Related Experiment Video
Updated: Aug 8, 2025
Measuring Fast Calcium Fluxes in Cardiomyocytes
Published on: November 29, 2011
High-speed 2D light-sheet fluorescence microscopy enables quantification of spatially varying calcium dynamics in
Liuba Dvinskikh1,2,3, Hugh Sparks1, Kenneth T MacLeod2
1Department of Physics, Imperial College London, London, United Kingdom.
Insights
Light-sheet microscopy reveals how t-tubule structure impacts cardiomyocyte calcium release. Findings show spatial variations in calcium dynamics, crucial for understanding cardiac function and arrhythmias.
Area of Science:
- Cardiovascular Physiology
- Cellular Biology
- Biophysics
Background:
- Disrupted calcium (Ca2+) release synchrony and t-tubule disorganization in cardiomyocytes impair contractile function and promote arrhythmias.
- Confocal microscopy, while common for Ca2+ dynamics, has limitations in speed and phototoxicity compared to advanced imaging techniques.
Purpose of the Study:
- To investigate the relationship between cardiomyocyte microstructure, specifically t-tubule organization, and intracellular Ca2+ dynamics using light-sheet fluorescence microscopy.
- To characterize Ca2+ spark morphology and map Ca2+ transient propagation in relation to cell structure with high spatiotemporal resolution.
Main Methods:
- Utilized a custom dual-channel light-sheet fluorescence microscope for 2D timelapse imaging of Ca2+ and sarcolemma in cardiomyocytes.
- Employed para-nitroblebbistatin to immobilize electrically stimulated, dual-labeled cardiomyocytes, enabling imaging at 395 fps with sub-micron resolution.
- Performed automated image analysis to quantify Ca2+ spark properties and map Ca2+ transient kinetics across the cell.
Main Results:
- Left ventricle myocytes exhibited Ca2+ sparks with greater amplitude compared to right ventricle myocytes.
- Calcium transients reached half-maximum amplitude approximately 2 ms faster in the cell center than at the cell ends.
- Ca2+ sparks co-localized with t-tubules displayed significantly longer duration, larger area, and greater mass than those not associated with t-tubules.
Conclusions:
- High-resolution light-sheet microscopy allows detailed 2D mapping and quantification of cardiomyocyte Ca2+ dynamics.
- Significant spatial variations in Ca2+ release characteristics exist across individual cardiomyocytes.
- The findings underscore the critical dependence of Ca2+ release synchrony and spark properties on the underlying t-tubule network structure.
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.
