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.

Superresolution fluorescence microscopy of multiple fluorophores still requires development. Here we present simultaneous three-colour stimulated emission depletion (STED) nanoscopy relying on a single STED beam at 620 nm. Toggling the STED beam between two or more power levels (“multilevelSTED”) optimizes resolution and contrast in all colour channels, which are intrinsically co-aligned and well separated. Three-colour recording is demonstrated by imaging the nanoscale cytoskeletal organization in cultured hippocampal neurons. The down to ~35 nm resolution identified periodic actin/betaII spectrin lattices along dendrites and spines; however, at presynaptic and postsynaptic sites, these patterns were found to be absent. Both our multicolour scheme and the 620 nm STED line should be attractive for routine STED microscopy applications.

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.