sSTED-FCS data of the lipid mobility inside and outside Gag assembly sites in Jurkat T cells were acquired at 37°C using fluorescent Gag signal as a guide. Fluorescent lipid signal intensity fluctuation carpets (scanning an orbit multiple times) were recorded using the Imspector software with the following parameters: orbital scan frequency, 3.23 kHz; scan orbit length, 1.5 μm; pixel dwell time, 10 μs; total measurement time, 10 s; pixel size, 50 nm per pixel, 10-μW excitation power (back aperture), 640 nm; and observation spot diameter, 100 nm (full width at half maximum) FWHM (as determined by SLB calibration measurements; section S1). The scan frequency selected for these experiments was set sufficiently high to enable the extraction of the diffusion dynamics of fluorescent lipid analogs in the cell plasma membrane (14).

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