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.

We introduce fluorogenic tubulin probes based on the recently reported fluorescent dyes (510R, 580CP, GeR and SiR) and chemotherapy agents – taxanes (docetaxel, cabazitaxel and larotaxel). The cytotoxicity of the final probe, its staining performance and specificity strongly depend on both components. We found correlation between the aggregation efficiency (related to the spirolactonization of fluorophore) and cytotoxicity. Probe optimization allowed us to reach 29 ± 11 nm resolution in stimulated emission depletion (STED) microscopy images of the microtubule network in living human fibroblasts. Application to living fruit fly (Drosophila melanogaster) tissues highlighted two distinct structures: microtubules and tracheoles. We identified 6-carboxy isomers of 580CP and SiR dyes as markers for chitin-containing taenidia, a component of tracheoles. STED microscopy revealed correlation between the taenidia periodicity and the diameter of the tracheole. Combined tubulin and taenidia STED imaging showed close interaction between the microtubules and respiratory networks in living tissues of the insect larvae.

Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6′-carboxy isomers and not the 5′-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of ¹H NMR spectra to discriminate between 6′- and 5′-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae.

Rationale: Recently, abundant axial tubule (AT) membrane structures were identified deep inside atrial myocytes (AMs). Upon excitation, ATs rapidly activate intracellular Ca²⁺ release and sarcomeric contraction through extensive AT junctions, a cell-specific atrial mechanism. While AT junctions with the sarcoplasmic reticulum contain unusually large clusters of ryanodine receptor 2 (RyR2) Ca²⁺ release channels in mouse AMs, it remains unclear if similar protein networks and membrane structures exist across species, particularly those relevant for atrial disease modeling. Objective: To examine and quantitatively analyze the architecture of AT membrane structures and associated Ca²⁺ signaling proteins across species from mouse to human. Methods and Results: We developed superresolution microscopy (nanoscopy) strategies for intact live AMs based on a new custom-made photostable cholesterol dye and immunofluorescence imaging of membraneous structures and membrane proteins in fixed tissue sections from human, porcine, and rodent atria. Consistently, in mouse, rat, and rabbit AMs, intact cell-wide tubule networks continuous with the surface membrane were observed, mainly composed of ATs. Moreover, co-immunofluorescence nanoscopy showed L-type Ca²⁺ channel clusters adjacent to extensive junctional RyR2 clusters at ATs. However, only junctional RyR2 clusters were highly phosphorylated and may thus prime Ca²⁺ release at ATs, locally for rapid signal amplification. While the density of the integrated L-type Ca²⁺ current was similar in human and mouse AMs, the intracellular Ca²⁺ transient showed quantitative differences. Importantly, local intracellular Ca²⁺ release from AT junctions occurred through instantaneous action potential propagation via transverse tubules (TTs) from the surface membrane. Hence, sparse TTs were sufficient as electrical conduits for rapid activation of Ca²⁺ release through ATs. Nanoscopy of atrial tissue sections confirmed abundant ATs as the major network component of AMs, particularly in human atrial tissue sections. Conclusion: AT junctions represent a conserved, cell-specific membrane structure for rapid excitation-contraction coupling throughout a representative spectrum of species including human. Since ATs provide the major excitable membrane network component in AMs, a new model of atrial “super-hub” Ca²⁺ signaling may apply across biomedically relevant species, opening avenues for future investigations about atrial disease mechanisms and therapeutic targeting.

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.