Microfluidic cultivation devices that facilitate O₂ control enable unique studies of the complex interplay between environmental O₂ availability and microbial physiology at the single-cell level. Therefore, microbial single-cell analysis based on time-lapse microscopy is typically used to resolve microbial behavior at the single-cell level with spatiotemporal resolution. Time-lapse imaging then provides large image-data stacks that can be efficiently analyzed by deep learning analysis techniques, providing new insights into microbiology. This knowledge gain justifies the additional and often laborious microfluidic experiments. Obviously, the integration of on-chip O₂ measurement and control during the already complex microfluidic cultivation, and the development of image analysis tools, can be a challenging endeavor. A comprehensive experimental approach to allow spatiotemporal single-cell analysis of living microorganisms under controlled O₂ availability is presented here. To this end, a gas-permeable polydimethylsiloxane microfluidic cultivation chip and a low-cost 3D-printed mini-incubator were successfully used to control O₂ availability inside microfluidic growth chambers during time-lapse microscopy. Dissolved O₂ was monitored by imaging the fluorescence lifetime of the O₂-sensitive dye RTDP using FLIM microscopy. The acquired image-data stacks from biological experiments containing phase contrast and fluorescence intensity data were analyzed using in-house developed and open-source image-analysis tools. The resulting oxygen concentration could be dynamically controlled between 0% and 100%. The system was experimentally tested by culturing and analyzing an E. coli strain expressing green fluorescent protein as an indirect intracellular oxygen indicator. The presented system allows for innovative microbiological research on microorganisms and microbial ecology with single-cell resolution.

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