Quantitative Confocal Microscopy for Grouping of Dose–Response Data: Deciphering Calcium Sequestration and Subsequent Cell Death in the Presence of Excess Norepinephrine

Manohar, Kuruba and Gare, Suman and Chel, Soumita and Dhyani, Vaibhav and Giri, Lopamudra (2021) Quantitative Confocal Microscopy for Grouping of Dose–Response Data: Deciphering Calcium Sequestration and Subsequent Cell Death in the Presence of Excess Norepinephrine. SLAS Technology, 26 (5). pp. 454-467. ISSN 2472-6303

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Abstract

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. © Society for Laboratory Automation and Screening 2021.

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IITH Creators:

IITH Creators	ORCiD
Giri, Lopamudra	http://orcid.org/0000-0002-2352-7919

Item Type:

Article

Additional Information:

The authors thank Dr. Narasimhan Gautam for providing the research facilities at Washington University in St. Louis, and we also thank the valuable contributions of Vani Kalyanraman in setting the initial stages of the project on live cell imaging. We acknowledge Dr. Soumya Jana for his valuable contribution in framing the computational methods. The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded in part by BT/PR22239/NNT/28/1269/2017. This work was also funded by BT/PR21261/MED/31/348/2016. We also thank Department of Science and Technology for providing the Fellowship granted to Kuruba Manohar.

Uncontrolled Keywords:

calcium imaging; cell lysis; confocal microscopy; fuzzy c-means clustering; GPCR-targeting drug

Subjects:

Chemical Engineering

Divisions:

Department of Chemical Engineering

Depositing User:

. LibTrainee 2021

Date Deposited:

21 Sep 2022 13:09

Last Modified:

21 Sep 2022 13:09