*4.2. Confocal Microscopy*

Myocytes were loaded with dyes for >20 min on a glass-bottom Petri dishes, incubated in HEPES-bu ffered solution (same composition as the storage solution) at 23 ◦C, and imaged with a LSM-510 inverted confocal microscope using a Plan-Neofluar 63 ×/1.25N.A. oil immersion lens (Carl Zeiss Inc., Jena, Germany). Scans were recorded in a single channel mode with excitation at 543 nm (for tetramethyl rhodamine methyl ester (TMRM; Molecular Probes, Inc., Eugene, OR, USA), nonyl acridine orange (NAO; Sigma-Aldrich, St. Louis, MI, USA), and mitotracker Deep Red (MTDR; ThermoFisher Scientific, Waldham, MA, USA)), collecting simultaneous fluorescence emission with LP560, LP 505, and LP650 nm, respectively. The confocal pinhole was set to obtain spatial resolutions of 0.4 μm in the horizontal plane and 1.0 μm in the axial dimension. Image processing was performed using Fiji software (U.S. National Institutes of Health, Bethesda, MD, USA). Frame scan along z-direction was performed to cover the entire space occupied with nuclei, which was identified as space of a spheroid shape (usually two per cell) poor in mitochondria. Time series mode along a single *<sup>x</sup>*–*y* plane was performed with 5 s intervals between scans.
