Voltage‐Programmed Sequential Fluorescence Encoding (VPSFE) Enables Multiplexed In Situ Proteo‐Imaging with Electric‐Field Turing Patterns

Abstract

Abstract 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.