OC_Finder: Osteoclast Segmentation, Counting, and Classification Using Watershed and Deep Learning

Osteoclasts are multinucleated cells that exclusively resorb bone matrix proteins and minerals on the bone surface. They differentiate from monocyte/macrophage lineage cells in the presence of osteoclastogenic cytokines such as the receptor activator of nuclear factor-κB ligand (RANKL) and are stained positive for tartrate-resistant acid phosphatase (TRAP). In vitro osteoclast formation assays are commonly used to assess the capacity of osteoclast precursor cells for differentiating into osteoclasts wherein the number of TRAP-positive multinucleated cells is counted as osteoclasts. Osteoclasts are manually identified on cell culture dishes by human eyes, which is a labor-intensive process. Moreover, the manual procedure is not objective and results in lack of reproducibility. To accelerate the process and reduce the workload for counting the number of osteoclasts, we developed OC_Finder, a fully automated system for identifying osteoclasts in microscopic images. OC_Finder consists of cell image segmentation with a watershed algorithm and cell classification using deep learning. OC_Finder detected osteoclasts differentiated from wild-type and Sh3bp2 ( KI/+ ) precursor cells at a 99.4% accuracy for segmentation and at a 98.1% accuracy for classification. The number of osteoclasts classified by OC_Finder was at the same accuracy level with manual counting by a human expert. OC_Finder also showed consistent performance on additional datasets collected with different microscopes with different settings by different operators. Together, successful development of OC_Finder suggests that deep learning is a useful tool to perform prompt and accurate unbiased classification and detection of specific cell types in microscopic images.