Bright and photostable fluorescent dyes with large Stokes shift are rare, though they are indispensable in optical microscopy, biology and chemistry. The rapid progress in super-resolution microscopy based on stimulated emission depletion (STED) phenomenon encouraged us to design and prepare new coumarins and a hybrid carborhodol dye. Variation of electron-withdrawing groups at C-3 and/or C-4 enabled us to create promising coumarin dyes possessing a 3-(2-pyridyl) group (λabs/λem = 432/512 nm in aqueous phos-phate buffer), a 3-(pyrido[1,2-a]pyrrolo[2,1-c]pyrazinium) group (489/587 nm in MeOH) and a fused quinoline ring (453/617 nm in aqueous phosphate buffer). The new dyes were decorated with a polar phosphate group which provided sufficient solubility in aqueous solutions and a carboxylic group which was required for bioconjugation.
The hybrid carborhodol dye was obtained by a combination of (carbo)fluorescein and carbopyronine fluorophores. Due to the broad absorption and emission spectra of the carborhodol in the conjugated form (586/613 nm in aqueous phosphate buffer), the effective Stokes shift is larger in comparison with small Stokes shifts of the parent dyes. This allowed the use of carborhodol in two-color imaging schemes as well as in STED micros-copy with a 775 nm depletion laser.
