A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins

Journal name:
Nature Chemistry
Year published:
Published online


The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proved to be extremely difficult. Here, we introduce a biocompatible near-infrared silicon–rhodamine probe that can be coupled specifically to proteins using different labelling techniques. Importantly, its high permeability and fluorogenic character permit the imaging of proteins in living cells and tissues, and its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.

At a glance


  1. SiR dyes used for SNAP-, CLIP-, Halo-tag and tetrazine labelling.
    Figure 1: SiR dyes used for SNAP-, CLIP-, Halo-tag and tetrazine labelling.

    a, Structures of SiR dyes, TMR and the formation of spirolactone of SiR-carboxyl. b, Normalized integral of absorption spectra of the zwitterion region of SiR and TMR derivatives in waterdioxane mixtures as a function of dielectric constant. Note that absorbance at ɛ = 80 (0% of dioxane) is affected by the aggregation of fluorophores. BG-TMR represents TMR coupled to BG (Supplementary Fig. S2)11. c, Absorption spectra of 2.5 µM SiR-SNAP measured in ethanol (red), Tris-buffered saline (TBS) buffer with (dashed black) and without (solid black) 0.1% sodium dodecyl sulfate (SDS).

  2. Three-colour confocal fluorescence microscopy of the tagged proteins.
    Figure 2: Three-colour confocal fluorescence microscopy of the tagged proteins.

    ad, SNAP (red in c), CLIP (red in a,b) and Halo-tagged proteins (red in d) in living HeLa cells expressing EGFP-α-tubulin (green) and H2B-mCherry (blue)16. The characteristic staining of the fusion proteins demonstrates the suitability of SiR-carboxyl derivatives for live-cell imaging. Z-stacks of images were deconvolved using the Huygens Essentials package and presented as MIPs. Scale bar, 10 µm.

  3. Ex vivo labelling of a rat brain with SiR-SNAP.
    Figure 3: Ex vivo labelling of a rat brain with SiR-SNAP.

    a, Scheme of in utero electroporation. Plasmid DNA is injected into an E16 rat embryo in utero through a micropipette and then electroporated with electrodes. The red square corresponds to the region shown in (b). b, SNAP and GFP plasmids (ratio 1:1) were introduced into a subset of neural progenitors at E16 by in utero electroporation. At E19, brains were sectioned and stained with SiR-SNAP and Hoechst. Scale bar, 200 µm. c, Images of the electroporated cortical neurons at a higher magnification (yellow box in b). The excellent overlap of the GFP and SiR-SNAP signals demonstrates the specificity of the labelling. Scale bar, 50 µm.

  4. Live-cell GSDIM/STORM imaging of nuclear localized H2B-SNAP-SiR.
    Figure 4: Live-cell GSDIM/STORM imaging of nuclear localized H2B-SNAP-SiR.

    a, Wide-field image of H2B-SNAP-SiR does not allow the detection of substructures. b, Single frame image showing the stochastic fluorescence (blinking) of individual molecules. c, GSDIM/STORM images reconstructed from 10,000 raw images (c′ was taken ten minutes after c). The enhancement in resolution permitted the detection of substructures. Scale bar, 5 µM (inset, 500 nm).

  5. Confocal and STED imaging of Cep41 protein localization in living U2OS cells.
    Figure 5: Confocal and STED imaging of Cep41 protein localization in living U2OS cells.

    a, Schematic presentation of the centrosome structure. b, Confocal two-colour imaging of SNAP-Cep41 (red)-expressing cells stained with SiR-SNAP. Nuclear DNA was stained with Hoechst 33342 (blue). Scale bar, 10 µm. c, Comparison of confocal (left) and STED microscopy (middle) images of Cep41-SNAP bound to microtubules, along with an intensity line profile (right) obtained by averaging the profiles of seven different microtubule sections in the image. Scale bar, 500 nm. d, Comparison of confocal (left) and STED (middle) microscopy images of SNAP-Cep41 localized at the centrosome with an intensity line profile (right) along the white dotted line marked in the images. The full width at half maximum (FWHM) of the imaged structures was obtained by fitting fluorescence-intensity profiles to Gauss or Lorentz distributions (OriginPro 8.1, http://www.originlab.com/). Two separated Lorentz distributions are indicated by grey dashed lines for the STED profile fitting. Distance between the peaks of the double Lorentz fitting was taken as the diameter of the structure. The diffuse signal visible in the top-left corner is the second centriole, which is located outside the focal plane. A corresponding two-colour image of SNAP-Cep41 and the centrosomal marker GFP-Centrin2 is presented in Supplementary Fig. S8. Scale bar, 500 nm. Numbers are presented as the fitted value ± standard error of the fit.

