ATP-Independent Water-Soluble Luciferins Enable Non-Invasive High-Speed Video-Rate Bioluminescence Imaging of Mice

NanoLuc luciferase and its derivatives are attractive bioluminescent reporters recognized for their efficient photon production and ATP independence. However, utilizing them for in vivo imaging poses notable challenges. Low substrate solubility has been a prominent problem, limiting in vivo brightness, while substrate instability hampers consistent results and handling. To address these issues, we developed a range of caged PEGylated luciferins with improved stability and water solubility of up to 25 mM, resulting in substantial bioluminescence increases in mouse models. This advancement has created the brightest and most sensitive luciferase-luciferin combination, enabling high-speed video-rate imaging of freely moving mice with brain-expressed luciferase. Furthermore, we developed a bioluminescent Ca ²⁺ indicator with exceptional sensitivity to physiological Ca ²⁺ changes and paired it with a new substrate to showcase non-invasive, video-rate imaging of Ca ²⁺ activity in a defined brain region in awake mice. These innovative substrates and the Ca ²⁺ indicator are poised to become invaluable resources for biological and biomedical fields.