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Mitophagy is an evolutionally conserved cellular process that eliminates dysfunctional and excess mitochondria, thereby facilitating mitochondrial quality control and metabolic recycling. In addition, mitophagy is essential for cellular homeostasis and tissue development, and mitophagic dysfunction is related to various pathologies including neurodegenerative diseases and cancer. Thus, accurate quantitative measurement of mitophagy is one of the hot topics in the field of mitochondrial research. Fluorescence microscopical technology, one of the most widely used technologies at present, can well explain the occurrence and activity of mitophagy.