Far-Red Emitting Fluorescent Dyes for Optical Nanoscopy: Fluorinated Silicon-Rhodamines (SiRF Dyes) and Phosphorylated Oxazines

• Published in: Chemistry : a European journal Vol. 21; no. 38; pp. 13344 - 13356
• Main Authors: Kolmakov, Kirill, Hebisch, Elke, Wolfram, Thomas, Nordwig, Lars A., Wurm, Christian A., Ta, Haisen, Westphal, Volker, Belov, Vladimir N., Hell, Stefan W.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 14.09.2015; WILEY‐VCH Verlag; Wiley; Wiley Subscription Services, Inc
• Subjects: Beams (radiation); Chemistry; Chemistry, Multidisciplinary; Depletion; Dyes; dyes/pigments; Emittance; Fluorescence; Fluorination; Imaging; Microscopy; Physical Sciences; Science & Technology; Stimulated emission; structure-activity relationships; super-resolution microscopy; synthesis design; WATER-SOLUBILIZATION; HIGHLY FLUORESCENT; pigments; XANTHENE DYES; SUPERRESOLUTION MICROSCOPY; ANALOGS; SHIFTS; dyes; super-resolution microscopy; NEAR-INFRARED FLUOROPHORES; structure-activity relationships; fluorescence; LIVE-CELL; ABSORPTION; DERIVATIVES; synthesis design; dyes/pigments
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.201501394

Far‐red emitting fluorescent dyes for optical microscopy, stimulated emission depletion (STED), and ground‐state depletion (GSDIM) super‐resolution microscopy are presented. Fluorinated silicon–rhodamines (SiRF dyes) and phosphorylated oxazines have absorption and emission maxima at about λ≈660 and 680 nm, respectively, possess high photostability, and large fluorescence quantum yields in water. A high‐yielding synthetic path to introduce three aromatic fluorine atoms and unconventional conjugation/solubilization spacers into the scaffold of a silicon–rhodamine is described. The bathochromic shift in SiRF dyes is achieved without additional fused rings or double bonds. As a result, the molecular size and molecular mass stay quite small (<600 Da). The use of the λ=800 nm STED beam instead of the commonly used one at λ=750–775 nm provides excellent imaging performance and suppresses re‐excitation of SiRF and the oxazine dyes. The photophysical properties and immunofluorescence imaging performance of these new far‐red emitting dyes (photobleaching, optical resolution, and switch‐off behavior) are discussed in detail and compared with those of some well‐established fluorophores with similar spectral properties. Edge of vision: Fluorinated silicon–rhodamines (SiRF dyes) and phosphorylated oxazines are applied in stimulated emission depletion (STED) and ground‐state depletion microscopy (GSDIM) as new far‐red‐emitting fluorophores (see figure). Their photophysical properties and immunofluorescence imaging performance with a redshifted STED beam are discussed in detail and compared to those of some well‐established near‐IR emitting dyes.