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CF™ Dyes for Multi-Color Super-Resolution Microscopy

Recent publications comparing synthetic dyes for super-resolution imaging have shown CF™ dyes give the best performance for multiple methods. The superior brightness, photostability, and photochemical switching properties of certain CF™ dyes are ideal for 3-D SIM, multi-color 3-D STORM, and other super-resolution and single molecule imaging techniques.

As Lehmann and colleagues report in their recent publication in the Journal of Biophotonics, “the spectrally close dyes CF647 and CF680 comprise an optimal dye pair for spectral demixing-based, registration free multicolor dSTORM with low crosstalk. Combining this dye pair with the separately excited CF568 we performed 3-color dSTORM to image the relative nanoscale distribution of components of the endocytic machinery and the cytoskeleton.”

To learn more, click on one of the references below or download our Super-Resolution Microscopy Flyer. We now offer single label secondary antibody conjugates, with one dye molecule per antibody, for STORM applications. Find out more about CF™ dyes.

 

Comparison of conventional wide-field microscopy (left) with STORM (right) using CF™ dye conjugates. Fixed cells were stained with mouse anti-tubulin antibody followed by CF™ dye conjugated anti-mouse secondary antibody (top row: CF™647, middle row: CF™660C, bottom row: CF™680). See the Super-Resolution Microscopy Flyer for details of STORM imaging. Images courtesy of Sam Kenny and Professor Ke Xu, College of Chemistry, University of California, Berkeley.
Comparison of conventional wide-field microscopy (left) with STORM (right) using CF™ dye conjugates. Fixed cells were stained with mouse anti-tubulin antibody followed by CF™ dye conjugated anti-mouse secondary antibody (top row: CF™647, middle row: CF™660C, bottom row: CF™680). See the Super-Resolution Microscopy Flyer for details of STORM imaging. Images courtesy of Sam Kenny and Professor Ke Xu, College of Chemistry, University of California, Berkeley.

 

 

CF568 STORM
CF™568 (left) produces better images than Cy®3b (right) in 3-D STORM microscopy. Fixed cells were stained with mouse anti-tubulin antibody followed by dye-conjugated anti-mouse secondary antibodies. See the Super-Resolution Microscopy Flyer for details of STORM imaging. Images courtesy of Sam Kenny and Professor Ke Xu, College of Chemistry, University of California, Berkeley.

 

References for CF™ Dyes in Super-Resolution Microscopy

DyeAbs/Em maximaExtinction coefficientSuper resolution applicationReferences
CF™405M408/45241,000SIM Markaki, Y. et al. (2013). Fluorescence In Situ Hybridization Applications for Super-Resolution 3D Structured Illumination Microscopy. Methods Mol Biol 950, 43-64.

Miron, E. et. al. (2016). In Vivo and In Situ Replication Labeling Methods for Super-resolution Structured Illumination Microscopy of Chromosome Territories and Chromatin Domains. in Mark C. Leake (ed.), Chromosome Architecture: Methods and Protocols, Methods in Molecular Biology, vol. 1431, 127-140.
CF™488A490/51570,000TIRFZanetti-Domingues, L.C. et al. (2013). Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding. PLoS ONE 8(9): e74200.
CF™535ST535/56895,000STORMCollaborator communication; for more information contact techsupport@biotium.com.
CF™568562/583100,000TIRF, STORMKaranasios, E. et al. (2016). Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles. Nature Communications 7:12420 | DOI: 10.1038/ncomms12420.

Lehmann, M. et al. (2015). Novel organic dyes for multicolor localization-based super-resolution microscopy. J Biophotonics DOI 10.1002/jbio.201500119

Turkowyd, B. et al. (2016). From single molecules to life: microscopy at the nanoscale. Anal Bioanal Chem DOI 10.1007/s00216-016-9781-8

Zanetti-Domingues, L.C. et al. (2013). Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding. PLoS ONE 8(9): e74200.

