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Deep-tissue SWIR imaging using rationally designed small red-shifted near-infrared fluorescent protein

An Author Correction to this article was published on 02 February 2023

This article has been updated


Applying rational design, we developed 17 kDa cyanobacteriochrome-based near-infrared (NIR-I) fluorescent protein, miRFP718nano. miRFP718nano efficiently binds endogenous biliverdin chromophore and brightly fluoresces in mammalian cells and tissues. miRFP718nano has maximal emission at 718 nm and an emission tail in the short-wave infrared (SWIR) region, allowing deep-penetrating off-peak fluorescence imaging in vivo. The miRFP718nano structure reveals the molecular basis of its red shift. We demonstrate superiority of miRFP718nano-enabled SWIR imaging over NIR-I imaging of microbes in the mouse digestive tract, mammalian cells injected into the mouse mammary gland and NF-kB activity in a mouse model of liver inflammation.

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Fig. 1: Properties of red-shifted miRFP718nano in comparison with parental miRFP670nano.
Fig. 2: miRFP718nano as a reporter of inflammation.

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Data availability

The data supporting the findings of this study are available within the article and its Supplementary Information. All other data that support the findings of the study are available from the corresponding author upon request. The main plasmids constructed in this study, their maps and sequences are deposited to Addgene. For the structure of miRFP670nano: PDB ID 6MGH, For the structure of miRFP709 PDB ID: 5VIQ,

Code availability

The miRFP718nano nucleotide sequence in GenBank is MW627296.1, The miRFP718nano structural data were deposited at the Protein Data Bank (PDB ID 7LSD),

Change history


  1. Frangioni, J. V. In vivo near-infrared fluorescence imaging. Curr. Opin. Chem. Biol. 7, 626–634 (2003).

    Article  CAS  Google Scholar 

  2. Scholkmann, F. et al. A review on continuous wave functional near-infrared spectroscopy and imaging instrumentation and methodology. Neuroimage 85, 6–27 (2014).

    Article  Google Scholar 

  3. Welsher, K. et al. A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice. Nat. Nanotechnol. 4, 773–780 (2009).

    Article  CAS  Google Scholar 

  4. Smith, A. M., Mancini, M. C. & Nie, S. Bioimaging: second window for in vivo imaging. Nat. Nanotechnol. 4, 710–711 (2009).

    Article  CAS  Google Scholar 

  5. Feng, Z. et al. Perfecting and extending the near-infrared imaging window. Light Sci. Appl. 10, 197 (2021).

    Article  CAS  Google Scholar 

  6. Shcherbakova, D. M., Stepanenko, O. V., Turoverov, K. K. & Verkhusha, V. V. Near-infrared fluorescent proteins: multiplexing and optogenetics across scales. Trends Biotechnol. 36, 1230–1243 (2018).

    Article  CAS  Google Scholar 

  7. Oliinyk, O. S., Chernov, K. G. & Verkhusha, V. V. Bacterial phytochromes, cyanobacteriochromes and allophycocyanins as a source of near-infrared fluorescent probes. Int. J. Mol. Sci. 18, 1691 (2017).

    Article  Google Scholar 

  8. Oliinyk, O. S., Shemetov, A. A., Pletnev, S., Shcherbakova, D. M. & Verkhusha, V. V. Smallest near-infrared fluorescent protein evolved from cyanobacteriochrome as versatile tag for spectral multiplexing. Nat. Commun. 10, 279 (2019).

    Article  Google Scholar 

  9. Oliinyk, O. S. et al. Single-domain near-infrared protein provides a scaffold for antigen-dependent fluorescent nanobodies. Nat. Methods 19, 740–750 (2022).

    Article  CAS  Google Scholar 

  10. Shcherbakova, D. M. et al. Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging. Nat. Commun. 7, 12405 (2016).

    Article  CAS  Google Scholar 

  11. Yu, D. et al. A naturally monomeric infrared fluorescent protein for protein labeling in vivo. Nat. Methods 12, 763–765 (2015).

    Article  Google Scholar 

  12. Komatsu, N. et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656 (2011).

    Article  CAS  Google Scholar 

  13. Shcherbakova, D. M. Near-infrared and far-red genetically encoded indicators of neuronal activity. J. Neurosci. Methods 362, 109314 (2021).

    Article  Google Scholar 

  14. Shcherbakova, D. M., Cox Cammer, N., Huisman, T. M., Verkhusha, V. V. & Hodgson, L. Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET. Nat. Chem. Biol. 14, 591–600 (2018).

    Article  CAS  Google Scholar 

  15. Li, L., Hsu, H. C., Verkhusha, V. V., Wang, L. V. & Shcherbakova, D. M. Multiscale photoacoustic tomography of a genetically encoded near-infrared FRET biosensor. Adv. Sci. 8, e2102474 (2021).

    Article  Google Scholar 

  16. Shemetov, A. A. et al. A near-infrared genetically encoded calcium indicator for in vivo imaging. Nat. Biotechnol. 39, 368–377 (2021).

    Article  CAS  Google Scholar 

  17. Baloban, M. et al. Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies. Chem. Sci. 8, 4546–4557 (2017).

    Article  CAS  Google Scholar 

  18. Chang, B. et al. A phosphorescent probe for in vivo imaging in the second near-infrared window. Nat. Biomed. Eng. 6, 629–639 (2021).

  19. Jeong, S. et al. Multiplexed in vivo imaging using size-controlled quantum dots in the second near-infrared window. Adv. Healthc. Mater. 7, e1800695 (2018).

