Self-Blinking Dye Restores Efficient Use of Nanobodies in Single-Molecule Localization Microscopy.

Direct stochastic optical reconstruction microscopy (dSTORM) relies on controlled fluorophore blinking to achieve nanometer-scale resolution, yet the field's 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 (SMLM) 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 subnanometer 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.