Quadscan sted microscope

The images shown in Fig. 4 were taken on an Abberior Instruments QuadSCAN STED microscope, equipped with a 775 nm STED laser, a 640 nm excitation line and avalanche photodiode (APD) gated detection.
The images shown in Supplementary Fig. 3 were taken on Leica SP8 gSTED × 3 microscope equipped with HC PL APO CS2 × 100/1.40 oil objective and 775 nm STED laser. Excitation white light laser was set to 633 nm and signal was detected by HyD detector set to 650–700 nm interval with 0.3–6.0 ns time gating. Pinhole was set to 1 a.u., images were acquired with three times line averaging and pixel size in xy plane was 20 × 20 nm.

The experiments were performed using a Leica TCS SP5 STED microscope (Leica Microsystems, Mannheim, Germany), through a 100 × , 1.4 NA PL APO CS oil objective (Leica Microsystems), as described^{48 (link)}; or using a modified Quad Scan STED Microscope (Abberior) with a 100 × , 1.4 NA UPlanSApo oil objective (Olympus). Actin was labeled using ATTO647N phalloidin (Sigma Aldrich), using a previously published protocol^{49 (link)}. ATTO647N was excited using a pulsed diode laser at 635 nm (Leica Microsystems), and depleted using a 750 nm wavelength, provided by a Spectra-Physics Mai Tai tunable laser (pulsed at 80 MHz; Titanium Sapphire, Newport Spectra-Physics, Irvine, CA). For all stainings 3 independent experiments were analyzed and the total number of cells are presented in Supplementary Table S2.