A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500–630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye–ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure–property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one‐ and two‐color images of living cells with an optical resolution of 40–60 nm.

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine (“BG”) with a charged sulfonate (“SBG”). This chemical manipulation can be applied to any SNAP-tag substrate, improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic superresolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

Helle und photostabile Rhodamine und Carbopyronine mit Absorptionsmaxima im Bereich von λ=500–630 nm wurden entwickelt, die nach Anwendung von HaloTag‐Technologie, gefolgt von einer Färbung mit 1 μm Lösungen der Farbstoff‐Ligand‐Konjugate, die spezifische Markierung von Zellskelettfilamenten ermöglichen. Die Synthese, die photophysikalischen Eigenschaften und die Fluorogenität sowie die Struktur‐Eigenschafts‐Beziehungen der neuen Farbstoffe werden diskutiert. STED‐Fluoreszenzmikroskopie lieferte Ein‐ und Zweifarbenbilder von lebenden Zellen mit einer optischen Auflösung von 40–60 nm.

Fluorescent actin probes made of 6′-carbopyronines and jasplakinolide are insensitive to efflux pumps and stain F- and G-actin efficiently in living cells, allowing high quality 2D and 3D nanoscopy of dynamic actin structures.

The postsynaptic density component PSD-95 undergoes activity-dependent plasticity mechanisms that rely on protein synthesis and structural remodeling. How synaptic activity can influence these dynamics at the single synapse level remains unclear. Here we combine genome-editing, pulse-chase experiments, STED and 3D MINFLUX nanoscopy on hippocampal neuronal cultures to study the integration of newly translated PSD-95 molecules at postsynaptic sites and their rearrangement within individual clusters at near-molecular resolution. We show that the amount of newly translated PSD-95 recruited to individual synapses scales with synaptic size, and modulates in a bidirectional manner, resulting in less new protein following excitatory and more new protein following inhibitory stimulation. Furthermore, we show that within synaptic clusters PSD-95 has a dispersed organization that is largely robust to long-lasting changes in activity. Altogether, this work sheds new light on the mechanisms underlying plasticity at the single synapse level, adding previously inaccessible information.

The design and preparation of synthetic binders (SBs) applicable for small biomolecule sensing in aqueous media remains very challenging. SBs designed by the lock‐and‐key principle can be selective for their target analyte but usually show an insufficient binding strength in water. In contrast, SBs based on symmetric macrocycles with a hydrophobic cavity can display high binding affinities but generally suffer from indiscriminate binding of many analytes. Herein, a completely new and modular receptor design strategy based on microporous hybrid materials is presented yielding zeolite‐based artificial receptors (ZARs) which reversibly bind the neurotransmitters serotonin and dopamine with unprecedented affinity and selectivity even in saline biofluids. ZARs are thought to uniquely exploit both the non‐classical hydrophobic effect and direct non‐covalent recognition motifs, which is supported by in‐depth photophysical, and calorimetric experiments combined with full atomistic modeling. ZARs are thermally and chemically robust and can be readily prepared at gram scales. Their applicability for the label‐free monitoring of important enzymatic reactions, for (two‐photon) fluorescence imaging, and for high‐throughput diagnostics in biofluids is demonstrated. This study showcases that artificial receptor based on microporous hybrid materials can overcome standing limitations of synthetic chemosensors, paving the way towards personalized diagnostics and metabolomics.