Line scans were acquired on a confocal laser scanning microscope, Leica SP8, with a white-light laser at an acquisition speed of 1,800 Hz with a line size of 256 pixels. All measurements were conducted with an HC PLAP CS2 40× 1.3 numerical aperture (NA) oil immersion objective (Leica). The pixel size was 50 nm, and the number of acquired lines was between 3 × 105 and 6 × 105. The infrared laser-based autofocus of the microscope (Adaptive Focus Control; Leica) was enabled during the acquisition to stabilize the focal position, as described in ref. 40. The ligand was excited at 633 nm with a laser power of 1 or 5%, which corresponded to 0.5 or 3 µW at the sample, respectively. To determine the laser power, a PM100A power meter (Thorlabs) with an S120VC (Thorlabs) photodiode power sensor head was used. Lines were placed either along the PM over the full length or crossing the TTs. Here, the confocal beam is repeatedly scanned at high speed over the same portion of the sample to extract diffusion data from the raw line scans by calculating the autocorrelation function.

Images of cells were acquired on the Leica SP8 with the same objective using either 633 nm (going up to 10% laser power, which corresponds to 6 µW) or 405 nm. Emission was detected on hybrid detectors in photon-counting mode detecting in the range of either 460 to 540 nm (CFP) or 650 to 750 nm (JE1319). Beam waists were determined either by extraction from mean square displacement curves (along the PM) or based on the observation of the profiles of fluorescent microspheres (Tetraspeck; Thermofisher Scientific) as in ref. 40, resulting in a lateral waist of ω0(633 nm) = 0.33 µm and an axial waist of ωz(633 nm) = 1.12 µm (only for β2-AR in wt cells PM and TT).

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