Enabling oxygen-controlled microfluidic cultures for spatiotemporal microbial single-cell analysis

Keitaro Kasahara; Markus Leygeber; Johannes Seiffarth; Karina Ruzaeva; Thomas Drepper; Katharina Nöh; Dietrich Kohlheyer

doi:10.3389/fmicb.2023.1198170

Enabling oxygen-controlled microfluidic cultures for spatiotemporal microbial single-cell analysis

Front Microbiol. 2023 Jun 20:14:1198170. doi: 10.3389/fmicb.2023.1198170. eCollection 2023.

Authors

Keitaro Kasahara^#¹, Markus Leygeber^#¹, Johannes Seiffarth^{1

2}, Karina Ruzaeva^{1

3}, Thomas Drepper⁴, Katharina Nöh¹, Dietrich Kohlheyer¹

Affiliations

¹ IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich GmbH, Jülich, Germany.
² Computational Systems Biotechnology (AVT.CSB), RWTH Aachen University, Aachen, Germany.
³ Aachen Institute for Advanced Study in Computational Engineering Science (AICES), RWTH Aachen University, Aachen, Germany.
⁴ Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich GmbH, Jülich, Germany.

^# Contributed equally.

Abstract

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.

Keywords: FLIM; PDMS; RTDP; automated image analysis; microbial single-cell analysis; microfluidic; oxygen control.

Grants and funding

KK was supported by Japan Student Services Organization (JASSO) Student Exchange Support Program. JS was supported by the President's Initiative and Networking Funds of the Helmholtz Association of German Research Centres [SATOMI ZT-I-PF-04-011]. KR was supported by the Helmholtz School for Data Science in Life, Earth and Energy (HDS-LEE) and received funding from the Helmholtz Association of German Research Centres. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - 491111487.