Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine–dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine–dye conjugates with minimal distance between tetrazine and the fluorophore. Two synthetic routes give access to a series of cell-permeable and -impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as the dominant excited-state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multicolor and super-resolution STED, and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.

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In this work, the effect of extra-cellular conductivity on electroporation-mediated molecular delivery efficiency is investigated. A numerical model combining the Smoluchowski equation for membrane permeabilization and the Nernst-Planck equations for ion transport is used to simulate the evolution of ion concentration spatially and temporally. The results are compared with and used to interpret trends observed from previous experimental measurements. Agreements are found which suggest the critical importance of electrophoretic transport. This mechanism controls delivery efficiency on the quantitative level. Meanwhile, a simple formula is developed to predict the molecular content delivered via electrophoresis. The formula can be used as a compact model which provides good approximation to the full numerical model while avoiding the computational cost.