Kirill A. Kolmakov : Red-emitting dyes for microscopy and nanoscopy: design and optimization

Good imaging performance of fluorophores in stimulated emission depletion (STED) microscopy, fluorescence correlation spectroscopy (FCS), ground state depletion microscopy (GSDIM),fluorescence lifetime imaging (FLIM) and related techniques demands high photostability, large absorption coefficients and fluorescence quantum yields. No less important are predictable lifetimes of the excited states and specific binding to biological substrates. These features result in high image brightness and contrast, which in turn correlate with high optical resolution. For practical use of the dye probes, properties like hydrolytic stability, sufficient amino reactivity, and solubility in aqueous media, simple handling and “user-friendliness” become deciding. All these features are often contradicting. The new approach to dye design that allows meeting all these requirements is based on the vision of a dye molecule as a scaffold with multiple variable positions and is largely influenced by combinatorial chemistry. This approach is applicable not only to the dye molecule as a whole but also to its key fragments, i. e., linkers, spacers and additional hydrophilic functional groups. Rhodamine, Si-rhodamine, carbopyronine and oxazine dyes with absorption and emission maxima in the range of 600 - 660 nm and 620 - 680 nm, respectively, were the subjects of a systematic study. We created hydrolytically stable water-soluble red-emitting dye markers of required polarities, hydrophilicities and net charges. These dyes have a wide scope of functional groups and applicable in two-color STED microscopy and FLIM due to their different fluorescence lifetimes. Also, red-emitting fluorescent dyes are available in a colorless caged (masked) form, which unfolds the initial color and fluorescence upon irradiation with UV/visible light or with a STED beam at 750-800 nm (two-photon uncaging).