This project belongs to the field of ldquo; New technical methods in biochemistry and cell biology rdquo;.&& Real-time tracking of intracellular redox states is an important technical challenge facing basic research on life mechanisms, bioengineering and disease diagnosis and treatment applications. This project carries out research on key scientific issues such as redox molecular recognition, high-performance fluorescent sensing configurations, and photo-controlled molecular switch principles, and develops a variety of high-performance redox fluorescent probes and photo-controlled gene expression systems, realizing the imaging and control of intracellular redox homeostasis in time and space, providing innovative tools for basic research in life sciences related to redox homeostasis, and can be widely used in application fields such as bioengineering and drug discovery. The main research results are as follows: fluorescent probe encoding the redox state gene of coenzyme I and high spatio-temporal resolution imaging. Using allosteric action to couple the recognition of redox molecules by protein-based receptors with fluorescent chromophores, we have taken the lead in developing a series of genetically encoded fluorescent probes to specifically monitor the redox state of NADH, realizing dynamic monitoring and imaging of redox steady state in living cells, subcells and living animals. Using this technology, compounds that efficiently kill tumors were discovered through high-throughput screening, and their mechanism of action was identified. This invention provides important innovative tools and means for people to better analyze cellular redox homeostasis and drug discovery research. In situ imaging of protein thiol redox fluorescent probes. Taking advantage of the specific binding characteristics of adjacent thiol groups in proteins and trivalent arsenic, a specific fluorescent probe for the detection of adjacent dithiol groups in proteins has been innovatively developed, and its signal-to-noise ratio has been optimized in series through the principle of fluorescence resonance energy transfer. These small-molecule fluorescent probes are used to realize dynamic real-time tracking of o-dithiol-containing proteins in living cells, and reveal the important regulatory role of mitochondrial reactive oxygen species in these sulfhydryl modification forms, which is useful for the analysis of redox signal transduction in organisms and the development of biological processes and biomanufacturing technologies. Provide new research ideas. Technology for precise regulation of gene expression and cell redox state. A simple, stable and truly practical light-controlled gene expression system has been established to regulate the expression of target genes with high precision in time and space, and for the first time, light has controlled gene expression in mammalian organs; it has been discovered that regulation of intracellular redox states can effectively improve the expression efficiency of important protein drugs. This achievement can provide a powerful tool for basic research in life sciences, and can also be widely used in application fields such as bioengineering and cutting-edge medical care. The 8 representative papers of this project were all published in first-class journals such as Nature Methods, Nature Protocols, Cell Metabolism (2 papers), Angew Chem Int Ed, J Am Chem Soc, Cell Research, etc., with an average impact factor of 16.7 and were cited 500 times in SCI journals such as Science; the maximum number of single papers was 206 times. During the project, 4 China invention patents were authorized, 4 foreign invention patents were applied, 4 have been authorized, and 12 China invention patents have been authorized, 5 have been authorized. The project leader is the first China scientist introduced in the special article of Nature Methods. The cutting-edge methods of project development have gained important influence among international colleagues. They have been evaluated as ldquo; research in the field of subversive oxidation and reduction rdquo; by international colleagues. They have been tracked and applied by more than 500 research groups around the world. The research results of others applying the technology invented by this project have been successively published in authoritative journals such as Science.&& In recent years, the main completers of the project have been selected as academicians of China Academy of Engineering, Changjiang Scholars of the Ministry of Education, Young Changjiang Scholars, Leading Talents in Scientific and Technological Innovation of the Ten Thousand Talents Plan and multiple Shanghai City Talent Plans, and have received funding from the National Outstanding Youth Fund and the Outstanding Youth Fund.