CF dye

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.[1][2] 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.[3]

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.[3] These dyes have been used in super-resolution microscopy, where several variants have been validated for techniques including STORM, MINFLUX, and STED microscopy.[4][5]

History and development

Development began around 2007[2][6] 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.[3] 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.[3]

In 2009, 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.[3][7] 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.[7]

In 2022, a collaboration with researchers at UC Berkeley yielded CF583R and CF597R, which are rhodamine-based dyes optimized for STORM microscopy.[7]

Chemistry

CF dyes are synthesized through chemical modifications of established coumarin, rhodamine, and cyanine dye scaffolds.[7] The dyes employ a dual strategy of sulfonation and pegylation.[3] Sulfonation introduces sulfonate groups (–SO₃⁻) to improve water solubility, while pegylation adds polyethylene glycol (PEG) chains that sterically shield charged groups and reduce dye aggregation.[3]

The PEG moieties inhibit π-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.[3]

Rhodamine-based near-infrared CF dyes (designated with an "R" suffix) utilize rhodamine–imidazole 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.[3][7] The rigid xanthene core of rhodamines confers resistance to photobleaching relative to the flexible polymethine bridge found in cyanine dyes.[7]

Applications

Applications include immunofluorescence microscopy, flow cytometry, western blotting, in vivo imaging, fluorescence in situ hybridization, expansion microscopy, and apoptosis detection.[8][9][10]

Super-resolution microscopy

The dyes have been evaluated in peer-reviewed studies for use in super-resolution microscopy techniques.[4][7][11] 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.[4] CF583R and CF597R enable localization precision of approximately 10 nm laterally and 20 nm axially.[7]

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).[5] CF640R and CF680R have been validated for stimulated emission depletion (STED) microscopy.[12] Several dyes have been employed in structured illumination microscopy (SIM).[13] CF660C and CF680 have been validated for MINFLUX nanoscopy using standard GLOX+MEA photoswitching buffers.[14]

Representative spectral and validation data

Spectral properties and reported super-resolution validations for selected CF dyes
DyeEx (nm)Em (nm)ε (M⁻¹cm⁻¹)Notes
CF35034744818,000UV excitable
CF405S40443133,000405 nm excitable
CF405M40845241,000405 nm excitable
CF405L39554524,000405 nm excitable, large Stokes shift
CF43042649840,000405 nm excitable, green emission
CF44044051540,000405 nm excitable, green emission
CF45045053840,000
CF488A49051570,000Validated for STORM, TIRF[15]
CF503R50354290,000
CF514514~530105,000
CF532532~55096,000
CF535ST53556895,000Rhodamine-based; designed for STORM
CF543543~560100,000
CF550R550~570100,000
CF555555565150,000
CF568562583100,000Validated for STORM[16]
CF570570~590150,000
CF583583606150,000
CF583R586609100,000Rhodamine-based; validated for STORM[7]
CF594593614115,000
CF597R597619115,000Validated for STORM[7]
CF620R620~642115,000
CF633630~650100,000
CF640R642662105,000Rhodamine-based; validated for STED[12]
CF647650665240,000Validated for STORM[4]
CF647Plus652668240,000
CF660C667685200,000Validated for STORM, MINFLUX[4][14]
CF660R660682100,000Rhodamine-based
CF680681698210,000Validated for STORM[4]
CF680R680701140,000Rhodamine-based; validated for STED[12]
CF700696~719240,000
CF710712736115,000
CF725729750120,000
CF740~740~760105,000Rhodamine-based
CF750755777250,000Validated for STORM[17]
CF770770797220,000
CF790784806210,000
CF800797816210,000
CF820822835253,000
CF850852870—
CF870876896—
RPE-Astral™616496, 546, 566617—FRET tandem dye for flow cytometry[14]
RPE-Astral™775496, 546, 565774—FRET tandem dye for flow cytometry[14]
APC-Astral™813633, 638813—FRET tandem dye for flow cytometry[14]

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.[3][18]

