Fast, Streamlined In Situ Genotyping of Bacterial Strains for Single-Cell and Subcellular Dynamics Profiling

Abstract Temporal dynamics, such as cell-cycle oscillations, transcriptional bursting, and the regulated assembly and disassembly of phase-separated condensates, constitute an important layer of regulation in biology. Yet, existing technologies, including DNA sequencing, plate readers, and fluorescence microscopy, face a fundamental trade-off between temporal resolution, single-cell and subcellular information, and experimental throughput. This trade-off not only fragments our understanding of fundamental cellular processes but also impedes large-scale screening for dynamic phenotypes. Efforts to mitigate such limitations are beginning to emerge. One promising approach combines live-cell time-lapse imaging of pooled bacterial libraries in microfluidic chips with sequential fluorescence in situ hybridization (seqFISH) to connect dynamic phenotypes exhibited by individual cells with their corresponding genotypes. However, widespread adoption of this strategy has been constrained by two technical challenges: (1) compromised in situ genotyping efficiency due to nonspecific fluorescent probe aggregation in confined microfluidic growth chambers, and (2) the reliance on high-copy-number plasmids to ensure genotyping sensitivity, which often distorts dynamic phenotypes and perturbs normal physiology during live-cell imaging. Here, we address both limitations with an integrated and physiologically compatible solution. We introduce a simple surface-modification strategy to suppress fluorescent probe aggregation in microfluidic chips, enabling robust in situ genotyping to be completed overnight. In parallel, we implement barcoded copy-number-tunable plasmids to decouple the conflicting requirements of live-cell imaging and post hoc genotyping, thereby preserving delicate subcellular dynamics while maintaining genotyping sensitivity. Together, these improvements transform image-based bacterial screening into a faster and minimally perturbative workflow, expanding the scalability and versatility of high-throughput single-cell and subcellular dynamics screening for both fundamental and synthetic biology research.