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Updated: Jul 24, 2025

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

Keitaro Kasahara1, Markus Leygeber1, Johannes Seiffarth1,2

  • 1IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich GmbH, Jülich, Germany.

Frontiers in Microbiology
|July 6, 2023
PubMed
Summary

This study presents a microfluidic system for precise oxygen control during single-cell microbial analysis. It enables detailed observation of microbial responses to varying oxygen levels using time-lapse microscopy and deep learning.

Keywords:
FLIMPDMSRTDPautomated image analysismicrobial single-cell analysismicrofluidicoxygen control

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Area of Science:

  • Microbiology
  • Microbial Physiology
  • Biotechnology

Background:

  • Microfluidic devices offer precise control over environmental conditions for single-cell microbial studies.
  • Time-lapse microscopy coupled with deep learning is crucial for analyzing complex microbial behavior at the single-cell level.
  • Integrating oxygen measurement and control into microfluidic systems presents significant technical challenges.

Purpose of the Study:

  • To develop a comprehensive experimental approach for spatiotemporal single-cell analysis of microorganisms under controlled oxygen availability.
  • To enable innovative microbiological research and microbial ecology studies with single-cell resolution.

Main Methods:

  • Utilized a gas-permeable polydimethylsiloxane microfluidic chip and a 3D-printed mini-incubator for oxygen control.
  • Employed fluorescence lifetime imaging microscopy (FLIM) with an oxygen-sensitive dye (RTDP) to monitor dissolved oxygen.
  • Analyzed image data stacks (phase contrast and fluorescence) using open-source image-analysis tools.
  • Dynamically controlled oxygen concentration from 0% to 100%.

Main Results:

  • Successfully controlled and monitored dissolved oxygen levels within microfluidic growth chambers.
  • Demonstrated the system's efficacy by culturing and analyzing an *E. coli* strain with green fluorescent protein.
  • Validated the integration of microfluidics, oxygen control, time-lapse microscopy, and image analysis for microbial studies.

Conclusions:

  • The presented system provides a robust platform for investigating the intricate relationship between oxygen availability and microbial physiology at the single-cell level.
  • This approach facilitates novel insights into microbial responses and ecological dynamics under precisely controlled oxygen conditions.
  • The developed system supports advanced microbiological research by enabling detailed spatiotemporal analysis of microorganisms.