AFM-optimized single-cell level LA-ICP-MS imaging for quantitative mapping of intracellular zinc concentration in immobilized human parietal cells using gelatin droplet-based calibration

Background: Quantitative bioimaging of trace elements at the single-cell level is crucial for understanding cellular processes, including metal uptake and distribution. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has emerged as a gold standard for elemental bioimaging due to its high sensitivity and spatial resolution. However, calibration remains challenging due to the lack of homogeneous biological standards. This study addresses these challenges by introducing a gelatin-based calibration strategy optimized for Zn mapping in human parietal cells. By minimizing heterogeneity in gelatin standards and optimizing laser ablation conditions, the approach ensures accurate and reproducible results for cellular bioimaging. Results: A gelatin-based calibration strategy for LA-ICP-MS was developed to quantify intracellular Zn at a single-cell level in human parietal cells. Preparation conditions for gelatin standards were optimized to minimize heterogeneity, eliminating the need for entire droplet ablation and significantly reducing analysis time. Atomic force microscopy (AFM) was employed to optimize laser ablation conditions and determine ablated volumes, ensuring quantitative Zn detection. The method demonstrated high linearity (R² > 0.99) and reproducibility. Application of the calibration strategy to ZnCl₂-treated parietal cells revealed Zn distribution at a cellular level, visualized using a 5 μm laser beam. Integration with bright field imaging enabled the exclusion of apoptotic cells and debris, ensuring robust analysis. Validation with bulk ICP-MS showed excellent agreement, confirming the method's reliability and potential for high-resolution bioimaging. Significance: This work introduces a robust and reproducible calibration strategy for quantitative elemental bioimaging using LA-ICP-MS. It details the preparation of a gelatin matrix with a homogeneous element distribution, serving as an alternative to using biological material and significantly reducing analysis time. Laser ablation parameters were optimized using AFM to ensure quantitative ablation, which is necessary for calibration through LA-ICP-MS imaging. This approach provides a powerful tool for studying trace element dynamics in single cells and holds potential for diverse biological and biomedical applications.