CF dyes (trademarked as CF Dyes by Biotium) are a class of fluorescent dyes developed for biological research applications, including fluorescence microscopy, flow cytometry, and in vivo imaging. First introduced in the late 2000s, these dyes are characterized by a chemical strategy combining pegylation with sulfonation to achieve high water solubility while minimizing non-specific binding.
Varieties include 42 fluorophores spanning excitation wavelengths from 347 nm (ultraviolet) to 876 nm (near-infrared), built on four core chemical scaffolds: coumarin, pyrene, rhodamine, and cyanine. These dyes have been used in super-resolution microscopy, where several variants have been validated for techniques including STORM, MINFLUX, and STED microscopy.
Development began around 2007 in response to limitations observed in existing commercial fluorophores, particularly the tendency of heavily sulfonated dyes to exhibit non-specific binding to positively charged cellular components. To address these issues, researchers developed a design strategy combining sulfonation with polyethylene glycol (PEG) modification, the details of which are described in a 2014 U.S. patent.
In 2009, researchers reported the development of a rhodamineâÂÂimidazole substitution strategy in which the benzene ring commonly used for conjugation was replaced with an imidazolium group. This modification produced a red shift in emission wavelength while preserving the photostability of the rhodamine xanthene core, extending the usable spectral range of rhodamine dyes toward the near-infrared region.
In 2022, a collaboration with researchers at UC Berkeley yielded CF583R and CF597R, which are rhodamine-based dyes optimized for STORM microscopy.
CF dyes are synthesized through chemical modifications of established coumarin, rhodamine, and cyanine dye scaffolds. The dyes employ a dual strategy of sulfonation and pegylation. Sulfonation introduces sulfonate groups (âÂÂSOâÂÂâ») to improve water solubility, while pegylation adds polyethylene glycol (PEG) chains that sterically shield charged groups and reduce dye aggregation.
The PEG moieties inhibit ÃÂ-stacking between adjacent dye molecules, reducing H-aggregate formation. H-aggregation is a cause of fluorescence quenching when multiple dye molecules are attached to a single antibody, limiting the useful degree of labeling (DOL) in antibody conjugates.
Rhodamine-based near-infrared CF dyes (designated with an "R" suffix) utilize rhodamineâÂÂimidazole substitution chemistry, as described in Wang et al. (2022), to extend emission wavelengths beyond the traditional ~600 nm limit while retaining the photostability characteristic of the rhodamine scaffold. The rigid xanthene core of rhodamines confers resistance to photobleaching relative to the flexible polymethine bridge found in cyanine dyes.
Applications include immunofluorescence microscopy, flow cytometry, western blotting, in vivo imaging, fluorescence in situ hybridization, expansion microscopy, and apoptosis detection.
The dyes have been evaluated in peer-reviewed studies for use in super-resolution microscopy techniques. A systematic evaluation of 28 commercial dyes by Lehmann and colleagues (2016) identified CF647 and CF680 as an optimal dye pair for spectral demixing-based, registration-free multicolor dSTORM in combination with CF568, due to low spectral crosstalk. CF583R and CF597R enable localization precision of approximately 10 nm laterally and 20 nm axially.
Research from Diekmann and colleagues at EMBL demonstrated that CF660C exhibits photostability during extended imaging sessions, enabling acquisition of approximately one million frames covering entire mitotic cells (40 à40 à6 üm volumes). CF640R and CF680R have been validated for stimulated emission depletion (STED) microscopy. Several dyes have been employed in structured illumination microscopy (SIM). CF660C and CF680 have been validated for MINFLUX nanoscopy using standard GLOX+MEA photoswitching buffers.
Key patents covering CF Dye technology include US8709830B2 ("Fluorescent dyes, fluorescent dye kits, and methods of preparing labeled molecules"), EP2223086B1 (priority date 2007), and international application WO2012129128A1.