sSTED-FCS measurements were performed on the Abberior Instrument Expert Line STED super-resolution microscope (Abberior Instruments GmbH, Göttingen, Germany) using 485- and 640-nm pulsed excitation laser sources and a pulsed STED laser operating at 775 nm and an 80-MHz repetition rate. The fluorescence excitation and collection were performed using a 100×/1.40 numerical aperture (NA) UPlanSApo oil immersion objective (Olympus Industrial, Southend-on-Sea, UK). All acquisition operations were controlled by Imspector software (Abberior Instruments GmbH), and point FCS data for the calibration of the observation spot sizes were recorded using a hardware correlator (Flex02-08D, correlator.com, operated by the company’s software).

Confocal microscope FCS measurements were performed on a Zeiss LSM 780 (Zeiss, Iena, Germany) using a HeNe 633-nm laser as the excitation source. Fluorescence excitation and collection were performed using a 63×/1.40-NA PlanApo oil immersion objective (Zeiss). Acquisition operations were controlled by the Zeiss Zen software. Point FCS was used to calibrate the observation spot with the LSM 780 internal hardware correlator.

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