High-throughput quantitation of pathogen-induced calcium signals captured through live-cell fluorescence microscopy

Area of Science:

• Cellular Biology
• Microbiology
• Biophysics

Background:

• Intracellular pathogens manipulate host cell calcium for survival and replication.
• Live-cell microscopy with calcium indicators is crucial for studying cellular calcium dynamics.
• Lack of specific inhibitors for pathogen-induced calcium signals hinders research.

Purpose of the Study:

• To develop quantitative imaging pipelines for characterizing pathogen-induced calcium signals.
• To create computational tools for automated analysis of cellular calcium dynamics during infection.
• To enable robust quantification of previously unmeasurable signal parameters.

Main Methods:

• Utilized live-cell microscopy with genetically encoded calcium indicators.
• Developed automated computational tools in ImageJ for signal quantitation.
• Employed rotavirus as a model pathogen and used fluorescently tagged recombinant strains.
• Normalized calcium signals to infectivity for accurate analysis.

Main Results:

• Created a suite of computational tools for automated quantification of intra- and inter-cellular calcium signals.
• Enabled automated detection of infected cells and signal normalization.
• Tools are freely available, adaptable, and require minimal user input.
• Allowed extraction of previously unquantifiable signal parameters, enhancing characterization speed and breadth.

Conclusions:

• The developed ImageJ tools provide a robust and efficient method for analyzing pathogen-induced calcium dynamics.
• These tools facilitate deeper understanding of host-pathogen interactions at the cellular level.
• The freely available nature of the tools promotes broader adoption and advancement in the field.