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

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

Detection of Specific Templates in Calcium Spiking in HeLa Cells Using Hierarchical DBSCAN: Clustering and Visualization of CellDrug Interaction at Multiple Doses*

One of the major challenges in analyzing large scale intracellular calcium spiking data obtained through fluorescent imaging is to identify various patterns present in time series data. Such an analysis identifying the distinct frequency and amplitude encoding during cell-drug interaction study is expected to provide new insights into the drug action patterns over a time course. Here, we present the HDBSCAN clustering algorithm to find a clustering pattern present in calcium spiking obtained by confocal imaging of single cells. Our methodology uncovers the specific templates present in dynamic responses obtained through treatment with multiple doses of the drug. First, we attempt to visualize the clustering pattern present in time-series data corresponding to various doses of the drug. Secondly, we show that the HDBSCAN can be used for the detection of specific signatures corresponding to low and high cell density regions selected from in vitro experiments. To the best of our knowledge, this is the first attempt to optimize the clustering of calcium dynamics using HDBSCAN. Finally, we emphasize that HDBSCAN offers a high-level grasp on systems biology data, including complex spiking pattern and can be used as a visual analytic tool for drug dose selection

Confocal Imaging of Intercellular Calcium in HeLa Cells for Monitoring Drug-Response: Biophysical Framework for Visualization of the Time-Lapse Images

Recent advancements in biomedical imaging focus on fluorescent imaging using laser scanning confocal microscopy. However, high-resolution imaging of cellular activity remains considerably expensive for both in vitro and in vivo model. In this context, integration of mathematical modeling and imaging data analysis to predict the cellular activity may aid understanding of cell signaling. Here we performed dynamic imaging using confocal microscopy and propose a model considering cell to cell connectivity that can predict the effect of the drug on Ca2+ oscillations. The proposed model consists of a large number of ordinary differential (ODE) equations and uses the concept of adjoint matrix containing coupling factors to capture the activity of cells with the random arrangement. The results show that the cell-to-cell connection plays a crucial role in controlling the calcium oscillations through a diffusion-based mechanism. The present simulation tool can be used as a generalized framework to generate and visualize the time-lapse videos required for in vitro drug testing for various drug dose

Three‐dimensional imaging and quantification of real‐time cytosolic calcium oscillations in microglial cells cultured on electrospun matrices using laser scanning confocal microscopy

The development of a minimally invasive, robust, and inexpensive technique that permits real-time monitoring of cell responses on biomaterial scaffolds can improve the eventual outcomes of scaffold-based tissue engineering strategies. Towards establishing correlations between in situ biological activity and cell fate, we have developed a comprehensive workflow for real-time volumetric imaging of spatiotemporally varying cytosolic calcium oscillations in pure microglial cells cultured on electrospun meshes. Live HMC3 cells on randomly oriented electrospun fibers were stained with a fluorescent dye and imaged using a laser scanning confocal microscope. Resonance scanning provided high-resolution in obtaining the time-course of intracellular calcium levels without compromising spatial and temporal resolution. Three-dimensional reconstruction and depth-coding enabled the visualization of cell location and intracellular calcium levels as a function of sample thickness. Importantly, changes in cell morphology and in situ calcium spiking were quantified in response to a soluble biochemical cue and varying matrix architectures (i.e., randomly oriented and aligned fibers). Importantly, raster plots generated from spiking data revealed calcium signatures specific to culture conditions. In the future, our approach can be used to elucidate correlations between calcium signatures and cell phenotype/activation, and facilitate the rational design of scaffolds for biomedical applications

Three‐dimensional imaging and quantification of real‐time cytosolic calcium oscillations in microglial cells cultured on electrospun matrices using laser scanning confocal microscopy

The development of a minimally invasive, robust, and inexpensive technique that permits real-time monitoring of cell responses on biomaterial scaffolds can improve the eventual outcomes of scaffold-based tissue engineering strategies. Towards establishing correlations between in situ biological activity and cell fate, we have developed a comprehensive workflow for real-time volumetric imaging of spatiotemporally varying cytosolic calcium oscillations in pure microglial cells cultured on electrospun meshes. Live HMC3 cells on randomly oriented electrospun fibers were stained with a fluorescent dye and imaged using a laser scanning confocal microscope. Resonance scanning provided high-resolution in obtaining the time-course of intracellular calcium levels without compromising spatial and temporal resolution. Three-dimensional reconstruction and depth-coding enabled the visualization of cell location and intracellular calcium levels as a function of sample thickness. Importantly, changes in cell morphology and in situ calcium spiking were quantified in response to a soluble biochemical cue and varying matrix architectures (i.e., randomly oriented and aligned fibers). Importantly, raster plots generated from spiking data revealed calcium signatures specific to culture conditions. In the future, our approach can be used to elucidate correlations between calcium signatures and cell phenotype/activation, and facilitate the rational design of scaffolds for biomedical applications

