Accurate Quantification of Metals in Individual <i>Synechocystis</i> sp. PCC 6803 Cells by Single-Cell ICP-MS: Dual-Calibration and Sample Stabilization Strategies

Single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) is an emerging technique to investigate metal heterogeneity in individual cells. However, due to the absence of consistent calibration and suitable stabilization strategy for cells, accurate quantification of cellular heterogeneity and the content of metals remains a challenge. Herein, an accurate quantification method for the content and heterogeneous distribution of metals among individual microalgae cells was developed based on SC-ICP-MS using dual-calibration strategies and robust pretreatment methods. Gold nanoparticles (AuNPs) were used as calibration for measuring metal contents in single cells, but it would lead to a 13.6-63.1% underestimation of cell numbers due to inaccurate detection of cells' transport efficiency. To avoid this inaccuracy, we proposed an additional calibration strategy to measure cellular transport efficiency and cell numbers using endogenous Mg, enabling a more accurate assessment of cell heterogeneity. Then, an effective pretreatment method was optimized through fixation of cells with glutaraldehyde for 1 h to maintain the cellular stability and obtain accurate results, with satisfactory recoveries for cell number (98.4%) and Mg contents (91.7%), even after long-time storage. After optimization, the proposed method showed high sensitivity and repeatability in both cellular metal contents (Mg, Hg, Cd, and Co) and cell number, with detection limits (LODs) to be 0.14-0.53 fg/cell and 5.5 × 10³ cells/mL, respectively. Finally, the proposed method was successfully used for detecting various metals and their heterogeneity in <i>Synechocystis</i> sp. PCC 6803 cells provided an accurate and robust tool for investigating the uptake and heterogeneous distribution of metals in microalgae.