(A) Computer-generated depiction of the PMMA mold design. The top part containing eight pairs of vertical posts is shown in black. Eight holes positioned equidistant between each pair of posts allows for gas and media exchange. Two larger sized vertical posts at either end of the top part serve to fix the top and bottom portion (translucent white) together and ensure vertical posts are properly positioned. The bottom portion is affixed to a microscopy grade glass (B) Images of remodeled C2C12 muscle tissues at 7 days of differentiation anchored to the end of the posts captured to provide (left image) a view looking up into a well in which the top and bottom parts are fashioned together (left; ×4 objective) or (right image) from the side looking at the top part to visualize six pairs of posts with tissues at the bottom. (C) Schematic workflow used to raise 3D skeletal muscle tissues in vitro. (D–F) Representative confocal microscopy longitudinal (D; whole mount, flattened stack, ×40 water immersion objective, PMMA chamber, (E); whole mount, flattened stack, ×20 air objective, PDMS chamber) and transverse (F; cryosection, single snap, ×40 water immersion objective) images of multinucleated myotubes within a 14-day-old muscle tissue immunostained for sacomeric alpha-actinin (SAA, green) and counterstained with Hoechst 33342 to visualize nuclei (blue). (G) Timeseries of myoblast fusion within a 3D skeletal muscle tissue on day 4 of differentiation, demonstrating the possibility of high-resolution imaging during living tissue formation. Lifeact-GFP (green) and H2B-mCherry (red) was stably introduced into AB1167 cells. (H) Super-resolution dSTORM imaging of SAA-stained myofibrillar structures present inside a myotube, recorded with ×100 oil immersion objective.