GPCR mediated control of calcium dynamics: A systems perspective

G-protein coupled receptor (GPCR) mediated calcium (Ca2+)-signaling transduction remains crucial in designing drugs for various complex diseases including neurodegeneration, chronic heart failure as well as respiratory diseases. Although there are several reviews detailing various aspects of Ca2+-signaling such as the role of IP3 receptors and Ca2+-induced-Ca2+-release, none of them provide an integrated view of the mathematical descriptions of GPCR signal transduction and investigations on dose-response curves. This article is the first study in reviewing the network structures underlying GPCR signal transduction that control downstream [Cac2+]-oscillations. The central theme of this paper is to present the biochemical pathways, as well as molecular mechanisms underlying the GPCR-mediated Ca2+-dynamics in order to facilitate a better understanding of how agonist concentration is encoded in Ca2+-signals for Gαq, Gαs, and Gαi/o signaling pathways. Moreover, we present the GPCR targeting drugs that are relevant for treating cardiac, respiratory, and neuro-diseases. The current paper presents the ODE formulation for various models along with the detailed schematics of signaling networks. To provide a systems perspective, we present the network motifs that can provide readers an insight into the complex and intriguing science of agonist-mediated Ca2+-dynamics. One of the features of this review is to pinpoint the interplay between positive and negative feedback loops that are involved in controlling intracellular [Cac2+]-oscillations. Furthermore, we review several examples of dose-response curves obtained from [Cac2+]-spiking for various GPCR pathways. This paper is expected to be useful for pharmacologists and computational biologists for designing clinical applications of GPCR targeting drugs through modulation of Ca2+-dynamics

3D imaging and quantification of PLL coated fluorescent ZnO NP distribution and ROS accumulation using laser scanning confocal microscopy

Investigations on nanomedicine involve conventional two dimensional (2D) imaging techniques for observing the nanoparticle internalization at a single time point where various phases of internalization can be overlooked. In contrast, three dimensional (3D) imaging of fluorescent nanoparticles with anticancer potential can be used for obtaining the time course of cellular retention of particles, and cells can be followed for days. This article demonstrates the application of laser scanning confocal microscopy to quantify poly-l-lysine coated fluorescent ZnO nanoparticle retention and reactive oxygen species (ROS) generation using volumetric imaging. Synthesis of these particles allows monitoring of ROS formation, internalization, and cytotoxicity using the same imaging platform that offers an advantage over measurement using various instruments. PLL-coated ZnO particles' ability to induce a significant reduction in cell-viability suggests its potential as a therapeutic agent. The proposed framework opens up a new avenue for investigating mechanistic aspects of ZnO adsorption and the evaluation of therapeutic efficiency. © 2022 American Institute of Chemical Engineers

FDA approved L-type channel blocker Nifedipine reduces cell death in hypoxic A549 cells through modulation of mitochondrial calcium and superoxide generation

As hypoxia is a major driver for the pathophysiology of COVID-19, it is crucial to characterize the hypoxic response at the cellular and molecular levels. In order to augment drug repurposing with the identification of appropriate molecular targets, investigations on therapeutics preventing hypoxic cell damage is required. In this work, we propose a hypoxia model based on alveolar lung epithelial cells line using chemical inducer, CoCl2 that can be used for testing calcium channel blockers (CCBs). Since recent studies suggested that CCBs may reduce the infectivity of SARS-Cov-2, we specifically select FDA approved calcium channel blocker, nifedipine for the study. First, we examined hypoxia-induced cell morphology and found a significant increase in cytosolic calcium levels, mitochondrial calcium overload as well as ROS production in hypoxic A549 cells. Secondly, we demonstrate the protective behaviour of nifedipine for cells that are already subjected to hypoxia through measurement of cell viability as well as 4D imaging of cellular morphology and nuclear condensation. Thirdly, we show that the protective effect of nifedipine is achieved through the reduction of cytosolic calcium, mitochondrial calcium, and ROS generation. Overall, we outline a framework for quantitative analysis of mitochondrial calcium and ROS using 3D imaging in laser scanning confocal microscopy and the open-source image analysis platform ImageJ. The proposed pipeline was used to visualize mitochondrial calcium and ROS level in individual cells that provide an understanding of molecular targets. Our findings suggest that the therapeutic value of nifedipine may potentially be evaluated in the context of COVID-19 therapeutic trials. © 202