Epifluorescence and confocal images were acquired using a Zeiss Axiovert 200 microscope and an Olympus FV-1000 confocal microscope, respectively. 3D reconstruction and analysis of confocal images were performed with IMARIS software (Bitplane). Muscle contraction measurement was performed in a stage-top incubator (INUBG2TF-WSKM-SET, Tokai Hit) on Axiovert 200, and image sequences were captured using AxioVision. Automated tracking of the local deformation of the skeletal muscle cells and deflection of the pillars upon light excitation were carried out using ImageJ and Python script with an OpenCV package (fig. S8, A and B). Formulas for calculating muscle contraction force from pillar displacements are provided in fig. S8. The presence of gel did not affect the calculation of muscle contraction force as shown previously (18). Image analysis regarding the muscle width and axon outgrowth was conducted using ImageJ. Fusion index for skeletal muscle cells is defined as the number of myogenin-expressing myotubes with greater than two nuclei divided by the total number of nuclei. To quantify nAChR clusters, quantitative fluorescence imaging was performed using an open-source software package in CellProfiler (Broad Institute). The automated image cytometry system identifies and measures objects’ size, shape, pixel intensity, and topology, and the resulting data are corrected using a background-subtraction algorithm. nAChR clusters in CellProfiler were defined using agrin-treated myotubes [Alexa 488–conjugated anti-agrin antibody (ab85174), Abcam]. Before the measurement, skeletal myotubes were prestained by Alexa 488–conjugated anti-agrin antibody in culture medium (1:100) for 1 hour at 37°C in the device. Agrin, normally secreted by MNs, stabilizes and aggregates nAChRs into distinguishable cluster regions (>65% intensity), whereas outside these regions, nAChRs largely exist in dispersed microclusters (<65% intensity) denoted by lower-intensity pixels. In this manner, nAChR area was calculated for individual muscle fiber bundles.

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