Large Image

Abstract

Responses to diverse environmental stimuli are fundamental, evolutionarily conserved processes that enable organisms to survive. High‑precision and quantitative analysis of these responses and associated cell-to-cell variability requires the simultaneous and efficient measurement of a large number of cells. Here, we constructed a high‑throughput imaging system (HT system) to improve the throughput of chemotaxis response measurements in Escherichia coli, consisting of a fluorescence microscope to observe hundreds of cells and a semi-automated analysis program termed the high‑throughput analyzer (HT analyzer) implemented in Python with available algorithms. The HT analyzer identified cells within a single chamber and performed a comprehensive analysis of multiple cellular parameters based on fluorescence intensity derived from fluorescently labeled target proteins. Using the HT system, we successfully performed efficient measurement of chemotactic responses in single E. coli cells within a cell population. We simultaneously observed up to approximately 100 E. coli cells within a single chamber and detected the transient assembly of GFP‑fused CheB—an adaptation enzyme of the chemotaxis signaling system—at the cell poles in response to L‑isoleucine stimulation, followed by dissociation upon completion of adaptation. The duration of CheB localization increased with isoleucine concentration from 1 to 15 mM and reached saturation at higher concentrations. Notably, this localization duration exhibited reproducible cell‑to‑cell variability, both across experimental trials and within a single trial. These results demonstrate that the HT system enables quantitative, single‑cell analysis of responses across large cell populations.