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Methylene blue (MB) is a biocompatible and environmentally friendly material that has been widely used in clinical and biomedical research fields for over a century. In addition, it also has wide applications in other fields, such as dyeing and finishing industry, aquaculture industry, photocatalysis and food industry. This landmark publication is unique as it covers MB-mediated in vivo fluorescence, photoacoustic imaging and light-induced cancer therapy. The optical properties and photothermal effect of MB are also systematically described.