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bioRxiv · 10.64898/2025.12.17.694880

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.
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Abstract

Direct stochastic optical reconstruction microscopy (dSTORM) relies on controlled fluorophore blinking to achieve nanometer-scale resolution, yet the fields benchmark dye, Alexa Fluor 647, underperforms when conjugated to nanobodies, limiting the practical use of minimal-linkage labeling strategies. Here, we show that the self-blinking dye JF635b overcomes this limitation by maintaining robust, photostable blinking upon conjugation to nanobodies under buffer-independent conditions. This enables reliable single-molecule localization microscopy without the need for complex switching buffers. Using JF635b-labeled nanobodies, we demonstrate consistent performance across multiple imaging modalities, including wide-field dSTORM, fluorescence lifetime SMLM, and MINFLUX nanoscopy, achieving localization precisions from [~]15 nm down to the sub-nanometer regime. In addition, JF635b supports long-term sample preservation and efficient blinking even in pure water, enabling minimally perturbative imaging conditions. Together, these results establish self-blinking dSTORM as a robust and accessible platform for quantitative nanoscopy across experimental contexts.

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Basak, S., Inamdar, K., Pollak, Y. G., Albert, L., Jans, D. C., Jakobs, S., Enderlein, J., Tsukanov, R., Opazo, F.. 2025-12-19. Self-blinking dye restores efficient use of nanobodies in single-molecule localization microscopy.. https://doi.org/10.64898/2025.12.17.694880

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