Reversible Quantum Dot-Driven Multiplex Technology for <i>In Situ</i> Subcellular Structure Imaging

Traditional immunofluorescence methods and organic fluorescent dyes can achieve localized staining analysis of organelle structures, but they often face challenges, such as fluorescence signal quenching, limited detection throughput, and complex antibody modification. To address these issues, we developed ssDNA-quantum dot imaging encoding (ssQIE), an in situ imaging method that combines quantum dots with DNA nanotechnology. This method, which uses ssDNA labels to modify antibodies and captures quantum dot-DNA coupled complexes instead of traditional organic fluorescent dye signals, shows signal amplification capability during imaging, allows multiplexing of single-channel excitation, and is suitable for multiple rounds of cyclic imaging under mild conditions. In this study, we applied ssQIE to in situ imaging of organelles and compared the results to those based on conventional immunofluorescence and CODEX methods. The unique optical properties of quantum dots provide a guarantee for the good imaging performance of the ssQIE. In addition, we applied ssQIE to image key organelles in the ferroptosis state to analyze the abundance and distribution of these organelles. Therefore, ssQIE provides a breakthrough solution for the limited modification of antibody species and fluorescence channels, demonstrating a broad prospect for the combination of quantum dot nanomaterials and DNA nanotechnology.