Quantitative confocal microscopy for grouping of dose–response data:
Deciphering calcium sequestration and subsequent cell death in presence
of excess norepinephrine
Abstract
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.