and 3D quantification used to acquire the spatial distribution of mitotic cells along the epididymal tubule. <b>(A, A′)</b> A projected raw image of the immunofluorescence of Pax2, a marker for the epididymal tubule, as well as that for phospho histone H3, a mitosis marker, in the epididymis dissected at E16.5. <b>(B)</b> The Pax2-positive cells were extracted from the image <b>(A)</b> as an epididymal tubule via 3D image processing. <b>(C)</b> Mitotic cells located only in the tubule were obtained by masking <b>(A′)</b> with <b>(B)</b>. <b>(D)</b> A superimposed image of the extracted epididymal tubule (black), extracted mitotic cells (magenta), and the centerline of the tubule was automatically obtained. The centerline was the result of image skeletonization <b>(B)</b>. A set of circles with lines colored in blue represent the shortest path to a position on the centerline from each mitotic cell. Note that an automatic count of mitotic cells along the tubule was performed in 3D, although the image was projected onto 2D for visualization. <b>(E, E′)</b> Spatial distribution of mitotic cells in the epididymal tubule along the centerline of the tubule at E15.5 and E16.5. The histogram integrates the raw data in which the position of the mitotic cell is represented as a pulse. The value on the vertical histogram axis was normalized to that in a 10-μm section. <i>n</i> = 12. The error bars represent s.d.