Fig. 6: DNA-PAINT imaging of Drosophila retinal epithelium on an SDC-OPR microscope up to 9 µm penetration depth.
a Left: Schematic of 3rd instar Drosophila eye disc and sample mounting for SDC-OPR imaging, described in Methods. Right: Phalloidin-stained eye disc (top). The peripodial membrane (ppm), luminal space (*), and retinal epithelium (retina) are highlighted with green, magenta, and orange masks, respectively (bottom). b SPC-OPR 3D projection of E-cadherin-GFP with 0.5 μm z-step, showing 10 μm depth with color-coded axial position. c Representative SDC-OPR DNA-PAINT images of E-cadherin at different penetration depths (0, 4, 5, and 9 µm). E-cadherin-GFP was labeled with DNA-conjugated anti-GFP antibodies (see Methods). Panels include localization precision (σSMLM) for each penetration depth. d Zoomed-in view of the highlighted region from (c), showing density-based clustering analysis (DBSCAN) of the E-cadherin DNA-PAINT image. Histogram displays the maximum distances within clusters, derived from the sample shown in (c) for z = 0 µm and z = 5 µm, and the value of the smallest pick of the histogram (Min unit). e Collagen-IV imaging of the basement membrane showing three example cells outlined (full image can be found in Supplementary Fig. 14). σSMLM: 4.6 nm. Collagen-IV is labeled with GFP and DNA-labeled anti-GFP nanobody was used for DNA-PAINT imaging. Central panels i, ii and iii provide zoomed views of three examples vesicles. The histogram quantifies the number of Collagen IV molecules present in all analyzed vesicles, with qPAINT quantification indicating an average (µ) of 46 ± 27 molecules per vesicle. ξ is the qPAINT index used for calibration. See methods. Scale bar: 100 nm. All Drosophila imaging was repeated in three independent experiments. Source data of histograms are provided as a Source Data file. Schematic of panel (a) was created with BioRender.com.
