Spinning disk confocal and Lightning microscopy

Spinning disk confocal imaging was performed with a Quorum spinning disk confocal head on a Zeiss Axiovert 200M microscope, equipped with a 63× NA 1.4 oil objective and 25× NA 0.8 multi-immersion objective, a 1.5× tube lens, and a back-thinned EM-CCD camera (C9100–13, Hamamatsu). Acquisition settings were controlled using Volocity software and were acquired at equal laser and exposure settings within experiments across compared conditions. Lightning microscopy was performed on a Leica SP8 Lightning Confocal DMI6000 microscope utilizing a 100× 1.4 NA (O, STED) objective and two HyD detectors. Acquisitions were driven by a Leica motorized XY stage and Leica SuperZ Galvo with Adaptive Focus Control. Acquisition settings were controlled using the Leica LAS, Lightning Module software, with matched settings within experiments.