Discrimination of Microalgal Cellular Metabolic Heterogeneity and

Microalgae play a crucial role in geological processes on Earth, including carbon and nitrogen cycling and oxygen production. Microalgae are mostly unicellular organisms that exhibit considerable phenotypic diversity in response to environmental changes. Emerging methods for single-cell metabolite analysis offer new avenues for exploring microalgae-environmental interactions. Among these, label-free mass cytometry (CyESI-MS) has enabled high-throughput metabolite detection in both animal and microalgal cells. However, sensitivity challenges persist. In this paper, a novel MicroCyESI-MS system that replaces the pneumatic electrospray system of CyESI with a microspray device is proposed. This replacement resulted in a 10-fold increase in sensitivity and significantly reduced sample consumption while simplifying the apparatus. In addition, this advancement allowed for the detection of hundreds of metabolites from individual Chlamydomonas reinhardtii cells (7-10 μm in diameter) as well as Microcystis aeruginosa (1-3 μm in diameter). The strong light-induced differentiation of subtypes in Haematococcus pluvialis was also revealed by this technique. Moreover, through the integration of a 13C stable isotope probe, MicroCyESI-MS demonstrated the capacity to detect 13C metabolic flux in single microalgal cells, highlighting its potential for investigating microbial carbon sequestration mechanisms.