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Series of three-dimensional structured illumination microscopy (3DSIM) of the rat cardiomyoblast cell-line H9c2 adapted to either glucose (GLU) or galactose (GAL). The outer mitochondrial membranes are labeled with the double tag mCherry-EGFP-OMP-25TM (dTag), i.e., both a red and green fluorescent protein variant. dTag can be used as a sensor for acidity (or lysosomal degradation), as the green fluorescence is lost sooner in an acidic environment than the red fluorescence. Where indicated, the samples are additionally labeled with LysoTracker Deep Red (LTDR). The aim of the study was to test the feasibility of multi-color 3DSIM for the examination of mitochondria-derived vesicles (MDVs) in both living and fixed cells.