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High-speed 2D light-sheet fluorescence microscopy enables quantification of spatially varying calcium dynamics in ventricular cardiomyocytes

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Title: High-speed 2D light-sheet fluorescence microscopy enables quantification of spatially varying calcium dynamics in ventricular cardiomyocytes
Authors: Dvinskikh, L
Sparks, H
MacLeod, K
Dunsby, C
Item Type: Journal Article
Abstract: Introduction: Reduced synchrony of calcium release and t-tubule structure organization in individual cardiomyocytes has been linked to loss of contractile strength and arrhythmia. Compared to confocal scanning techniques widely used for imaging calcium dynamics in cardiac muscle cells, light-sheet fluorescence microscopy enables fast acquisition of a 2D plane in the sample with low phototoxicity. Methods: A custom light-sheet fluorescence microscope was used to achieve dual-channel 2D timelapse imaging of calcium and the sarcolemma, enabling calcium sparks and transients in left and right ventricle cardiomyocytes to be correlated with the cell microstructure. Imaging electrically stimulated dual-labelled cardiomyocytes immobilized with para-nitroblebbistatin, a non-phototoxic, low fluorescence contraction uncoupler, with sub-micron resolution at 395 fps over a 38 μm × 170 µm FOV allowed characterization of calcium spark morphology and 2D mapping of the calcium transient time-to-half-maximum across the cell. Results: Blinded analysis of the data revealed sparks with greater amplitude in left ventricle myocytes. The time for the calcium transient to reach half-maximum amplitude in the central part of the cell was found to be, on average, 2 ms shorter than at the cell ends. Sparks co-localized with t-tubules were found to have significantly longer duration, larger area and spark mass than those further away from t-tubules. Conclusion: The high spatiotemporal resolution of the microscope and automated image-analysis enabled detailed 2D mapping and quantification of calcium dynamics of n = 60 myocytes, with the findings demonstrating multi-level spatial variation of calcium dynamics across the cell, supporting the dependence of synchrony and characteristics of calcium release on the underlying t-tubule structure.
Issue Date: 14-Feb-2023
Date of Acceptance: 27-Jan-2023
URI: http://hdl.handle.net/10044/1/102844
DOI: 10.3389/fphys.2023.1079727
ISSN: 1664-042X
Publisher: Frontiers Media
Start Page: 1
End Page: 14
Journal / Book Title: Frontiers in Physiology
Volume: 14
Copyright Statement: Copyright © 2023 Dvinskikh, Sparks, MacLeod and Dunsby. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Publication Status: Published
Article Number: 1079727
Online Publication Date: 2023-02-14
Appears in Collections:Physics
National Heart and Lung Institute
Faculty of Natural Sciences

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