Fig. 3: Time-resolved photoswitching fingerprint analysis in cells.

a–c, Molecular structures of the pentameric GABA-A (PDB 6HUG) and tetrameric GluK2 receptor (PDB 5KUF) with incorporation sites of ncAAs shown as black circles (blue, γ2 subunit GABA-A (a); red, dimeric α2 GABA-A (b); orange, homotetrameric GluK2 (c)) and corresponding dSTORM images of HEK293T membrane sections showing fluorescence signals of individual receptors (5 nm pixel⁻¹). The ncAAs were labeled by click chemistry with Met-Tet-Cy5. In the GABA-A^S181TAG mutant the distance between the two fluorophores in the α2 subunits is roughly 5 nm. In the GluK2^S398TAG mutant the distance between the four Cy5 molecules is roughly 7 nm (refs. ^41,42). The samples were measured 3–5 times independently. Scale bars, 500 nm. d, Relative occurrence of lifetimes of the off-state (Off-time), and number of on-states (On-events) detected from individual receptors in dSTORM experiments (n = 3–5). e, Number of on-events (localizations) detected per frame as a function of time during 10 min dSTORM experiments of membrane receptors (n = 3–5). f, FLIM images of HEK293T cells expressing monomeric γ2 subunit of GABA-A (left, blue), dimeric α2 subunit of GABA-A (middle, red), and homotetrameric GluK2 receptors (right, orange) click-labeled with Met-Tet-Cy5 measured by confocal TCSPC imaging in photoswitching buffer at an irradiation intensity of 2.5 kW cm⁻². To minimize photobleaching of fluorophores FLIM images were recorded at 5 µs of integration time per pixel. No intensity threshold was applied. Scale bars, 2 µm. g, Average fluorescence decays from n = 8–13 FLIM images of HEK293T cells expressing receptors labeled with one, two and four Cy5 fluorophores.