    Article  Google Scholar 

  20. Robinson, J. T. et al. In vivo fluorescence imaging in the second near-infrared window with long circulating carbon nanotubes capable of ultrahigh tumor uptake. J. Am. Chem. Soc. 134, 10664–10669 (2012).

    Article  CAS  Google Scholar 

  21. Hugenholtz, F. & de Vos, W. M. Mouse models for human intestinal microbiota research: a critical evaluation. Cell. Mol. Life Sci. 75, 149–160 (2018).

    Article  CAS  Google Scholar 

  22. Hamano, N., Inada, T., Iwata, R., Asai, T. & Shingu, K. The alpha2-adrenergic receptor antagonist yohimbine improves endotoxin-induced inhibition of gastrointestinal motility in mice. Br. J. Anaesth. 98, 484–490 (2007).

    Article  CAS  Google Scholar 

  23. Wang, D. et al. Trans-illumination intestine projection imaging of intestinal motility in mice. Nat. Commun. 12, 1682 (2021).

    Article  CAS  Google Scholar 

  24. Liu, T., Zhang, L., Joo, D. & Sun, S. C. NF-κB signaling in inflammation. Signal Transduct. Target Ther. 2, 17023 (2017).

    Article  Google Scholar 

  25. Osorio, F. G., de la Rosa, J. & Freije, J. M. Luminescence-based in vivo monitoring of NF-κB activity through a gene delivery approach. Cell Commun. Signal 11, 19 (2013).

    Article  CAS  Google Scholar 

  26. Fushimi, K. & Narikawa, R. Phytochromes and cyanobacteriochromes: photoreceptor molecules incorporating a linear tetrapyrrole chromophore. Adv. Exp. Med Biol. 1293, 167–187 (2021).

    Article  CAS  Google Scholar 

  27. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  28. Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).

    Article  CAS  Google Scholar 

  29. Lamzin, V. S., Perrakis, A. & Wilson, K. S. in International Tables for Crystallography, Vol. F, Crystallography of Biological Macromolecules (eds Arnold E. et al.) 525–528 (Kluwer Academic Publishers, 2012).

  30. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D. Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  Google Scholar 

  31. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  32. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  33. Longo, P. A., Kavran, J. M., Kim, M. S. & Leahy, D. J. Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol. 529, 227–240 (2013).

    Article  CAS  Google Scholar 

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We thank N. Peitsaro and N. Aarnio from the Flow Cytometry Core Facility of the University of Helsinki for the technical assistance. This work was supported by grants from the US National Institutes of Health (NIH) (grant nos. GM122567, EB028143, NS111039 and NS115581), Chan Zuckerberg Initiative (grant no. 226178), Cancer Foundation Finland and Magnus Ehrnrooth Foundation. S.P. was supported in part by the NIH Intramural Research Program for the Vaccine Research Center of the National Institute of Allergy and Infectious Diseases.

Author information

Authors and Affiliations



O.S.O. developed and characterized the protein in vitro and in cultured cells. C.M. developed the SWIR imaging system, performed imaging and analyzed the data. C.T. developed the SWIR imaging system and measured fluorescence emission. H.S. performed animal surgeries. S.P. designed structural biology experiments and analyzed the data. M.B. purified the recombinant proteins and prepared cells for SWIR imaging. J.Y. planned and supervised the SWIR imaging experiments and analyzed the data. V.V.V. conceived, planned and supervised the whole project and together with J.Y., O.S.O. and S.P. wrote the manuscript. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Junjie Yao or Vladislav V. Verkhusha.

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Competing interests

The authors declare no competing interests.

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Peer review information

Nature Methods thanks Takeharu Nagai and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Structure of miRFP718nano and its red-shifted chromophore.

Overall structures of (a) miRFP670nano (PDB ID: 6MGH), (b) miRFP718nano, and (c) miRFP709 (PDB ID: 5VIQ). The biliverdin (BV) chromophores are shown in magenta. The PAS and GAF domains of miRFP709 are in cyan and yellow, respectively. The BV chromophores in (d) miRFP670nano, (e) miRFP718nano, and (f) miRFP709 bound to the respective Cys residues and their chemical formulas. Carbon, nitrogen, oxygen, and sulfur atoms are in white, blue, red, and yellow, respectively. Sticks representations show only rings A and B of the chromophores and Cys residues. In miRFP670nano, the BV chromophore (d) is bound to Cys86 via the C31 atom, miRFP718nano (e) and miRFP709 (f) have the same chromophore species bound to the Cys57 and Cys20, respectively. (g) Superposition of miRFP670nano (green) and miRFP718nano (yellow) structures. (h) miRFP718nano hydrogen bond network around the chromophores. (i) Stacking interactions between the chromophores and the surrounding residues in miRFP718nano. (j) The chromophores of miRFP718nano bound to the respective Cys57 residues in the 2Fo-Fc electron density map. The map is countered at 2.0σ-levels. (k) Superposition of the chromophores in miRFP670nano (green) and miRFP718nano (yellow). (l) Stabilizing mutations and hydrophobic clusters in miRFP718nano. The residues forming H-bonds are shown in green, hydrophobic clusters (one formed by residues Leu8, Ile11, Val12, Val26, Ile104, Leu114, Met140 and the other by Val15, Phe18, Leu19, Trp128, Phe132, Leu133) are in cyan and magenta.

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2, Figs. 1–16, Tables 1–4 and References.

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Oliinyk, O.S., Ma, C., Pletnev, S. et al. Deep-tissue SWIR imaging using rationally designed small red-shifted near-infrared fluorescent protein. Nat Methods 20, 70–74 (2023).

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