Live imaging based testing of ligands and identification of toxic dose range during in vitro studies is a time-intensive process due to the inherent heterogeneity present in cell responses. In this context, high magnification imaging and large-scale data visualization remains challenging for analysis of toxicity during the drug screening as well as selection of dose-range. To address this challenge, we propose the measurement of cytosolic calcium ion (Ca2+) using spinning disk confocal microscopy at a higher resolution for generation of imaging data that can be visualized using uniform manifold and projection (UMAP). First, we performed large scale experiments and showed norepinephrine induced increase in Ca2+ flux in HeLa cells for a large range of doses. Secondly, the time-series dataset was mapped in 2D plane using UMAP. We also show that the proposed framework can be used to depict the relative distribution of various responses corresponding to a range of drug doses. To the best of our knowledge, this is the first attempt for UMAP visualization of time-series dose-response and identification of Ca2+ signature in the toxic dose-range. Such quantitative microscopy can be used for prediction of toxic drug dose range, and identification of drugs that lead to lytic cell death.

Abstract Generally, investigations on nanomedicine involve conventional imaging techniques for obtaining static images on nanoparticle internalization at a single time point where various phases can be overlooked. In contrast, 3D live-cell imaging can be used for obtaining cellular retention of drugs at various phases, and cells can be followed for days. This article demonstrates the application of time-lapse microscopy in the investigation of Poly-L-lysine coated ZnO nanoparticle dynamics. In this work, a laser scanning confocal microscope has been employed to quantify the dynamics of internalization particles and reactive oxygen species generation (ROS) using volumetric imaging. Firstly, we show that simultaneous spatial mapping of nanoparticle uptake in MCF-7 cells and ROS in a single cell can be used to identify the interdependence between the accumulation of particles and ROS generation. Secondly, monitoring of ROS formation and cytotoxicity using the same imaging platform offers an advantage over monitoring these parameters using various instruments. Finally, the ability of the fluorescent particles in inducing a significant reduction in cell viability suggests its potential to be used as a therapeutic agent. The proposed framework opens up a new avenue of research for investigating mechanistic aspects of ZnO particle adsorption in vitro through long term imaging. Keywords: Fluorescent ZnO particle, Time-lapse microscopy, 3D Live-cell imaging, laser scanning confocal microscope, Reactive oxygen species