Self-healing dyes have emerged as a new promising class of fluorescent labels. They consist of two units, a fluorescent dye and a photostabilizer. The latter heals whenever the fluorescent dye is in danger of taking a reaction pathway toward photobleaching. We describe the underlying concepts and summarize the developmental history and state-of-the-art, including latest applications in high-resolution microscopy, live-cell, and single-molecule imaging. We further discuss remaining limitations, which are (i) lower photostabilization of most self-healing dyes when compared to solution additives, (ii) limited mechanistic understanding on the influence of the biochemical environment and molecular oxygen on self-healing, and (iii) the lack of cheap and facile bioconjugation strategies. Finally, we provide ideas on how to further advance self-healing dyes, show new data on redox blinking caused by double-stranded DNA, and highlight forthcoming work on intramolecular photostabilization of fluorescent proteins.

Zinc is an essential element in all domains of life. Nonetheless, how prokaryotes achieve selective acquisition of zinc from the extracellular environment remains poorly understood. Here, we elucidate a novel mechanism for zinc-binding in AdcA, a solute-binding protein of Streptococcus pneumoniae. Crystal structure analyses reveal the two-domain organization of the protein and show that only the N-terminal domain (AdcAN) is necessary for zinc import. Zinc binding induces only minor changes in the global protein conformation of AdcA and stabilizes a highly mobile loop within the AdcAN domain. This loop region, which is conserved in zinc-specific solute-binding proteins, facilitates closure of the AdcAN binding site and is crucial for zinc acquisition. Collectively, these findings elucidate the structural and functional basis of selective zinc uptake in prokaryotes. IMPORTANCE Zinc is an essential nutrient for the virulence of bacterial pathogens such as Streptococcus pneumoniae. Many Gram-positive bacteria use a two-domain lipoprotein for zinc acquisition, but how this class of metal-recruiting proteins acquire zinc and interact with the uptake machinery has remained poorly defined. We report the first structure of a two-domain lipoprotein, AdcA from S. pneumoniae, and use computational, spectroscopic, and microbiological approaches to provide new insights into the functional basis of zinc recruitment. Our findings reveal that AdcA employs a novel mechanism for zinc binding that we have termed the “trap-door” mechanism, and we show how the static metal-binding site of the protein, which confers its selectivity for zinc ions, is combined with a dynamic surface element to facilitate zinc recruitment and import into the bacterium. Together, these findings expand our understanding of how bacteria acquire zinc from the environment and provide a foundation for inhibiting this process, through antimicrobial targeting of the dynamic structural elements to block bacterial zinc scavenging.

Many life‐science techniques and assays rely on selective labeling of biological target structures with commercial fluorophores that have specific yet invariant properties. Consequently, a fluorophore (or dye) is only useful for a limited range of applications, e.g., as a label for cellular compartments, super‐resolution imaging, DNA sequencing or for a specific biomedical assay. Modifications of fluorophores with the goal to alter their bioconjugation chemistry, photophysical or functional properties typically require complex synthesis schemes. We here introduce a general strategy that allows to customize these properties during biolabelling with the goal to introduce the fluorophore in the last step of biolabelling. For this, we present the design and synthesis of ‘linker’ compounds, that bridge biotarget, fluorophore and a functional moiety via well‐established labeling protocols. Linker molecules were synthesized via the Ugi four‐component reaction (Ugi‐4CR) which facilitates a modular design of linkers with diverse functional properties and bioconjugation‐ and fluorophore attachment moieties. To demonstrate the possibilities of different linkers experimentally, we characterized the ability of commercial fluorophores from the classes of cyanines, rhodamines, carbopyronines and silicon‐rhodamines to become functional labels on different biological targets in vitro and in vivo via thiol‐maleimide chemistry. With our strategy, we showed that the same commercial dye can become a photostable self‐healing dye or a sensor for bivalent ions subject to the linker used. Finally, we quantified the photophysical performance of different self‐healing linker–fluorophore conjugates and demonstrated their applications in super‐resolution imaging and single‐molecule spectroscopy.

Many life‐science techniques and assays rely on selective labeling of biological target structures with commercial fluorophores that have specific yet invariant properties. Consequently, a fluorophore (or dye) is only useful for a limited range of applications, e.g., as a label for cellular compartments, super‐resolution imaging, DNA sequencing or for a specific biomedical assay. Modifications of fluorophores with the goal to alter their bioconjugation chemistry, photophysical or functional properties typically require complex synthesis schemes. We here introduce a general strategy that allows to customize these properties during biolabelling with the goal to introduce the fluorophore in the last step of biolabelling. For this, we present the design and synthesis of ‘linker’ compounds, that bridge biotarget, fluorophore and a functional moiety via well‐established labeling protocols. Linker molecules were synthesized via the Ugi four‐component reaction (Ugi‐4CR) which facilitates a modular design of linkers with diverse functional properties and bioconjugation‐ and fluorophore attachment moieties. To demonstrate the possibilities of different linkers experimentally, we characterized the ability of commercial fluorophores from the classes of cyanines, rhodamines, carbopyronines and silicon‐rhodamines to become functional labels on different biological targets in vitro and in vivo via thiol‐maleimide chemistry. With our strategy, we showed that the same commercial dye can become a photostable self‐healing dye or a sensor for bivalent ions subject to the linker used. Finally, we quantified the photophysical performance of different self‐healing linker–fluorophore conjugates and demonstrated their applications in super‐resolution imaging and single‐molecule spectroscopy.