Zhang, M. et al. (2015). Translocation of interleukin-1β into a vesicle intermediate in autophagy-mediated secretion. eLife 2015;10.7554/eLife.11205
CF™594ST593/614115,000STORMCollaborator communication; for more information contact techsupport@biotium.com. Please note: CF™594ST is a unique dye designed specifically for STORM. Our original CF™594 dye is not suitable for STORM.
CF™633630/650100,000TIRF, FIONA, gSHRImPBosch, P. J. et al. (2014). Evaluation of fluorophores to label SNAP-tag fused proteins for multicolor single-molecule tracking microscopy in live cells. Biophys J 107, 803-814.

Zanetti-Domingues, L.C. et al. (2013). Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding. PLoS ONE 8(9): e74200.

Kim, H. J., and Selvin, P. R. (2013). Fluorescence Imaging with One Nanometer Accuracy. SpringerReference Encyclopedia of Biophysics

Simonson, P. D.,Rothenberg, E., and Selvin, P. R. (2011). Single-molecule-based super-resolution images in the presence of multiple fluorophores. Nano Lett 11, 5090-5096. DOI:10.1021/nl203560r
CF™640R642/662105,000TIRF, FLImPBosch, P. J. et al. (2014). Evaluation of fluorophores to label SNAP-tag fused proteins for multicolor single-molecule tracking microscopy in live cells. Biophys J 107, 803-814.

Martin-Fernandez, M. L. et al. (2013). A 'pocket guide' to total internal reflection fluorescence. J Microsc 252, 16-22.

Zanetti-Domingues, L.C. et al. (2013). Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding. PLoS ONE 8(9): e74200.

Needham, S.R.,et al. (2015). Determining the geometry of oligomers of the human epidermal growth factor family on cells with <10 nm resolution. Biochem Soc Trans 43, 309–314.
CF™647650/665240,000STORMLehmann, M. et al. (2015). Novel organic dyes for multicolor localization-based super-resolution microscopy. J Biophotonics DOI 10.1002/jbio.201500119

Olivier, N. et al. (2013). Simple buffers for 3D STORM microscopy. Biomed Opt Express 4, 885-899.

Turkowyd, B. et al. (2016). From single molecules to life: microscopy at the nanoscale. Anal Bioanal Chem DOI 10.1007/s00216-016-9781-8
CF™660C667/685200,000STORMTurkowyd, B. et al. (2016). From single molecules to life: microscopy at the nanoscale. Anal Bioanal Chem DOI 10.1007/s00216-016-9781-8

Zhang, Z. et al. (2015). Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy. Nature Methods doi:10.1038/nmeth.3528
CF™680681/698210,000STORMFrüh, S.M. et al. (2015). Molecular architecture of native fibronectin fibrils. Nature Communications 6, 7275.

Lehmann, M. et al. (2015). Novel organic dyes for multicolor localization-based super-resolution microscopy. J Biophotonics DOI 10.1002/jbio.201500119

Platonova, E. et al. (2015). A Simple Method for GFP- and RFP-based Dual Color Single-Molecule Localization Microscopy. ACS Chem. Biol.10(6),1411–1416.

Platonova, E. et al. (2015). Single-molecule microscopy of molecules tagged with GFP or mRFP derivatives in mammalian cells using nanobody binders. Methods doi: http://dx.doi.org/10.1016/j.ymeth.2015.06.018

Turkowyd, B. et al. (2016). From single molecules to life: microscopy at the nanoscale. Anal Bioanal Chem DOI 10.1007/s00216-016-9781-8

Venkataramani, V. et al. (2016). SuReSim: simulating localization microscopy experiments from ground truth models. Nature Methods 13, 319-321. doi:10.1038/nmeth.3775

Winterflood, C.M. et al. (2015). Dual-Color 3D Superresolution Microscopy by Combined Spectral-Demixing and Biplane Imaging. Biophys J. 109, 3–6.

Zhang, Z. et al. (2015). Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy. Nature Methods doi:10.1038/nmeth.3528
CF™680R680/701140,000STED, single-molecule spectroscopyGörlitz, F. et al. (2014). A STED Microscope Designed for Routine Biomedical Applications. Progress Electromagnetics Res 147, 57-68.

König, I. et al. (2015). Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells. Nature Methods doi:10.1038/nmeth.3475.

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