Bright and stable near-infrared fluorescent protein for in vivo imaging

Abstract

Imaging biological processes in mammalian tissues will be facilitated by fluorescent probes with excitation and emission bands within the near-infrared optical window of high transparency1. Here we report a phytochrome-based near-infrared fluorescent protein (iRFP) with excitation and emission maxima at 690 nm and 713 nm, respectively. iRFP does not require an exogenous supply of the chromophore biliverdin and has higher effective brightness, intracellular stability and photostability than earlier phytochrome-derived fluorescent probes. Compared with far-red GFP-like proteins, iRFP has a substantially higher signal-to-background ratio in a mouse model due to its infrared-shifted spectra.

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Figure 1: In vitro properties of iRFP and IFP1.4.
Figure 2: Expression of iRFP in living mouse.
Figure 3: Comparison of iRFP with far-red GFP-like proteins in mouse phantom.

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Acknowledgements

We thank E. Giraud (Institute for Research and Development, France) for the plasmid encoding bacteriophytochrome from R. palustris, A. Ulijasz and R. Vierstra (both from University of Wisconsin) for the plasmid encoding the heme-oxygenase from Synechocystis sp. PCC6803 and B. Glick (University of Chicago), D. Chudakov and K. Lukyanov (both from Institute of Bioorganic Chemistry, Russia) for the plasmids encoding GFP-like far-red fluorescent proteins. We are grateful to D. Entenberg for the help with two-photon excitation measurements, A. Muesch and D. Cohen for the assistance with virus purification and providing GFP-encoding adenoviruses and R. Zheng for the help with size-exclusion chromatography (all from Albert Einstein College of Medicine). This work was supported by grants from the US National Institutes of Health, AI046985 and AI087625 to K.K., GM073913 to V.V.V. and S10RR027308 for purchase of the IVIS imager.

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G.S.F. developed the protein and together with K.D.P. characterized it in vitro. G.S.F. studied the protein in mammalian cells. G.S.F. and J.Z. analyzed and sorted cells using FACS. G.S.F., L.-M.T. and K.K. characterized protein expression in mice. V.V.V. designed and planned the project and together with G.S.F. wrote the manuscript.

Corresponding author

Correspondence to Vladislav V Verkhusha.

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The authors declare no competing financial interests.

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Supplementary Table 1 and Supplementary Figures 1–10 (PDF 1851 kb)

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Filonov, G., Piatkevich, K., Ting, L. et al. Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat Biotechnol 29, 757–761 (2011). https://doi.org/10.1038/nbt.1918

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