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Self-blinking dye restores efficient use of nanobodies in single-molecule localization microscopy.

Basak, S.; Inamdar, K.; Pollak, Y. G.; Albert, L.; Jans, D. C.; Jakobs, S.; Enderlein, J.; Tsukanov, R.; Opazo, F.

2025-12-19 biophysics
10.64898/2025.12.17.694880 bioRxiv
Show abstract

Direct STORM relies on robust fluorophore blinking; observations suggest that AlexaFluor647 underperforms when coupled to nanobodies, thereby undermining the advantages of nanobodies. We show that the self-blinking dye JF635b preserves excellent blinking behavior upon nanobody conjugation, enabling sparse, stable localizations in saline buffers. Using these self-blinking nanobodies, we obtained robust dSTORM, FL-SMLM, and MINFLUX images, achieving localization precision of [~]1 nm. Its simple implementation improves reproducibility and makes single-molecule localization microscopy more accessible.

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