Reversibly photoswitchable 1,2‐bis(2‐ethyl‐6‐phenyl‐1‐benzothiophene‐1,1‐dioxide‐3‐yl)perfluorocyclopentenes (EBT) having fluorescent “closed” forms were decorated with four or eight carboxylic groups and attached to antibodies. Low aggregation, efficient photoswitching in aqueous buffers, specific staining of cellular structures, and good photophysical properties were demonstrated. Alternating light pulses of UV and blue light induce numerous reversible photochemical transformations between two stables states with distinct structures. Using relatively low light intensities, EBTs were applied in biology‐related super‐resolution microscopy based on the reversible saturable (switchable) optical linear fluorescence transitions (RESOLFT) and demonstrated optical resolution of 75 nm.

Photostable and bright fluorescent dyes with large Stokes shifts are widely used as markers in far-field optical microscopy, but the variety of useful dyes is limited. The present study introduces new 3-heteroaryl coumarins decorated with a primary phosphate group (OP(O)(OH)(2)) attached to C-4 in 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused with the coumarin fluorophore. The general synthetic route is based on the Suzuki reaction of 3-bromocoumarines with hetarylboronic acids followed by oxidation of the methyl group at the C=C bond with SeO(2) (to an aldehyde), reduction with NaBH(4) (to an alcohol), and conversion into a primary phosphate. The 4 position in the coumarin system may be unsubstituted or bear a methyl group. Phosphorylated coumarins were found to have high fluorescence quantum yields in the free state and after conjugation with proteins (in aqueous buffers). In super-resolution light microscopy with stimulated emission depletion (STED), the new coumarin dyes provide an optical resolution of 40-60 nm with a low background signal. Due to their large Stokes shifts and high photostability, phosphorylated coumarins enable to combine multilabel imaging (using one detector and several excitation sources) with diffraction unlimited optical resolution.

Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetero)aryl- or (hetero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413-480 and 527-668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyridyl)ethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.

Fluorescence microscopy reveals the localization, spatial distribution, and temporal dynamics of the specifically labeled organelles in living cells. Labeling with exogenous conjugates prepared from fluorescent dyes and small molecules (ligands) is an attractive alternative to the use of fluorescent proteins, but proved to be challenging due to insufficient cell‐permeability of the probes, unspecific staining, or low dye brightness. We evaluated four green‐emitting rhodamine dyes and their conjugates intended for the specific labeling of lysosomes, mitochondria, tubulin, and actin in living cells. The imaging performance of the probes in living human fibroblasts has been studied by using confocal and stimulated emission depletion (STED) super‐resolution microscopy with a commercial 595 nm STED laser. Two bright and photostable dyes (LIVE 510 and LIVE 515) provide specific and versatile staining.