CF dye

CF® Dyes are a class of proprietary fluorescent dyes developed by Biotium, Inc. 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.

The CF Dye portfolio currently includes over 40 fluorophores spanning excitation wavelengths from 347 nm (ultraviolet) to 876 nm (near-infrared), built on four core chemical scaffolds: coumarin, pyrene, rhodamine, and cyanine. CF Dyes have been used in super-resolution microscopy, where several variants have been validated for techniques including STORM, MINFLUX, and STED microscopy.

History and development

Biotium was founded in 2001 in Fremont, California by Fei Mao, PhD, and Vivien Chen, MBA. Development of CF Dyes 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, Biotium researchers developed a chemical design strategy combining sulfonation with polyethylene glycol (PEG) modification.

The name "CF" originally stood for "Cyanine-based Fluorescent" dyes, but now represents "Clear Fluor," reflecting the signal-to-noise performance achieved through the combined pegylation-sulfonation strategy.

In 2009, Biotium researchers reported the development of a rhodamine–imidazole 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.

Biotium also developed nucleic acid stains that are safer for users than other common options: GelRed and GelGreen. The company helped invent Viability PCR (v-PCR) and the reagents propidium monoazide (PMA) and its successor technology, PMAxx™.

In August 2021, Biotium launched CF850 and CF870, fluorescent dyes with emission maxima beyond 850 nm. In 2022, a collaboration with Professor Ke Xu at UC Berkeley yielded CF583R and CF597R, green-excited rhodamine dyes optimized for STORM microscopy.

In 2023, Biotium introduced Astral Leap™ Tandem Dyes, engineered with optimized Förster Resonance Energy Transfer (FRET) capabilities for enhanced signal-to-noise in flow cytometry applications.

Chemistry

CF Dyes were synthesized through chemical modifications of established families of coumarin, rhodamine, and cyanine dyes. The dyes employ a dual strategy of sulfonation and pegylation. Sulfonation introduces sulfonate groups (–SO₃⁻) to improve water solubility, while pegylation adds polyethylene glycol (PEG) chains that physically shield charged groups and reduce dye aggregation. This approach achieves water solubility exceeding 100 mg/mL while maintaining only 1-2 net negative charges, compared to 3-6 charges in traditional heavily sulfonated dyes.

The PEG moieties sterically prevent π-stacking between adjacent dye molecules, reducing H-aggregate formation, a cause of fluorescence quenching when multiple dyes are attached to a single antibody. This property enables antibody conjugates to achieve higher degrees of labeling (DOL values of 4–10) without significant self-quenching.

Rhodamine-based near-infrared CF Dyes (designated with "R" suffix) utilize rhodamine-imidazole substitution chemistry to extend emission wavelengths beyond the traditional ~600 nm limit while maintaining the photostability characteristics of the rhodamine scaffold. The rigid xanthene structure of rhodamines resists photobleaching compared to the flexible polymethine bridge found in cyanine dyes.

Applications

CF Dyes are commercially available as reactive dyes and labeling kits for the preparation of fluorescent bioconjugates. They have been used to label proteins, including antibodies and lectins, as well as other biomolecules. Reported applications include immunofluorescence microscopy, flow cytometry, western blotting, in vivo imaging, fluorescence in situ hybridization, expansion microscopy, and apoptosis detection.

Near-infrared CF Dyes (CF680, CF770, CF790) have been used in quantitative western blotting on LI-COR Odyssey detection systems. In comparative studies, CF790 was reported to exhibit reduced non-specific binding compared to Alexa Fluor 790 due to lower net negative charge.

Super-resolution microscopy

Multiple CF 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 with low crosstalk." CF583R and CF597R, developed through the Biotium-Ke Xu collaboration, 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 × 40 × 6 μm volumes). Likewise, CF640R and CF680R have been validated for stimulated emission depletion (STED) microscopy. Several CF Dyes have been used for structured illumination microscopy (SIM). And more recently, CF660C and CF680 have been validated for MINFLUX nanoscopy using standard GLOX+MEA photoswitching buffers.

Technical specifications

Representative CF® Dye specifications

Patents

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.

See also

• Fluorophore
• Fluorescence microscopy
• Super-resolution microscopy