Figure 4: Comparison between automatic follicle counting from three-dimensional reconstruction images and manual follicle counting from H and E-stained slices. (a) Images acquired at distances of 52 (left), 107 (middle), and 162 μm (right) from the top surface are presented. The distance of half ovary was determined based on the DAPI signal. Scale bar = 300 μm. (b) Three-dimensional images reconstructed using Imaris (left). Automatically identified DDX4-positive cells (middle) are presented as red or blue spots (right). Scale bar = 300 μm. (c) Standard H and E-stained images of ovarian slices under × 4, ×10, ×20, and × 40 microscope magnifications. Primordial, primary, secondary, or atretic follicles are marked. Scale bar in C = 100 μm. (d) Comparison between automatic and manual counting results revealed no differences. H and E: Hematoxylin and eosin.