Quantitative super-resolution imaging with qPAINT

Abstract

Counting molecules in complexes is challenging, even with super-resolution microscopy. Here, we use the programmable and specific binding of dye-labeled DNA probes to count integer numbers of targets. This method, called quantitative points accumulation in nanoscale topography (qPAINT), works independently of dye photophysics for robust counting with high precision and accuracy over a wide dynamic range. qPAINT was benchmarked on DNA nanostructures and demonstrated for cellular applications by quantifying proteins in situ and the number of single-molecule FISH probes bound to an mRNA target.

Figure 1. qPAINT principle

(a) In DNA-PAINT, fluorescently labeled “imager” strands (P*) transiently bind from solution to complementary “docking” strands (P) attached to a target. Intensity vs. time traces show characteristic fluorescence on- and off-times (τ_b and τ_d, respectively). qPAINT uses the predictable blinking kinetics to deduct molecule numbers. (b) The number of binding sites can be calculated given a known probe influx rate ξ = k_on · c_i. Stochastic simulations of DNA-PAINT binding events show a linear relationship between simulated and “measured” numbers of binding sites (mean ± s.d.). The counting precision for a given number of sites is dependent on the probe influx rate ξ (green: 0.01 s⁻¹, orange: 0.005 s⁻¹).

Figure 2. qPAINT in vitro benchmarking

(a) DNA origami structure with 12 designed docking sites. (b) DNA-PAINT image of the structures. (c) Visual counting (x-axis), in silico simulation (gray), and in vitro experimental qPAINT data (orange and green) are in good agreement (97 % accuracy and 90 % precision for ξ = 0.02 s⁻¹ and 25 min imaging time; 99.6 % accuracy and 95.4 % precision for ξ = 0.03 s⁻¹ and 166 min imaging time, error bars, 1 s.d.). (d) Distributions plotted from data in c (green data points) demonstrate qPAINT’s ability to distinguish between integer numbers of binding sites (i.e. 9 vs. 10 vs. 11 vs. 12; Post Tukey test: F(3,605)=1032.52, p<0.01, Supplementary Fig. 7). (e) Dynamic range. Same DNA-PAINT dataset as in e (orange data points) reanalyzed by grouping four DNA origami structures together. (f) Comparison between visual counting (x-asis) and in vitro (orange) qPAINT analysis shows good agreement (98 % accuracy, 85 % precision, error bars, 1 s.d.). (g) In silico analysis of the counting error (coefficient of variation, c_v) dependency on the number of binding sites and imager strand influx rate. Tuning ξ (experimentally adjustable over a wide range) can reach optimal conditions with low counting errors (<10 %) for virtually any number of bindings sites. (h) Multiplexed qPAINT. Three distinct DNA origami structures (similar to c) with orthogonal docking strand sequences (red P1, green P3 and blue P5, error bars, 1 s.d.) were imaged sequentially using Exchange-PAINT. qPAINT analysis on 11 binding sites structures (inset) demonstrates qPAINT’s ability to count multiple distinct target species with comparable performance. Scale bars: 100 nm (b and h) and 500 nm (e)

Figure 3. qPAINT in situ

(a–c) qPAINT in situ benchmarking with Nup98. (a) DNA-PAINT image of Nup98 proteins in NPCs. Inset: Labeling and imaging schematic for NPC proteins. Single targets (marked with arrows) are used for influx rate calibration. (b) NPC structures displaying three, four, five, and six distinct Nup98 protein clusters were respectively grouped for qPAINT analysis. (c) Comparison between visual counting (x-axis) and qPAINT data (orange) shows good agreement (95 % accuracy and 84 % precision, error bars, 1 s.d.). (d–e) Brp qPAINT experiments. (d) DNA-PAINT image of Drosophila NMJs obtained by using secondary DNA-antibody conjugates, and primary monoclonal antibodies against Brp^Nc82. (i: DNA-PAINT, ii: diffraction-limited) Zoomed-in view of the highlighted area in d showing two separate CAZ-units (assemblies of Brp proteins into multiprotein clusters). (e) qPAINT quantification (n = 981) indicating that the average number of Brp molecules per CAZ-unit is 142 ± 39. (f–g) smFISH qPAINT experiments. (f) SUZ12 mRNAs molecules are tagged using single-stranded oligonucleotides with binding sequences unique to a part of the target mRNA (r1* – r64*), a fixed Cy3b label, and a single-stranded DNA-PAINT docking strand (p1). (g) qPAINT quantification (n = 301) yields ~45 probes bound to a single mRNA molecule (~90 probes for two mRNAs), revealing ~70 % hybridization efficiency of the FISH probes to the mRNA target. Scale bars: 500 nm (a, insets i and ii in d), 50 nm (b), 1 μm (d, f)