  6. Site-specific labelling of genetically encoded UAAs with SiR-tetrazine.
    Figure 6: Site-specific labelling of genetically encoded UAAs with SiR-tetrazine.

    a, Structures of UAA TCO and SCO. b, Intact E. coli cells expressing wild-type GFP (GFPWT), GFPTAG→TCO or GFPTAG→SCO were incubated with 20 µM SiR-tetrazine for ten minutes and analysed via SDS–PAGE. A fluorescent band (635 nm excitation) that indicates the successful labelling of GFP with SiR-tetrazine was observed only for GFPTAG→TCO, which confirms excellent bioorthogonality (black arrowhead indicates the height of the GFP). c, Live E. coli cells expressing GFPWT (upper row) and GFPTAG→TCO (lower row) were incubated for ten minutes with 20 µM SiR-tetrazine. After overnight washing, the cells were imaged for green (first column) or red (second column) fluorescence (third column shows the overlay of both channels with DIC image). Although GFP fluorescence was observed for all the samples, red fluorescence that originated from covalently reacted SiR-tetrazine was detected exclusively for GFPTAG→TCO (lower row), even though tRNAPyl/PylAF and TCO were also present during the expression in GFPWT cells (upper row). GFPTAG→SCO as an additional control is shown in Supplementary Fig. S9. Scale bar, 5 µm.


8 compounds View all compounds
  1. N-(10-(5-Carboxy-2-methylphenyl)-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)-N-methylmethanaminium
    Compound SiR-methyl N-(10-(5-Carboxy-2-methylphenyl)-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)-N-methylmethanaminium
  2. 4-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-(7-(dimethylamino)-3-(dimethyliminio)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10-yl)benzoate
    Compound SiR-SNAP 4-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-(7-(dimethylamino)-3-(dimethyliminio)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10-yl)benzoate
  3. 4-Carboxy-2-(7-(dimethylamino)-3-(dimethyliminio)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10-yl)benzoate
    Compound SiR-carboxyl 4-Carboxy-2-(7-(dimethylamino)-3-(dimethyliminio)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10-yl)benzoate
  4. 4-((4-(((4-Aminopyrimidin-2-yl)oxy)methyl)benzyl)carbamoyl)-2-(7-(dimethylamino)-3-(dimethyliminio)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10-yl)benzoate
    Compound SiR-CLIP 4-((4-(((4-Aminopyrimidin-2-yl)oxy)methyl)benzyl)carbamoyl)-2-(7-(dimethylamino)-3-(dimethyliminio)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10-yl)benzoate
  5. 2-(7-(Dimethylamino)-3-(dimethyliminio)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10-yl)-4-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)carbamoyl)benzoate
    Compound SiR-tetrazine 2-(7-(Dimethylamino)-3-(dimethyliminio)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10-yl)-4-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)carbamoyl)benzoate
  6. 4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(7-(dimethylamino)-3-(dimethyliminio)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10-yl)benzoate
    Compound SiR-Halo 4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(7-(dimethylamino)-3-(dimethyliminio)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10-yl)benzoate
  7. (2S)-2-Amino-6-((((E)-cyclooct-4-en-1-yloxy)carbonyl)amino)hexanoic acid
    Compound TCO (2S)-2-Amino-6-((((E)-cyclooct-4-en-1-yloxy)carbonyl)amino)hexanoic acid
  8. (2S)-2-Amino-6-(((cyclooct-2-yn-1-yloxy)carbonyl)amino)hexanoic acid
    Compound SCO (2S)-2-Amino-6-(((cyclooct-2-yn-1-yloxy)carbonyl)amino)hexanoic acid


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Author information

  1. These authors contributed equally to this work

    • Gražvydas Lukinavičius &
    • Keitaro Umezawa


  1. Ecole Polytechnique Fédérale de Lausanne, Institute of Chemical Sciences and Engineering (ISIC), Institute of Bioengineering, National Centre of Competence in Research (NCCR) in Chemical Biology, 1015 Lausanne, Switzerland

    • Gražvydas Lukinavičius,
    • Keitaro Umezawa,
    • Luc Reymond &
    • Kai Johnsson
  2. Ecole Polytechnique Fédérale de Lausanne, Laboratory of Experimental Biophysics, NCCR in Chemical Biology, 1015 Lausanne, Switzerland

    • Nicolas Olivier &
    • Suliana Manley
  3. Max-Planck-Institute for Biophysical Chemistry, Department NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany

    • Alf Honigmann,
    • Veronika Mueller &
    • Christian Eggeling
  4. European Molecular Biology Laboratory, Mouse Biology Unit, via Ramarini 32, 00015 Monterotondo (RM), Italy

    • Guoying Yang &
    • Paul Heppenstall
  5. European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

    • Tilman Plass,
    • Carsten Schultz &
    • Edward A. Lemke
  6. New England Biolabs Inc., 240 County Road, Ipswich, Massachusetts 01938, USA

    • Ivan R. Corrêa Jr
  7. Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

    • Zhen-Ge Luo
  8. Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, UK

    • Christian Eggeling


All authors planned the experiments and co-wrote the paper. K.U. designed the structure of SiR-carboxyl. K.U., L.R. and I.C. performed the chemical syntheses. G.L., K.U. and L.R. characterized the dyes. G.L., A.H. and V.M. performed the confocal and STED microscopy with subsequent data analysis. N.O. and S.M. performed the GSDIM/STORM imaging and data analysis. T.P., C.S. and E.A.L performed the amber suppression experiments and analysis. G.Y., Z-G.L. and P.H. performed the labelling in brain sections.

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