Fluorescent Dyes with Large Stokes Shifts of 80−200 nm for Optical Microscopy and Nanoscopy

Abstract

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.