Quantitative single-cell imaging of efflux pump heterogeneity and antibiotic response dynamics.

Understanding how bacterial cells survive antibiotic treatment requires tools capable of resolving dynamic physiological heterogeneity at single-cell resolution. Here, we present a high-throughput, lineage-resolved microfluidic imaging platform for quantifying the expression and spatial distribution of the AcrAB-TolC efflux pump in Escherichia coli during antibiotic exposure. The system is built around a custom-designed multilayer microfluidic device, fabricated using a direct-write photolithography protocol, which ensures uniform delivery of nutrients and antibiotics while confining thousands of individual cells in dead-end trenches. Brightfield imaging provides a label-free, high-temporal-resolution method for tracking cell growth and division, enabling accurate reconstruction of lineages over time. Efflux pump abundance is quantified using GFP-tagged AcrB, while a red fluorescent reporter driven by a constitutive promoter serves as a control to account for growth-dependent fluorescence artefacts. To analyse the sources of heterogeneity in efflux abundance and its consequences for antibiotic response dynamics, we developed a lineage-resolved analytical framework that compares cells of similar replicative age but differing spatial positions, and vice versa. This analysis was made possible by a novel machine-learning-based image processing pipeline, which enables robust cell segmentation, tracking, and lineage reconstruction across multiple fields of view. By extracting time-resolved single-cell data, this platform allows precise dissection of non-genetic variability in efflux activity and its role in determining survival outcomes, offering a powerful foundation for future quantitative studies of bacterial physiology under antibiotic stress.