Local mitochondrial networks in FDB fibers. (A) An isolated fiber (left) expressing mtDsRed (center). (B) Mitochondrial distribution. Specific subgroups of mitochondria are indicated. IMFM, intermyofibrillar mitochondria; N, nucleus; SSM, subsarcolemmal mitochondria; **, perinuclear mitochondria. (C) Image time series showing a representative FDB fiber before and after 2P photoactivation of mtPA-GFP (white dashed squares). Arrowheads show sites of diffusion, D, of mtPA-GFP into neighboring mitochondria. Line-scan images further illustrate transversal (c′) and longitudinal (c′) diffusion, D, of mtPA-GFP through interconnected mitochondria starting immediately after photoactivation and expanding continually, and a fusion event (F, orange arrowhead) appearing at a later time point as an abrupt expansion of the PA-GFP signal. (D) 3D reconstruction of a photoactivated area a few seconds (left) and 3 min after photoactivation (right) to illustrate matrix continuity and discrete mixing events (orange and white arrowheads), respectively. Notably, the discrete event could not be caused by mitochondrial movement because the mtPA-GFP and mtDsRed fluorescence changes had opposite directions (not depicted).