DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution microscopy method that can acquire high-fidelity images at nanometer resolution. It suffers, however, from high background and slow imaging speed, both of which can be attributed to the presence of unbound fluorophores in solution. Here we present two-color fluorogenic DNA-PAINT, which uses improved imager probe and docking strand designs to solve these problems. These self-quenching single-stranded DNA probes are conjugated with a fluorophore and quencher at the terminals, which permits an increase in fluorescence by up to 57-fold upon binding and unquenching. In addition, the engineering of base pair mismatches between the fluorogenic imager probes and docking strands allowed us to achieve both high fluorogenicity and the fast binding kinetics required for fast imaging. We demonstrate a 26-fold increase in imaging speed over regular DNA-PAINT and show that our new implementation enables three-dimensional super-resolution DNA-PAINT imaging without optical sectioning.
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The majority of datasets generated during and/or analyzed during the current study are available at the Zenodo repository (https://doi.org/10.5281/zenodo.6315337). Remaining raw datasets are available from the corresponding author on reasonable request.
PYME is available at https://python-microscopy.org/. The PYME modules that we have developed are shared at https://github.com/bewersdorflab. Codes for simulating multi-emitters (Supplementary Fig. 2) and for screening docking strands (Supplementary Note 3) are available at the GitHub repository (https://github.com/bewersdorflab/fluorogenic-dna-paint-manuscript-supplement).
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We thank L. Fuentes, K. Hu, Z. Marin and F. Schueder for helpful discussions. This work was primarily supported by a 4D Nucleome grant from the National Institutes of Health (NIH; U01 DA047734 to J.B. and D.B.) and the Wellcome Trust (203285/B/16/Z). J.B. acknowledges support from NIH grant P30 DK045735 (to R. Sherwin). C.L. acknowledges support from an NIH Director’s New Innovator award (GM114830), an NIH grant (GM132114) and Yale University faculty startup funding. N.D.W. was supported by an NIH training grant (T32 EB019941).
J.B. discloses financial interest in Bruker, Hamamatsu Photonics and panluminate. J.B. is co-inventor on a US patent (9,769,399) related to the 4Pi-SMS system and image analysis used in this work. Y.Z. and J.B. are co-inventors on a US patent (11,209,367) related to 4Pi-SMS microscopy. The remaining authors declare no competing interests.
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Nature Methods thanks Matthew Baker and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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List of imager probes and docking strands used in this study.
Fluorogenic DNA-PAINT uses imager probes and docking strands with internal mismatches. Complementary base pairings are colored in blue whereas mismatches in red.
The full dataset from which Fig. 4a-c were generated. (a) Fast 3D fluorogenic DNA-PAINT imaging of immunolabeled microtubules in COS-7 cells under widefield illumination at multiple time points. A reasonable image can be acquired in 30 s. (b) Bleaching is negligible, causing only a small reduction (30%) in blinking rate over an hour. (c) 3D resolution as quantified by Fourier shell correlation (FSC) improves with longer imaging duration as more blinking events are detected. The resolution reaches 34.3 nm after 1 hr. (d) The localization precision peaks at < 5 nm for all three dimensions (X: 1.7 nm, Y: 1.7 nm, Z: 4.5 nm). (e) Fitting an exponential decay function to blink durations (blinks that are only 1 frame in duration were ignored for fitting) estimates the mean off-rate at 46.7 s-1.
Extended Data Fig. 4 Time series of fast 2-color fluorogenic DNA-PAINT imaging without optical sectioning.
The full dataset from which Fig. 5b-f were generated, rendered at various timepoints. (a-d) Fast 2-color fluorogenic DNA-PAINT imaging of immunolabeled endoplasmic reticulum (ii; [imager probe A] = 10 nM) and mitochondria (iii; [imager probe B] = 1 nM) in U-2 OS cells under widefield illumination (100 Hz frame rate). There is no well-defined minimum imaging time as it depends on a multitude of factors including the biological question being addressed. The timepoint we reported in the main text (600 s) is more densely sampled than typical single-molecule localization microscopy images. Negligible changes are observed with prolonged imaging (d; 1,200 s) which would suggest oversampling.
Extended Data Fig. 5 Analysis of fast 2-color fluorogenic DNA-PAINT imaging without optical sectioning.
Detailed analysis of the full 20-minute 2-color fluorogenic DNA-PAINT dataset from which Fig. 5 and Extended Data Fig. 4 were generated. Image colored by the correlation parameter (C) based on Coordinate-Based Colocalization (CBC) analysis at low (a) and high (b) magnification. (c) Histogram of the correlation parameter, C. A value of zero indicates a lack of correlation between the two color channels (C = -0.09 ± 0.30; nblink = 2,322,207; two-sided one-sample Wilcoxon signed-rank test against zero, T-statistic=9×1011, p < 0.001). (d) Minimal bleaching is observed over a 20-minute timeframe (~20%). (e) The lateral localization precision peaks at < 5 nm for both channels. (f) Blink durations fitted with an exponential decay function to estimate the binding off-rate (blinks that are only 1 frame in duration were ignored for fitting).
Extended Data Fig. 6 Additional examples of fast 2-color fluorogenic DNA-PAINT imaging without optical sectioning.
Fast 2-color fluorogenic DNA-PAINT imaging of immunolabeled endoplasmic reticulum (green; [imager probe A] = 10 nM) and mitochondria (magenta; [imager probe B] = 1 nM) in U-2 OS cells under widefield illumination (100 Hz frame rate for 10 minutes) (n = 5 including the dataset presented in detail in Fig. 5 and Extended Data Figs. 4,5).
Supplementary Notes 1–3, Tables 1–4 and Figs. 1–3.
Fast 3D fluorogenic DNA-PAINT imaging (imager probe A concentration = 10 nM, 100 Hz, 10 min) of microtubules in a COS-7 cell without optical sectioning under widefield illumination. The hollow center of microtubules can be observed in both the xy and xz planes when viewing 30-nm-thick cross-sections. Scale bar, 1 µm.
a, Raw images from fast astigmatic 3D fluorogenic DNA-PAINT imaging (imager probe A concentration = 10 nM, 100 Hz) of microtubules in a COS-7 cell under widefield illumination. b, Live kymograph of the blinking within the dashed box in a.
a, Images from fast two-color fluorogenic DNA-PAINT using imager probe A (10 nM, Cy3B, green) and imager probe B (1 nM, ATTO 643, magenta) to image the endoplasmic reticulum and mitochondria, respectively (100 Hz; raw images from the two-color channels were transformed and aligned for display). b, Heatmap of pixel intensities. Negligible spectral cross-talk between the fluorophores (Cy3B and ATTO 643) was observed as indicated by the well-resolved populations (green and magenta dashed lines, respectively).
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Chung, K.K.H., Zhang, Z., Kidd, P. et al. Fluorogenic DNA-PAINT for faster, low-background super-resolution imaging. Nat Methods 19, 554–559 (2022). https://doi.org/10.1038/s41592-022-01464-9