Development of a single-cell ICP-MS method for element analysis in Pisum sativum leaf protoplasts and chloroplasts

Heterogeneity of cellular and subcellular elemental distribution is poorly captured by conventional techniques due to limited sensitivity, throughput, and resolution. Single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) enables quantitative single-cell element analysis but remains challenging in multicellular plants because of plant cell complexity and lack of protocols. Herein, this study established a robust SC-ICP-MS method for analyzing elemental heterogeneity in Pisum sativum leaf protoplasts and chloroplasts. An optimized fixation protocol (1% (v/v) glutaraldehyde for 15 min for protoplasts; 2.5% (v/v) for 30 min for chloroplasts) was applied to preserve structural integrity, with endogenous P and Mg identified as specific indicator elements for protoplasts and chloroplasts, respectively. To reduce interference from broken cells, a broken-signal correction method was employed during data processing. Following isolation, purification, and glutaraldehyde fixation, Pisum sativum leaf protoplasts and chloroplasts were subjected to SC-ICP-MS analysis. Quantitative analysis revealed significant elemental heterogeneity, with P as the most abundant in protoplasts (67.1-135 fg cell^-1) and Mg as the most abundant in chloroplasts (33.6-41.5 fg cell^-1). This technique advances single-cell element analysis in plants, enabling new insights into nutrient distribution, metal accumulation, and cellular responses to environmental stress beyond conventional techniques.