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The variety of available mitochondrial quantification tools makes it difficult to select the most reliable and accurate quantification tool. Here, we performed elaborate analyses on five open source ImageJ tools. Excessive clustering of mitochondrial structures was observed in four tools, caused by the global thresholding applied by these tools. The Mitochondrial Analyzer, which uses adaptive thresholding, outperformed the other examined tools, with accurate structural segregation and identification. Additionally, we showed that the Mit