Fret ab wizard

Prepared samples were placed into an Attofluor™ Cell Chamber (Fisher Scientific GmbH, Schwerte, Germany). Coverslips were washed once with HBSS (Hank’s Balanced Salt Solution) and the chamber was filled with 500 mL HBSS. The chamber was mounted onto a Leica SP8 confocal laser-scanning microscope. Cells were imaged using the Leica FRET-AB wizard with a HC PL APO CS2 40x/1.3 numerical aperture oil immersion objective. A 1.5 mW white light laser was set to 85% and a 560 nm laser line was used at 5% power for the donor imaging. For the acceptor imaging a 652 nm laser line at 2% power was used and for the bleaching step increased to 50% over 10 frames. 512 × 512 pixel images of the bottom cell-membrane expressing SNAP-tagged receptor-constructs were acquired with a hybrid detector in standard mode. Emission of donor channel was recorded within 575–640 nm and acceptor channel was acquired between 658 nm–776 nm. The zoom factor was set to 5.5 x resulting in a pixel-size of 0.103 µm and the laser scanning speed was set to 400 Hz. There were at maximum 2 cells taken for analysis per image. FRET efficiencies were calculated with the manufacturer’s Wizard tool based on the provided formula (see Supplementary Fig. 10). Potential vesicles close to the cell surface were excluded by the drawing of the region of interest.