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Recent developments in fluorescence microscopy call for novel small‐molecule‐based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live‐cell single‐molecule localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live‐cell experiments. At the same time, quality of super‐resolution imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, we present the design and synthesis of a small‐molecule label comprising both fluorogenic and self‐blinking features. Bioorthogonal click chemistry ensures fast and highly selective attachment onto a variety of biomolecular targets. Along with spectroscopic characterization, we demonstrate that the probe improves quality and conditions for regular and single‐molecule localization microscopy on live‐cell samples., Blink and you'll miss it: A rationally designed small‐molecule label featuring fluorogenic and self‐blinking features has been designed and synthesized for bioorthogonal chemistry and super‐resolution microscopy in living cells.

High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization. AFM routinely resolves atomic steps, however on soft biological samples, the forces between the tip and the sample deform the fragile membrane, thereby distorting the otherwise high axial resolution of the technique. Here we present scanning ion-conductance microscopy (SICM) as an alternative approach for topographical imaging of soft biological samples, preserving high axial resolution on cells. SICM is complemented with live-cell compatible super-resolution optical fluctuation imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. Finally, we employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction-resolution., Methods for imaging the 3D cell surface often require physical interaction. Here the authors report the combination of scanning ion conductance microscopy (SICM) and live-cell super-resolution optical fluctuation imaging (SOFI) for the non-invasive topographical imaging of soft biological samples.

High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization. AFM routinely resolves atomic steps, however on soft biological samples, the forces between the tip and the sample deform the fragile membrane, thereby distorting the otherwise high axial resolution of the technique. Here we present scanning ion-conductance microscopy (SICM) as an alternative approach for topographical imaging of soft biological samples, preserving high axial resolution on cells. SICM is complemented with live-cell compatible super-resolution optical fluctuation imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. Finally, we employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffractional resolution.

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labelling via bioorthogonal tetrazine chemistry confers minimal label size and rapid labelling 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 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 dominant excited state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multi-colour and super-resolution STED and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.

Bioorthogonal reactions have emerged as a versatile tool in life sciences. The inverse electron demand Diels–Alder reaction (DAinv) stands out due to the availability of reactants with very fast kinetics. However, highly reactive dienophiles suffer the disadvantage of being less stable and prone to side reactions. Herein, we evaluate the extent of acceleration of rather unreactive but highly stable dienophiles by DNA-templated proximity. To this end, we developed a modular synthetic route for a novel bifunctional fluorogenic tetrazine rhodamine probe that we used to determine the reaction kinetics of various dienophiles in a fluorescence assay. Under proximity-driven conditions the reaction was found to be several orders of magnitude faster, and we observed almost no background reaction when proximity was not induced. This fundamental study identifies a minimally sized fluorogenic tetrazine dienophile reactant pair that has potential to be generally used for the visualization of biomolecular interactions with temporal and spatial resolution in living systems.

Bioorthogonal chemistry holds great potential to generate difficult‐to‐access protein–protein conjugate architectures. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels–Alder cycloaddition with inverse electron demand (DAinv). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines. We expect our work to substantially enhance antibody applications such as immunodetection and protein toxin‐based targeted cancer therapies., A highly efficient method for protein functionalization with commonly used bioorthogonal motifs for Diels–Alder cycloaddition with inverse electron demand (DAinv) is presented. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras.

Bioorthogonal chemistry techniques enable the selective and targeted manipulation of living systems. In order to yield universally applicable techniques, it is of great importance for bioorthogonal reactions to take place rapidly, selectively, and with the formation of only benign side products. One of the reactions that match these criteria well is the inverse electron demand Diels-Alder reaction (DA

Bioorthogonal chemistry techniques enable the selective and targeted manipulation of living systems. In order to yield universally applicable techniques, it is of great importance for bioorthogonal reactions to take place rapidly, selectively, and with the formation of only benign side products. One of the reactions that match these criteria well is the inverse electron demand Diels-Alder reaction (DAinv) between tetrazines and strained dienophiles. However, even this prime technique comes with the disadvantage of its reactants having limited stability under physiological conditions. In our protocol, an unreactive and therefore stable DAinv diene/dienophile pair reacts rapidly using DNA hybridization as secondary rate-accelerating process. Due to the fluorogenicity of the presented tetrazine rhodamine conjugate, this method enables the selective screening and evaluation of reactant pairs for proximity-mediated bioorthogonal chemistry.

Following the success of the siderophore-inspired 1,2-hydroxypyridonate (HOPO) and 2-hydroxisophthalamide (IAM) chromophores in Eu(III) and Tb(III) luminescence, we designed three new ligands bearing both chromophores. Syntheses of the octadentate ligands 3,4,3-LI-IAM-1,2-HOPO and 3,4,3-LI-1,2-HOPO-IAM, where the chromophores are attached to different positions in the (LI=linear) spermine backbone, are reported in addition to a tetradentate ligand based on 1,5-diaminopentane. The Gd(III) complexes were prepared and revealed localized triplet states typical for the IAM and HOPO chromophores. Photophysical characterization of the Eu(III) and Tb(III) complexes revealed that the chromophores need to reside at a primary amine of the spermine backbone to be efficient in lanthanide excitation. These systems help us to understand the antenna effect in siderophore inspired chromophores and could be potential targets for sensing and biological imaging applications.