Voltage-Programmed Sequential Fluorescence Encoding (VPSFE) Enables Multiplexed In Situ Proteo-Imaging with...

DNA barcode-based immunolabeling has revolutionized single-cell protein profiling. However, conventional multiplexed imaging methods are hindered by laborious probe exchange procedures involving buffer washing-based probe removal and prolonged hybridization cycles (requiring tens of minutes to 1.5 h per cycle), which limit throughput, specificity, and universality. Here, we introduce an electrophoresis-based in situ probe removal method that achieves high-specificity iterative probe imaging without washing steps, utilizing 2-min low-voltage electrophoresis for excess probes removal and 3-min high-voltage electrophoresis for hybridized probes dissociation. The robustness was validated through 19 rounds of cyclic electrophoresis, 10 rounds of repetitive imaging, and simultaneous processing of 14 probes (µM-level) across five rounds of multiprobe exchange, demonstrating exceptional specificity and efficiency. Applied to sequential color coding-based multiplexed imaging, this approach establishes voltage-programmed sequential fluorescence encoding (VPSFE), enabling multiplexed imaging of epithelial-mesenchymal transition (EMT)-related proteins. Furthermore, we developed a VPSFE-based Turing pattern coding strategy for multiplexed detection that requires only a single multicolor probe hybridization step. Using three voltage conditions and three fluorescence channels, this system generates 27 unique fluorescence Turing patterns to encode 27 distinct targets. This electric-field Turing pattern coding strategy represents a novel probe exchange-free approach for rapid, universal, and highly specific multiplexed in situ imaging.