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
Dye Ex (nm) Em (nm) ε (M⁻¹cm⁻¹) Notes
CF350 347 448 18,000 UV excitable
CF405S 404 431 33,000 405 nm excitable
CF405M 408 452 41,000 405 nm excitable
CF405L 395 545 24,000 405 nm excitable, large Stokes shift
CF430 426 498 40,000 405 nm excitable, green emission
CF440 440 515 40,000 405 nm excitable, green emission
CF450 450 538 40,000
CF488A 490 515 70,000 Validated for STORM, TIRF[15]
CF503R 503 542 90,000
CF514 514 ~530 105,000
CF532 532 ~550 96,000
CF535ST 535 568 95,000 Rhodamine-based; designed for STORM
CF543 543 ~560 100,000
CF550R 550 ~570 100,000
CF555 555 565 150,000
CF568 562 583 100,000 Validated for STORM[16]
CF570 570 ~590 150,000
CF583 583 606 150,000
CF583R 586 609 100,000 Rhodamine-based; validated for STORM[7]
CF594 593 614 115,000
CF597R 597 619 115,000 Validated for STORM[7]
CF620R 620 ~642 115,000
CF633 630 ~650 100,000
CF640R 642 662 105,000 Rhodamine-based; validated for STED[12]
CF647 650 665 240,000 Validated for STORM[4]
CF647Plus 652 668 240,000
CF660C 667 685 200,000 Validated for STORM, MINFLUX[4][14]
CF660R 660 682 100,000 Rhodamine-based
CF680 681 698 210,000 Validated for STORM[4]
CF680R 680 701 140,000 Rhodamine-based; validated for STED[12]
CF700 696 ~719 240,000
CF710 712 736 115,000
CF725 729 750 120,000
CF740 ~740 ~760 105,000 Rhodamine-based
CF750 755 777 250,000 Validated for STORM[17]
CF770 770 797 220,000
CF790 784 806 210,000
CF800 797 816 210,000
CF820 822 835 253,000
CF850 852 870 —
CF870 876 896 —
RPE-Astral™616 496, 546, 566 617 — FRET tandem dye for flow cytometry[14]
RPE-Astral™775 496, 546, 565 774 — FRET tandem dye for flow cytometry[14]
APC-Astral™813 633, 638 813 — 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. ^ a b 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. ^ a b c d e f g h i j 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. ^ a b c d e f 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. ^ a b 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. ^ a b c d e f g h i j 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. ^ a b c 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. ^ a b c d e 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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