Single-Cell, Time-Lapse Reactive Oxygen Species Detection in E. coli

Area of Science:

• Microbiology
• Cell Biology
• Biochemistry

Background:

• Traditional bulk assays for reactive oxygen species (ROS) in bacteria lack spatial and temporal resolution.
• Understanding ROS dynamics at the single-cell level is crucial for correlating cellular phenotypes with environmental perturbations.

Purpose of the Study:

• To develop and validate novel single-cell, time-lapse assays for detecting ROS in live Escherichia coli (E. coli).
• To enable in situ detection of ROS in individual bacterial cells, linking ROS production to specific cellular responses.

Main Methods:

• Utilized flow systems integrated with fluorescence microscopy for real-time observation.
• Employed fluorogenic dyes (CellROX Green, Amplex® Red/APEX2) that accumulate intracellularly for ROS detection.
• Applied assays to both homogeneous and heterogeneous bacterial populations.

Main Results:

• Successfully detected intracellular ROS in single E. coli cells using the developed time-lapse assays.
• Demonstrated the capability to correlate observed cellular symptoms with in situ ROS levels.
• Validated the use of CellROX Green and Amplex® Red/APEX2 for sensitive ROS detection.

Conclusions:

• The developed single-cell assays provide unprecedented spatial and temporal insights into bacterial ROS production.
• These assays offer a powerful tool for studying the role of ROS in bacterial responses to various stimuli, including antibiotics.
• Facilitates a deeper understanding of bacterial physiology and stress responses at the individual cell level.