See also

References

  1. ↑ Goetz C, Hammerbeck C, Bonnevier J. "Flow Cytometry Basics for the Non-Expert." Techniques in Life Science and Biomedicine for the Non-Expert (2018).
  2. 1 2 Kist TB. "Fluorescent dye labels and stains: A database of photophysical properties." In Fluorescent Dye Labels and Stains: A Database of Photophysical Properties (2023).
  3. 1 2 3 4 5 6 7 8 9 10 United States Patent and Trademark Office. Patent US8709830B2: "Fluorescent dyes, fluorescent dye kits, and methods of preparing labeled molecules." Issued April 29, 2014. https://patents.google.com/patent/US8709830B2
  4. 1 2 3 4 5 6 Lehmann M, Lichtner G, Klenz H, Schmoranzer J. "Novel organic dyes for multicolor localization-based super-resolution microscopy." Journal of Biophotonics 9(1-2):161-170 (2016). https://doi.org/10.1002/jbio.201500119
  5. 1 2 Diekmann R, Kahnwald M, Schoenit A, Deschamps J, Matti U, Ries J. "Optimizing imaging speed and excitation intensity for single molecule localization microscopy." Nature Methods 17:909–912 (2020). https://doi.org/10.1038/s41592-020-0918-5
  6. ↑ Staff. "Fluorescence Reimagined: Improving Imaging with Chemistry." The Scientist (2026). https://www.the-scientist.com/fluorescence-reimagined-improving-imaging-with-chemistry-73887
  7. 1 2 3 4 5 6 7 8 9 10 Wang B, Xiong M, Susanto J, Li X, Leung WY, Xu K. "Transforming Rhodamine Dyes for (d)STORM Super-Resolution Microscopy via 1,3-Disubstituted Imidazolium Substitution." Angewandte Chemie International Edition 61(19):e202113612 (2022). https://doi.org/10.1002/ANIE.202113612
  8. ↑ Ferrer-Font L, Mehta P, Harmos P, Schmidt AJ, Chappell S, Price KM, Hermans IF, Ronchese F, Le Gros G, Larsen M, Peng L. "High-dimensional analysis of intestinal immune cells during helminth infection." eLife 9:e51678 (2020). https://doi.org/10.7554/eLife.51678
  9. ↑ Chen F, Tillberg PW, Boyden ES. "Optical imaging. Expansion microscopy." Science 347(6221):543-548 (2015). https://doi.org/10.1126/science.1260088
  10. ↑ Alvero AB, Mor G (Eds.). "Detection of Cell Death Mechanisms: Methods and Protocols." Humana Press (2021).
  11. ↑ Mao F, McGarraugh PG, Madrid AS, Leung WY, Roberts LM. "Nucleic acid modifying agents and uses thereof." U.S. Patent No. 10,570,463 B2. Washington, DC: U.S. Patent and Trademark Office (2020). https://patents.google.com/patent/US10570463B2/en
  12. 1 2 3 Heilemann M, van de Linde S, Schüttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M. "Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes." Angewandte Chemie International Edition 47(33):6172-6176 (2008). https://doi.org/10.1002/anie.200802376
  13. ↑ Bowler M, Kong D, Sun S, Nanjundappa R, Evans L, Farmer V, Holland A, Mahjoub MR, Sui H, Loncarek J. "High-resolution characterization of centriole distal appendage morphology and dynamics by correlative STORM and electron microscopy." Nature Communications 10(1):435 (2019). https://doi.org/10.1038/s41467-018-08216-4
  14. 1 2 3 4 5 Staff. "Growing Antibody Collection Featuring Astral Leap™ Tandem Dyes." FluoroFinder: New Fluorescent Dyes of 2024 (2024). https://fluorofinder.com/fluorescent-dyes-of-2024/
  15. ↑ Zanetti-Domingues LC, Martin-Fernandez ML, Needham SR, Rolfe DJ, Clarke DT. "A systematic investigation of differential effects of cell culture substrates on the extent of artifacts in single-molecule tracking." PLoS ONE 8(9):e74200 (2013). https://doi.org/10.1371/journal.pone.0074200
  16. ↑ Früh SM, Matti U, Spycher PR, Rubini M, Lickert S, Schlichthaerle T, Jungmann R, Vogel V, Hall H, Sapra KT. "Site-specifically-labeled antibodies for super-resolution microscopy reveal In Situ linkage errors." ACS Nano 15(8):12161-12170 (2021). https://doi.org/10.1021/acsnano.1c03677
  17. ↑ Turkowyd B, Virant D, Endesfelder U. "From single molecules to life: microscopy at the nanoscale." Analytical and Bioanalytical Chemistry 408:6885-6911 (2016). https://doi.org/10.1007/s00216-016-9781-8
  18. ↑ European Patent Office. Patent EP2223086B1: "Fluorescent dyes." Priority date 2007. https://patents.google.com/patent/EP2223086B1
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