Fig. 2

Visualization of DAT by STORM in CAD cells. TIRF-M (a, c, f, h), STORM (b, d, g, i, m, o), and widefield (l, n) images of CAD cells transiently expressing DAT with or without cholesterol depletion with 5 mM mβCD. DAT was visualized by immunolabeling with primary DAT antibody and Alexa405-Alexa647 conjugated-anti-rat secondary antibody. a, f TIRF-M view of DAT with (mβCD) or without cholesterol depletion (control). b, g Reconstructed STORM image of TIRF-M image in a or f. c, d, h, i Enlarged TIRF-M or STORM images corresponding to the boxed regions in a, b (control) or f, g (mβCD). e, j DBSCAN-based cluster map of the STORM images in d (control) and j (mβCD). Clustered localizations are shown by color-coding with non-clustered localizations in gray. k Fraction of particles (according to DBSCAN) clustered in nanodomains with (mβCD) or without cholesterol depletion (control). Data are % of localizations in cluster (means ± s.e.m., *p < 0.05, unpaired two-tailed t-test). l–o Visualization of DAT expressing CAD cells using inclined illumination to visualize a cross section through the cell. l Widefield image, m reconstructed STORM image of widefield image in l, n, o Enlarged TIRF-M or STORM images corresponding to the boxed regions in l, m. Red arrows mark DAT clustered in nanodomains. Data are based on 14–16 cells from three independent experiments. Scale bars a, b, f, g, l, m 5 μm, c–e, h–j 500 nm, n, o 400 nm