Subcellular Tandem‐Activated Signal Amplification for Spatially Precise Molecular Imaging in Mitochondria

Despite significant advances in DNA‐based signal amplification strategies for sensitive molecular imaging in live cells, achieving subcellular resolution remains challenging due to limited spatial precision. Here, we present a subcellular tandem‐regulated, spatially selective signal amplification technology for molecular imaging in mitochondria. This platform integrates ribosomal RNA (rRNA)‐activated target‐aptamer recognition with enzyme‐mediated cascade signal amplification, enabling in situ imaging of ATP within defined subcellular compartments (e.g., mitochondria) or membraneless regions (e.g., cytosol). The system facilitates in situ monitoring of ATP dynamics during drug intervention with enhanced spatial precision and sensitivity. Furthermore, by re‐engineering the cascade‐regulated sensor, we extended this approach to enable correlated imaging of mitochondrial ATP and microRNA. This strategy offers a powerful, modular tool for probing energy metabolism and regulatory networks across different subcellular environments.