Single-Cell ICP-MS: A Review of Analytical Bias Across the Workflow

Single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) has become an important tool for measuring elemental content in individual cells and revealing heterogeneity that is lost in bulk analysis. However, the reliability of these measurements is not determined by instrumental sensitivity alone. In practice, each stage of the workflow, from sample preparation and aerosol generation to transport efficiency, transient signal acquisition, and event discrimination, can alter the number of detected events and the calculated elemental mass per cell. This review discusses SC-ICP-MS from this workflow-centered perspective, focusing on the analytical bias introduced during sample handling, cell transport, instrumental acquisition, and data treatment. Attention is given to microflow introduction systems, collision/reaction cell strategies, ICP-MS/MS, ICP-TOF-MS, dwell-time selection, and Gaussian- and Poisson-based approaches for threshold definition. We also discuss how these factors affect the interpretation of endogenous elements, exogenous tracers, metal-labelled biomolecules, and particulate species in biological systems. Rather than treating single-cell distributions as direct representations of intracellular composition, we argue that the analytical workflow used to generate them should be considered when interpreting the data. This perspective is essential for improving comparability between studies and for moving SC-ICP-MS towards more reliable functional single-cell metallomics.