Adaptive focus control

At day 0, HEK293T TRE3G-GFP reporter cells were plated at 1 × 10⁵ cells per 24-well well (μ-Plate 24 well; ibidi). At day 1, 250 ng of each plasmid was transfected (see Fig. 2a and Supplementary Fig. 4). At day 2, 20 μM of CNO was added to appropriate wells and immediately imaged. Time-lapse microscopy was performed on a Leica DMi8 inverted microscope equipped with, Lumencor SOLA SMII 405, Leica DFC9000 GT camera and Oko-Lab cage incubation system at 37 ^oC with 5% CO₂. Leica Application Software was used to set up time-lapse imaging. Images from phase contrast, mCherry (filter cube TXR, No. 11525310), and GFP (filter cube GFP, Cat. No. 11525314) channels were taken every 0.5 h for 48 h with a 20x/0.40NA corr PH1 objective using Leica Adaptive Focus control. Image processing was performed in Fiji (ImageJ).

In order to extract the behavior of the diffusing species, linescan-FCS (Fluorescence Correlation Spectroscopy) was performed, in which a confocal beam is repeatedly scanned at high speed over the same portion of the sample. The resultant recorded kymographs or linescans are further analyzed by a custom-implemented code in MATLAB to extract information about the diffusion data of receptors from autocorrelation functions (as previously described in [15 (link)]).
In short, linescans were acquired on a commercial confocal laser scanning microscope, Leica SP8 WLL, with a white light laser (WLL). Via the HC PLAP CS2 40x 1.3 NA oil immersion objective (Leica) a pixel size of 50 nm was used to acquire about 6 × 10⁵ lines of 256 pixels each at a speed of 1800 Hz. In order to stabilize the focal position, the IR laser-based autofocus of the microscope (Leica, Adaptive Focus Control) was enabled, while the ligand JE1319 (Alexafluor 647-based) was excited at a wavelength of 633 nm with 5% or 50% laser power, upon which calibration corresponded to total power outputs in the µW-range. Hybrid detectors (HyD) in photon counting mode were applied to detect emissions in the range of 650–751 nm. The beam waist was extracted by the observation of the profiles of fluorescent microspheres (Tetraspeck, Thermo Fisher Scientific), as in [15 (link)], resulting in a lateral waist of ω₀^{(633 nm)}= 0.33 µm.