Unveiling microbial single-cell growth dynamics under rapid periodic oxygen oscillations

Microbial metabolism and growth are tightly linked to oxygen (O₂). Microbes experience fluctuating O₂ levels in natural environments; however, our understanding of how cells respond to fluctuating O₂ over various time scales remains limited due to challenges in observing microbial growth at single-cell resolution under controlled O₂ conditions and in linking individual cell growth with the specific O₂ microenvironment. We performed time-resolved microbial growth analyses at single-cell resolution under a temporally controlled O₂ supply. A multilayer microfluidic device was developed, featuring a gas supply above a cultivation layer, separated by a thin membrane enabling efficient gas transfer. This platform allows microbial cultivation under constant, dynamic, and oscillating O₂ conditions. Automated time-lapse microscopy and deep-learning-based image analysis provide access to spatiotemporally resolved growth data at the single-cell level. O₂ switching within tens of seconds, coupled with precise microenvironment monitoring, allows us to accurately correlate cellular growth with local O₂ concentrations. Growing Escherichia coli microcolonies subjected to varying O₂ oscillation periods show distinct growth dynamics characterized by response and recovery phases. The comprehensive growth data and insights gained from our unique platform are a crucial step forward to systematically study cell response and adaptation to fluctuating O₂ environments at single-cell resolution.