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

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.

Injectable Fluorescent Neural Interfaces for Cell-Specific Stimulating and Imaging

“…Simultaneously, mesoscale calcium imaging offers a noninvasive approach suitable for nonsurgical GCaMP imaging, capable of capturing large neuronal populations with a penetration depth of several hundred micrometers , (Figure E), aligning well with the proximity of injectable mesh electronics to neuronal populations. High-speed two-dimensional light-sheet fluorescence microscopy (LSFM) stands out for its ability to capture dynamic cellular events (Figure F) at ultrafast rates of up to 23,700 frames per minute (fpm). , This efficiency is achieved with a focused light sheet that illuminates the specimen perpendicular to the detection axis, reducing the overall exposure to excitation light. This selective illumination ensures that only the plane of interest within the specimen is exposed to the light sheet, reducing photobleaching and phototoxic effects that are common in conventional wide-field and confocal microscopy.…”

“…Highspeed two-dimensional light-sheet fluorescence microscopy (LSFM) stands out for its ability to capture dynamic cellular events (Figure 4F) at ultrafast rates of up to 23,700 frames per minute (fpm). 52,53 This efficiency is achieved with a focused light sheet that illuminates the specimen perpendicular to the detection axis, reducing the overall exposure to excitation light. This selective illumination ensures that only the plane of interest within the specimen is exposed to the light sheet, reducing photobleaching and phototoxic effects that are common in conventional wide-field and confocal microscopy.…”

Remote focusing for 2-photon microscopy to follow action potential propagation transmurally in acute rabbit cardiac slices

“…In [12], sarcomere length of cardiomyocytes was measured at 2.7 kHz rate scanning with RF; while it was done in an intact, Langendorff-perfused mouse heart, only cardiac structure, not electrical function was investigated. Electrophysiology of isolated cardiomyocytes and small cardiac preparations were probed with a bi-modal single-photon light sheet microscope (LSM) system [16][17][18]. While RF increased the throughput of the system, the geometry of oblique LSM does not accommodate larger preparations.…”

Single-sample image-fusion upsampling of fluorescence lifetime images

“…Scanning systems allow confocal or two-photon microscopy setups, giving excellent image resolution and aligning well with TCSPC methods that give rich fluorescence information. However, scanning also presents drawbacks, such as the lack of instantaneous complete field-of-view (FOV) information and long acquisition times that are incompatible with the rapid intracellular dynamics of living cells ( 25 , 26 ). Wide-field systems overcome these challenges by measuring temporal decay from the full FOV in parallel, often using time-gated cameras such as intensified charge-coupled devices ( 27 , 28 ), externally gated devices ( 29 , 30 ), or single-photon avalanche diode (SPAD) arrays ( 23 , 31 ).…”