Abstract
Oblique plane microscopy (OPM) is a form of light sheet microscopy that uses a single high numerical aperture microscope objective for both fluorescence excitation and collection. In this paper, measurements of the relative collection efficiency of OPM are presented. An OPM system incorporating two sCMOS cameras is then introduced that enables single isolated cardiac myocytes to be studied continuously for 22 seconds in two dimensions at 667 frames per second with 960 × 200 pixels and for 30 seconds with 960 × 200 × 20 voxels at 25 volumes per second. In both cases OPM is able to record in two spectral channels, enabling intracellular calcium to be studied via the probe Fluo‐4 AM simultaneously with the sarcolemma and transverse tubule network via the membrane dye Cellmask Orange. The OPM system was then applied to determine the spatial origin of spontaneous calcium waves for the first time and to measure the cell transverse tubule structure at their point of origin. Further results are presented to demonstrate that the OPM system can also be used to study calcium spark parameters depending on their relationship to the transverse tubule structure. figureWiley-VCH Verlag & Co.KGaA
Highlights
Calcium (Ca2+) dynamics within cardiac myocytes include Ca2+ sparks, spontaneous Ca2+ waves and stimulated global Ca2+ transients
The 3-D volume was acquired by scanning the piezo-electric actuator controlling O2 over a range of 100 μm and 100 evenly spaced images were recorded. 17 individual beads were identified in the raw image data and the PSF full-width at half maximum (FWHM) in the plane of
We have previously demonstrated the potential of Oblique plane microscopy (OPM) to image sparks in isolated cardiomyocytes [17], but significant advances to the system including multiple excitation wavelengths, high resolution and high speed scientific complementary metal–oxide–semiconductor (sCMOS) cameras and addition of a superfusion system have enabled us to use the system to study the 3-D origin of spontaneous calcium waves in the context of the t-tubule network for first time
Summary
Calcium (Ca2+) dynamics within cardiac myocytes include Ca2+ sparks, spontaneous Ca2+ waves and stimulated global Ca2+ transients. We and others have previously reported that the organization of t-tubule networks becomes significantly deranged in HF in both failing human hearts and animal models, in part due to a reduction in t-tubule density (detubulation) [6,7,8,9,10]. Whether these structural changes and enhanced arrhythmogenesis via an increase in Ca2+ waves are linked is difficult to elucidate using current microscopy techniques
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