Quantitative Confocal Microscopy for Grouping of Dose-Response Data: Deciphering Calcium Sequestration and Subsequent...

Fluorescent calcium (Ca²⁺) imaging is one of the preferred methods to record cellular activity during in vitro preclinical studies, high-content drug screening, and toxicity analysis. Visualization and analysis for dose-response data obtained using high-resolution imaging remain challenging, due to the inherent heterogeneity present in the Ca²⁺ spiking. To address this challenge, we propose measurement of cytosolic Ca²⁺ ions using spinning-disk confocal microscopy and machine learning-based analytics that is scalable. First, we implemented uniform manifold approximation and projection (UMAP) for visualizing the multivariate time-series dataset in the two-dimensional (2D) plane using Python. The dataset was obtained through live imaging experiments with norepinephrine-induced Ca²⁺ oscillation in HeLa cells for a large range of doses. Second, we demonstrate that the proposed framework can be used to depict the grouping of the spiking pattern for lower and higher drug doses. To the best of our knowledge, this is the first attempt at UMAP visualization of the time-series dose response and identification of the Ca²⁺ signature during lytic death. Such quantitative microscopy can be used as a component of a high-throughput data analysis workflow for toxicity analysis.