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
editDevelopment 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
editCF 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
editApplications 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
editThe 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
edit| 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
editSee also
editReferences
edit- ↑ Goetz C, Hammerbeck C, Bonnevier J. "Flow Cytometry Basics for the Non-Expert." Techniques in Life Science and Biomedicine for the Non-Expert (2018).
- 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).
- 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
- 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
- 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
- ↑ Staff. "Fluorescence Reimagined: Improving Imaging with Chemistry." The Scientist (2026). https://www.the-scientist.com/fluorescence-reimagined-improving-imaging-with-chemistry-73887
- 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
- ↑ 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
- ↑ Chen F, Tillberg PW, Boyden ES. "Optical imaging. Expansion microscopy." Science 347(6221):543-548 (2015). https://doi.org/10.1126/science.1260088
- ↑ Alvero AB, Mor G (Eds.). "Detection of Cell Death Mechanisms: Methods and Protocols." Humana Press (2021).
- ↑ 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
- 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
- ↑ 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
- 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/
- ↑ 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
- ↑ 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
- ↑ 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
- ↑ European Patent Office. Patent EP2223086B1: "Fluorescent dyes." Priority date 2007. https://patents.google.com/patent/EP2223086B1