Twelve-Hour Dynamic Monitoring of Microalgae Response to Oxidative Stress by Single-Cell Metabolomics Analysis

Abstract Cellular metabolism is inherently dynamic during stress responses, yet conventional single-cell metabolomics workflows remain largely limited to isolated snapshots. Here, we implemented an online, label-free CyESI-MS-based workflow for the continuous monitoring of single living microalgal cells, achieving a monitoring duration of 12 h with minute-scale temporal resolution across tens of thousands of living cells. We established a comprehensive data-processing pipeline for single-cell trajectory analysis. Using Chlamydomonas reinhardtii under H2O2-induced oxidative stress as a model, we performed continuous monitoring where chemical perturbation was introduced after a 1 h baseline recording. Across control and H2O2-treated experiments, the CyESI platform recorded 76,527 single-cell events, with a throughput of 21−36 cells/min and 15,000−26,000 cells per experiment, providing a stable single-cell data stream for dynamic metabolic analysis. This dataset revealed a temporal order of oxidative-stress metabolism in living microalgal cells, where early chlorophyll-associated pigment-state changes preceded pheophytin/pheophorbide-associated transitions and staged lipid-composition remodeling. Collectively, this work establishes continuous CyESI-based single-cell metabolomics as a robust platform for the dynamic monitoring of metabolic processes and cellular heterogeneity.