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

Abstract

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.

Multicolour nanoscopy of fixed and living cells with a single STED beam and hyperspectral detection

“…The dyes were sourced from the following suppliers: ATTO-TEC GmbH, Siegen, Germany (Atto594); Abberior GmbH, Göttingen, Germany (Abberior Star635P); gift of authors of ref. 24 (KK1441); Biotium Inc., Hayward, US (CF680R).…”

Silicon‐substituted Xanthene Dyes and Their Unique Photophysical Properties for Fluorescent Probes

“…These fluorophores of Si‐rhodamines have been further modified to obtain functional fluorophores. For super‐resolution stimulated emission depletion (STED) microscopy, SiR650 was fluorinated to obtain dyes with red‐shifted absorption and fluorescence maxima (Figure ; compound 21 , Table ) . Moreover, SiR680 (Figure ; compound 17 ) was modified to create a red‐shifted isologue of caged Q‐rhodamine (Rh Q : Q for tetrahydroquinoline) bearing nitroveratryl oxycarbonyl caging groups .…”

Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super‐Resolution STED Microscopy in Living Cells

“…The fluorogenic behavior of a dye can be tuned by the introduction of fluorine atoms in the alkyl chains (e.g., 500R ), the fluorophore core ( 515R , 620CP ), or the pendant aromatic ring 15. The principles of dye design exemplified in this study may enable the development of fluorogenic dyes of other classes (e.g.…”