A high-throughput microscopy method for single-cell analysis of event-time correlations in nanoparticle-induced cell...

Area of Science:

• Cell Biology
• Toxicology
• Biophysics

Background:

• Cell death timing is crucial for understanding cellular responses but often obscured in bulk measurements.
• Automated live-cell imaging offers potential for high-resolution temporal analysis of cellular events.

Purpose of the Study:

• To develop and apply a method for extracting event times of cell death markers from fluorescence time traces.
• To investigate the chronological sequence and kinetics of cell death pathways induced by nanoparticles in cancer cells.

Main Methods:

• Utilized automated live-cell imaging on single-cell arrays (LISCA).
• Monitored fluorescence time traces of lysosomal membrane permeabilization, mitochondrial outer membrane permeabilization, and oxidative burst.
• Analyzed pairwise marker combinations to determine event sequences and delays after nanoparticle exposure.

Main Results:

• A lysosomal cell death pathway was identified at low nanoparticle doses (25 µg/mL) in both A549 (lung) and Huh7 (liver) cancer cells.
• At higher doses (100 µg/mL), a concurrent mitochondrial pathway emerged in A549 cells, but not in Huh7 cells.
• Event-time correlations provided insights into pathway heterogeneity and signal transmission interdependencies.

Conclusions:

• The developed method enables detailed temporal analysis of cell death signaling pathways.
• Differential cell death pathway activation by nanoparticles highlights cell-type-specific responses.
• Understanding temporal dynamics is key to deciphering complex cellular signaling and toxicity mechanisms.