STED microscopy was performed on STAR-635–labeled F-actin and STAR-580–labeled myosin-II on an Abberior Expert Line 775 nm STED system. A pulsed 775-nm depletion laser and two pulsed excitation lasers (561 and 640 nm) were used to image myosin and actin, respectively. Spatial light modulator technology on the system allowed us to tune the STED beam between lateral and axial directions to improve xy and z resolutions. We characterized our fluorophores by measuring their full width at half maximum at different 775-nm power densities. We could achieve a resolution as good as ≈30 nm in xy and 60 nm in z. Actomyosin asters were imaged at 50% 3D settings, with a measured resolution of 70 and 100 nm in xy and 120 and 150 nm in z for actin-635 and myosin-II-580, respectively (fig. S5). Signal was collected with a 100 × 1.4 NA Olympus objective with an avalanche photodiode (APD) by Excelitas Technologies. Images were analyzed using ImageJ.

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