Fluorogenic Crystallization via Enzyme-Instructed Excited-State Intramolecular Proton Transfer for Dynamic Microenvironment Profiling

Understanding dynamic changes within cellular microenvironments is crucial for elucidating biological processes and developing targeted therapies. Here, we present a rapid and straightforward strategy for profiling tumor microenvironments via fluorogenic crystallization driven by enzyme-instructed excited-state intramolecular proton transfer (ESIPT). By engineering ESIPT-based fluorophores, we achieve selective crystallization with strong dual-emission ratiometric fluorescence signals that are easily visualized, offering a real-time readout of tumor microenvironmental variations. We demonstrate that this method enables the efficient detection of tumor microenvironmental features, including enzymatic activity and pH heterogeneity, across different cellular models. This approach provides a simple, efficient, and versatile tool for studying microenvironment dynamics with broad potential applications in disease diagnosis, drug discovery, and personalized medicine.