iiinova — where your digital self keeps evolving

Johannes Stein @jmstein.bsky.social · 13/08/2025

(16/n) Last but not least, massive thanks also to the many colleagues and experts whose support and advice helped make this project a success – and most importantly – great fun🙏!!!

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Johannes Stein @jmstein.bsky.social · 13/08/2025

(10/n) Using a simple custom-addition to our TIRF scope (700$, described in SI), we finally map nuclear antigens via 3D tkPAINT imaging based on astigmatism.

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Johannes Stein @jmstein.bsky.social · 13/08/2025

(9/n) Multiplexing and multimodal imaging? Yes. Nuclear tkPAINT works seamlessly with Exchange-PAINT and earlier multimodal cryosection protocols targeting proteins, nucleic acids, or both🔄🧬.

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Johannes Stein @jmstein.bsky.social · 13/08/2025

(8/n) Physical sectioning = no detergent needed🫧❌. tkPAINT allows imaging throughout ultrastructurally-preserved cells without permeabilization → detergent-sensitive antigens are retained. Sections are thus also promising samples for structural fluorescence imaging methods like MINFLUX & RESI.

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Johannes Stein @jmstein.bsky.social · 13/08/2025

(7/n) tkPAINT opens the door for spatial biology studies in both cultured cells & tissues at identical resolution – revealing, for instance, cell- and tissue-specific Pol II heterogeneities.

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Johannes Stein @jmstein.bsky.social · 13/08/2025

(6/n) …and favor molecular counting in the nucleus📊! With qPAINT we measure local Pol II antibody counts and estimate total nuclear abundance.

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Johannes Stein @jmstein.bsky.social · 13/08/2025

(5/n) More binding events enable: •⚡ Efficient kinetic filtering to remove non-specific localizations •🎯 High-fidelity antigen profiling down to single-antibody

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Johannes Stein @jmstein.bsky.social · 13/08/2025

(4/n) While TIRF ensures nuclear imaging of Pol II at down to 3-nm localization precision, ultrathin sections reduce the imaging volume, speeding up image acquisition⚡ and enhancing imager binding statistics 📈.

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Johannes Stein @jmstein.bsky.social · 13/08/2025

(3/n) Inspired by work by Eric Betzig, @heilemannlab.bsky.social, @apombo1.bsky.social and others, we adopted Tokuyasu cryosectioning to enable TIRF-based DNA-PAINT imaging throughout fixed HeLa cells without range constraint. We started with the nucleus & Pol II to explore tkPAINT’s potential.

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Johannes Stein @jmstein.bsky.social · 13/08/2025

(2/n) TIRF is an excellent imaging modality for DNA-PAINT🔬🧬✨: low background, high resolution, small imaging volume. Fantastic for DNA origami, ideal for cellular targets close to the cover glass. The drawback: its limited range.

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Johannes Stein @jmstein.bsky.social · 13/08/2025

(1/n) DNA-PAINT imaging inside the nucleus at single antibody resolution using TIRF? Ultrathin sectioning makes it happen! Grateful to share my postdoctoral work introducing “tomographic & kinetically-enhanced DNA-PAINT” or in brief: tkPAINT. Out in @pnas.org! doi.org/10.1073/pnas... 👇🧵

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Johannes Stein @jmstein.bsky.social · 21/07/2025

(7/n) We validated SST for cellular DNA-PAINT, confirming robust imaging and stable performance in fixed cells.

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Johannes Stein @jmstein.bsky.social · 21/07/2025

(6/n) SST enabled >24h imaging with stable localization precision and docking strands preservation. Also no performance loss after >1mo at room temp! Even better, SST doubled binding rates - allowing faster imaging or lower imager concentrations. Also ideal for RESI.

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Johannes Stein @jmstein.bsky.social · 21/07/2025

(5/n) We systematically compared SST (Sodium Sulfite + Trolox) to PPT using the PAINT-favorite dye Cy3b and also tested different triplet state quenchers (Trolox, DABCO). 💡 SST was brighter, more stable, and better protected Cy3b from photobleaching than PPT.

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Johannes Stein @jmstein.bsky.social · 21/07/2025

(2/n) DNA-PAINT resists photobleaching via imager replenishment, but reactive oxygen species (ROS) can still damage docking strands during long imaging (Blumhardt et al. 2019), limiting quality and duration. Good news: oxygen scavenging systems (OSS) can help prevent this.

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Johannes Stein @jmstein.bsky.social · 21/07/2025

(1/n) A simple, ultrastable, and cost-effective oxygen-scavenging system for long-term DNA-PAINT imaging Please check out our new study on the benefits of sodium sulfite as oxygen scavenging system for DNA-PAINT. 👇🧵 #SuperResolution #DNAPAINT #SMLM #Microscopy

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Johannes Stein @jmstein.bsky.social · 19/11/2024

Important work by E. Lučinskaitė, …, C. Soeller, A. Clowsley: ‘shielding’ DNA-conjugated antibodies with short complementary oligos during labeling & high salt (1 M NaCl) can reduce non-specific antibody sticking. onlinelibrary.wiley.com/doi/10.1002/...

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