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. Author manuscript; available in PMC: 2021 Apr 1.
Published in final edited form as: Nat Methods. 2020 Sep 14;17(10):1018–1024. doi: 10.1038/s41592-020-0929-2

Extended Data Figure 1: Strain-free (sf)-tPAINT probe modeling and testing shows significant advantages over conventional hairpin probes (strained-tPAINT probes).

Extended Data Figure 1:

(a) Schematic of binding of imagers to strain-free (sf-tPAINT) and conventional/strained probes. (b) Total energy and change in energy as a function of applied force as predicted by the kinetic model described in Supplementary Note 2. (c) Docking site occupancy as a function of applied F for strained (red), and sf-tPAINT probes (green) at 10 nM imager and (d) for varying imager concentration. Force impedes imager binding to strained but not sf-tPAINT probes. (e-h) Experimental validation that sf-tPAINT probes outperform strained probes. (e) Strain-free and (f) conventional (strained) probes tagged with Cy3B-BHQ2 report pN tension as fluorescence. Images representative of n = 6 (e) and n = 3 (f). (g) Strain-free and (h) conventional/strained probes were incubated with 10 nM Cy3B-imager and imaged in TIRF excitation. These images were reconstructed from 5000 frames of single molecule localizations. Data shown in g and h are from paired experiments using different fluidic channels on the same glass coverslip and using the same platelet donor. The results shown are representative of n = 3 experiments. (i-k) Quantification of single molecule localizations (μm−2 s−1) for strained and sf-tPAINT probes. Results are representative of n = 4 (11 images) independent experiments for strained tPAINT surfaces and n = 6 independent sf-tPAINT surfaces (8 images). Data in k is displayed as mean with 95% CI. Cell (strained tPAINT): Mean= 0.29, 95% CI 0.12–0.46; Background (strained tPAINT): Mean= 0.06, 95% CI 0.04–0.07; Cell (sf-tPAINT): Mean= 1.09, 95% CI 0.89–1.29; Background (sf-tPAINT): Mean= 0.22, 95% CI 0.16–0.27. To determine the statistical significance, student t-test was applied to the data and the exact p-values are calculated (2 tailed, 2 sample). Cell (strained tPAINT) vs Background (strained tPAINT): 1.5×10−2, Cell (sf-tPAINT) vs Background (sf-tPAINT): 7.2×10−7, Cell (strained tPAINT) vs Cell (sf-tPAINT): 2.3×10−6, Background (strained tPAINT) vs Background (sf-tPAINT): 8.4×10−5.