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

Journal name:
Nature Chemistry
Volume:
5,
Pages:
132–139
Year published:
DOI:
doi:10.1038/nchem.1546
Received
Accepted
Published online

Abstract

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

Figures

  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.

Compounds

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

References

  1. Hinner, M. & Johnsson, K. How to obtain labeled proteins and what to do with them. Curr. Opin. Biotechnol. 21, 766776 (2010).
  2. Schneckenburger, H. et al. Light exposure and cell viability in fluorescence microscopy. J. Microsc. 245, 311318 (2012).
  3. Pellett, P. A. et al. Two-color STED microscopy in living cells. Biomed Opt Express 2, 23642371 (2011).
  4. Wombacher, R. et al. Live-cell super-resolution imaging with trimethoprim conjugates. Nature Methods 7, 717719 (2010).
  5. Jones, S. A., Shim, S. H., He, J. & Zhuang, X. Fast, three-dimensional super-resolution imaging of live cells. Nature Methods 8, 499508 (2011).
  6. van de Linde, S., Heilemann, M. & Sauer, M. Live-cell super-resolution imaging with synthetic fluorophores. Annu. Rev. Phys. Chem. 61, 519540 (2012).
  7. Koide, Y., Urano, Y., Hanaoka, K., Terai, T. & Nagano, T. Evolution of group 14 rhodamines as platforms for near-infrared fluorescence probes utilizing photoinduced electron transfer. ACS Chem. Biol. 6, 600608 (2011).
  8. Egawa, T. et al. Development of a far-red to near-infrared fluorescence probe for calcium ion and its application to multicolor neuronal imaging. J. Am. Chem. Soc. 133, 1415714159 (2011).
  9. Koide, Y., Urano, Y., Hanaoka, K., Terai, T. & Nagano, T. Development of an Si-rhodamine-based far-red to near-infrared fluorescence probe selective for hypochlorous acid and its applications for biological imaging. J. Am. Chem. Soc. 133, 56805682 (2011).
  10. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nature Biotechnol. 21, 8689 (2003).
  11. Keppler, A., Pick, H., Arrivoli, C., Vogel, H. & Johnsson, K. Labeling of fusion proteins with synthetic fluorophores in live cells. Proc. Natl Acad. Sci. USA 101, 99559959 (2004).
  12. Wang, T. et al. Spirolactonized Si-rhodamine: a novel NIR fluorophore utilized as a platform to construct Si-rhodamine-based probes. Chem. Commun. 48, 87818783 (2012).
  13. Åkerlöf, G. & Short, A. O. The dielectric constant of dioxane–water mixtures between 0 and 80°. J. Am. Chem. Soc. 58, 12411243 (1936).
  14. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128136 (2008).
  15. Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373382 (2008).
  16. Held, M. et al. CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging. Nature Methods 7, 747754 (2010).
  17. Hell, S. W. Microscopy and its focal switch. Nature Methods 6, 2432 (2009).
  18. Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M. & Zhuang, X. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nature Methods 8, 10271036 (2011).
  19. Schnell, U., Dijk, F., Sjollema, K. A. & Giepmans, B. N. Immunolabeling artifacts and the need for live-cell imaging. Nature Methods 9, 152158 (2012).
  20. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793795 (2006).
  21. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 16421645 (2006).
  22. Folling, J. et al. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nature Methods 5, 943945 (2008).
  23. Heilemann, M. et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew. Chem. Int. Ed. 47, 61726176 (2008).
  24. Steinhauer, C., Forthmann, C., Vogelsang, J. & Tinnefeld, P. Superresolution microscopy on the basis of engineered dark states. J. Am. Chem. Soc. 130, 1684016841 (2008).
  25. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780782 (1994).
  26. Hein, B. et al. Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins. Biophys. J. 98, 158163 (2010).
  27. Berning, S., Willig, K. I., Steffens, H., Dibaj, P. & Hell, S. W. Nanoscopy in a living mouse brain. Science 335, 551 (2012).
  28. Morozova, K. S. et al. Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy. Biophys. J. 99, L13L15 (2010).
  29. David, R. Cell cycle: building the centriole. Nature Rev. Mol. Cell. Biol. 12, 342 (2011).
  30. Azimzadeh, J. & Marshall, W. F. Building the centriole. Curr. Biol. 20, R816R825 (2010).
  31. Bettencourt-Dias, M. & Glover, D. M. Centrosome biogenesis and function: centrosomics brings new understanding. Nature Rev. Mol. Cell. Biol. 8, 451463 (2007).
  32. Gache, V. et al. Xenopus meiotic microtubule-associated interactome. PLoS One 5, e9248 (2010).
  33. Korvatska, O. et al. Mutations in the TSGA14 gene in families with autism spectrum disorders. Am. J. Med. Genet. B Neuropsychiatr. Genet. 156, 303311 (2011).
  34. Lee, J. E. et al. CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium. Nature Genet. 44, 193199 (2012).
  35. Lang, K. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nature Chem. 4, 298304 (2012).
  36. Plass, T. et al. Amino acids for Diels–Alder reactions in living cells. Angew. Chem. Int. Ed. 51, 41664170 (2012).
  37. Lang, K. et al. Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels–Alder reactions. J. Am. Chem. Soc. 134, 1031710320 (2012).
  38. Liu, C. C. & Schultz, P. G. Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413444 (2010).
  39. Plass, T., Milles, S., Koehler, C., Schultz, C. & Lemke, E. A. Genetically encoded copper-free click chemistry. Angew. Chem. Int. Ed. 50, 38783881 (2011).
  40. Saito, T. & Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 240, 237246 (2001).
  41. Niu, L. & Yu, J. Investigating intracellular dynamics of FtsZ cytoskeleton with photoactivation single-molecule tracking. Biophys. J. 95, 20092016 (2008).
  42. Mueller, V. et al. STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells. Biophys. J. 101, 16511660 (2011).

Download references

Author information

  1. These authors contributed equally to this work

    • Gražvydas Lukinavičius &
    • Keitaro Umezawa

Affiliations

  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

Contributions

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary information (2,304 KB)

    Supplementary information

Movies

  1. Supplementary information (12,823 KB)

    Supplementary movie 1

  2. Supplementary information (837 KB)

    Supplementary movie 2

Additional data