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Existing DNA stains for live cell microscopy are either toxic, require illumination with blue light, or are not compatible with super-resolution microscopy. Here the authors develop SiRHoechst, a non-toxic far-red DNA stain that is compatible with super-resolution microscopy.

SNAP-tag is a widespread tool for labeling protein for bioimaging. Now, Kühn et al. report SNAP-tag2 with increased labeling kinetics and brightness, which translates into a better performance in live-cell super-resolution imaging.

Designing proteins whose activities can be switched on and off by effector molecules is a central challenge in protein engineering. Here, the authors use tethered chemical ligands with two mutually exclusive binding sites as a general method to modulate protein activity in response to specific effectors.

A target-identification strategy based on the yeast three-hybrid system and the SNAP-tag labeling technique identifies new targets for three small-molecule drugs and helps identify a new mechanism for the activity of the anti-inflammatory drug sulfasalazine involving inhibition of sepiapterin reductase.

Molecular recorders based on kinase activity-dependent protein labeling track specific kinase activities to understand their link to cellular phenotypes in heterogeneous cell populations and in vivo.

For decades chemists have focused on increasing the brightness of fluorophores. In super-resolution microscopy, however, fluorophores that preferentially exist in a non-fluorescent state, but occasionally re-arrange into a fluorescent form, can give better results.

HaloTag variants offer distinct brightness and fluorescence lifetimes compared with HaloTag7 when labeled with rhodamines. These variants were used for multiplexed imaging with a single fluorophore and to create lifetime-based cell cycle indicators.

The combination of synthetic ligands, luminescent proteins and binding proteins converts a well-established ligand-sensing system into a tunable and quantitative reporter for drug concentrations in blood, as demonstrated with six different drugs and using a simple digital camera.

Fluorescent probes for bioimaging need to exhibit bright fluorescence, be biocompatible and offer several alternatives for attachment to biomolecules of interest. Here, a near-infrared silicon–rhodamine fluorophore is introduced that can be coupled to intracellular proteins in live cells and tissues and can be exploited for super-resolution microscopy.

Activatable fluorophores are of interest for a wide range of applications but the need for caging groups complicates their development and application. Here, the authors report on a photoactivatable silicon rhodamine derivative and its application in live cell imaging and single-particle tracking.

Coenzyme A (CoA) is a ubiquitous and essential cofactor. A biosensor for visualizing cytosolic and mitochondrial CoA in living cells was developed to address central questions concerning CoA homeostasis.

It is difficult to develop suitable fluorescent probes for live-cell nanoscopy, but a general strategy is now reported that can transform regular fluorophores into fluorogenic probes with excellent cell permeability and low unspecific background signals. Using this approach, probes in a variety of colours were developed for different cellular targets and used for wash-free, multicolour, live-cell confocal and STED microscopy.

The tight interaction between the small molecule biotin and the tetrameric protein streptavidin is widely exploited for many different applications in protein science. In this issue, researchers present the design of a monovalent streptavidin tetramer with a single biotin binding site and demonstrate its enhanced properties over wild-type streptavidin for use in cell-surface protein labeling.

Snap-tag reporter mice allow flexible yet efficient targeting of chemical indicators to genetically labeled cells in vivo. With this strategy, cells can either be labeled fluorescently or ablated using the same reporter.

Super-resolution microscopy is a powerful tool for cellular studies but requires bright and stable fluorescent probes. Here, the authors report on a strategy to introduce quinoxaline motifs to conventional probes to make them brighter, more photostable, larger Stokes shift, and demonstrate the probes for biosensing applications.

Fluorescent proteins and HaloTag allow the flexible design of FRET-based biosensors with adjustable color using different fluorescent proteins or fluorophores and readout can be modified to fluorescence intensity, lifetime or bioluminescence.

There are several classes of sensory neuron that contribute to pain states. Here, the authors demonstrate that TrkB⁺ sensory neurons detect light touch under normal conditions in mice but contribute to hypersensitivity in models of chronic pain, and that ligand-guided laser ablation of TrkB⁺ sensory neurons in the mouse skin attenuates this hypersensitivity.

SLC25A51 is identified as a transporter of intact NAD⁺ into mammalian mitochondria and is required to maintain the mitochondrial NAD⁺ pool and respiratory function.

Glucagon-like peptide-1 receptor is an important regulator of appetite and glucose homeostasis. Here the authors describe super-resolution microscopy and in vivo imaging compatible fluorescent probes, which reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics in islets and brain.

Autofluorescent proteins have become indispensable in our quest to visualize molecular events in living cells. Further progress in the visualization and quantification of all biochemical activities of the cell will require the introduction of additional and complementary methods for sensing and probing biomolecules. Here I highlight some of the areas where the development of new probes and labeling methods is eagerly awaited and where chemical biologists could make important contributions.

Far-red fluorogenic probes for live-cell imaging of either actin or tubulin are described and used for super-resolution microscopy of various structures in a variety of cell types.

Engineered nanobody allows reversible control of activity in cells through the binding of small molecules.

Yu et al. report a bioluminescence- and paper-based assay for the rapid quantification of NAD⁺ levels in biological samples, such as blood and tissues.

The Innovative Medicines Initiative Consortium RESOLUTE has started to develop tools and produce data sets to de-orphanize transporters in the solute carrier protein (SLC) superfamily, thereby lowering the barrier for the scientific community to explore SLCs as an attractive drug target class.

Tetrahydrobiopterin (BH4) is an enzyme co-factor that is involved in the nervous system; it is shown here to also function in T cell activation and proliferation, with roles in autoimmunity, allergic inflammation and cancer.

Computational protein design is used to create a protein that binds the steroid digoxigenin (DIG) with high affinity and selectivity; the computational design methods described here should help to enable the development of a new generation of small molecule receptors for synthetic biology, diagnostics and therapeutics.

Single Xe clusters are superheated using an intense optical laser pulse and the structural evolution is imaged with a single X-ray pulse. Ultrafast surface softening on the nanometre scale is resolved within 100 fs at the vacuum/sample interface.