PEGylated ATP-Independent\nLuciferins for Noninvasive\nHigh-Sensitivity High-Speed Bioluminescence Imaging

Bioluminescence imaging (BLI) is a powerful, noninvasive\nimaging\nmethod for animal studies. NanoLuc luciferase and its derivatives\nare attractive bioluminescent reporters recognized for their efficient\nphoton production and ATP independence. However, utilizing them for\nanimal imaging poses notable challenges. Low substrate solubility\nhas been a prominent problem, limiting <i>in vivo</i> brightness,\nwhile the susceptibility of luciferins to auto-oxidation by molecular\noxygen in air increases handling complexity and poses an obstacle\nto obtaining consistent results. To address these issues, we developed\na range of caged PEGylated luciferins with increased auto-oxidation\nresistance and water solubility of up to 25 mM, resulting in substantial <i>in vivo</i> bioluminescence increases in mouse models. This\nadvancement has created the brightest and most sensitive luciferase-luciferin\ncombination, enabling high-speed video-rate imaging of freely moving\nmice with brain-expressed luciferase. These innovative substrates\noffer new possibilities for investigating a wide range of biological\nprocesses and are poised to become invaluable resources for chemical,\nbiological, and biomedical fields.