Fig. 1: Resolving the location of individual molecules in crowded subcellular structures.
a Schematic overview of quantitative single-molecule colocalization analysis (qSMCL) to evaluate nearest-neighbor distance (NND), molecular densities (mol/µm2), detection efficiencies (DEs), and labeling efficiencies (LEs). b Overlay of diffraction-limited (DL) and super-resolved (SR) DNA-PAINT image showing a talin-Halo447 expressing cell. Zoom into focal adhesion area reveals distinct talin localization clouds. Regions in the free membrane (MEM) are characterized by more disperse talin localization clouds. c The approach allows the separation of distinct talin-1 localization clouds at a distance of approximately 15 nm. d Histogram analysis demonstrates that the localization clouds shown in c can indeed be resolved (σPeak1 = 3.5 nm, σPeak2 = 5.6 nm); number of localizations (nlocs = 157). e Schematic illustration of a DNA origami calibration. f Transient binding events of dye-labeled imager strands to the complementary, immobilized docking strands, creating the characteristic “blinking” (ON/OFF) required for single-molecule localization microscopy. g DNA origami structures carrying single docking strands were placed next to talin-Halo447 cells to calculate the number of molecules per localization cloud. Inset: Zoom onto DNA origami structures. h Plotting the binding events over the number of recorded frames reveals similar binding traces in DNA origami and talin-Halo447 localization clouds. i Quantitative histogram analysis of absolute binding site numbers on DNA origami and talin localization clouds confirming that the observed talin localization clouds represent single talin-1 proteins (n = 8 cells). j Zoom-in of a 20 nm grid DNA origami displaying a single binding sequence (1xP3) per site and the corresponding binding event history. k Zoom-in of a DNA origami structure with three concatenated binding sequences (3xP3) per docking strand and the corresponding binding event history. l Analysis of the mean photon counts per individual localization event reveals highly similar values for single (j) and triple (k) binding sequences (n = 334 localization events). m qPAINT analysis confirmed either one or three binding sites per DNA origami localization cloud (n = 1). n Binding frequency of a single-labeled (due to <100% labeling efficiency) and a dual-labeled localization cloud. o qPAINT analysis reveals, as expected, that either one or two binding sites are detected (n = 1 cell). Scale bars: 7 µm (b), 370 nm (g), 110 nm (g inset), 70 nm (b insets), 50 nm (c, right), 30 nm (j, k), 9 nm (c, right). Source data are provided in the Source Data file.
