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Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein


A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

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Figure 1: Spectral characteristics of new far-red FPs.
Figure 2: Structural basis of red-shifting in mCardinal.
Figure 3: Comparison of far-red FPs for deep-tissue imaging.
Figure 4: Non-invasive longitudinal visualization of muscle regeneration in living mice.
Figure 5: Comparison of mCardinal with mNeptune1, iRFP and Clover GFP for non-invasive visualization of muscle regeneration in living mice.

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We thank F. Kanokwan for technical support, F. Fang and L. Lisowski for cell sorting, C. Ran and X. Chen for two-photon imaging, F. Zhang for hydrodynamic injection and S. Classen for help with data collection at ALS Beamline 12.3.1. We also thank K. Yusa (Wellcome Trust Sanger Institute) for pCMV-piggyBAC, M. Kay (Stanford University) for minicircle plasmid, N. Deliolanis and C. Vinegoni for helpful suggestions on mouse imaging, members of the laboratory of E. Mellins for help with protein purification and members of the Lin laboratory for advice and assistance. This work was supported by a seed grant from the Center for Biological Imaging at Stanford (J.C.), US National Institutes of Health (NIH) grants 1R01NS076860-01 (J.C., M.Z.L.), T32 HD007249 (R.D.H.) and 5R01AG020961-08 (S.Y.C., H.M.B.), the Florida State University Research Foundation (P.J.C., M.A.B., M.W.D.), the Chambers Family Foundation and the Pachyonychia Congenita Project (E.G.-G., C.H.C.), an Irvington Postdoctoral Fellowship from the Cancer Research Institute (J.S.B.), the University of Hawaii (N.J.A., H.-L.N.), the Howard Hughes Medical Institute (K.S., K.C.G.) and the Burroughs Wellcome Fund and the Rita Allen Foundation (M.Z.L.).

Author information




J.C. performed protein mutagenesis and characterization, cell imaging, mouse imaging and data analysis and cowrote the paper. S.Y.C. performed myoblast and stem cell purification and transfection. R.D.H. and E.G.-G. assisted with animal experiments. P.L. performed worm culture and made transgenic worms. P.J.C., M.A.B. and M.W.D. performed microscopy of FP fusions. A.J.L. prepared mCardinal proteins for crystallization. J.S.B. and N.J.A. obtained structures of mCardinal and mCardinal-V218E, respectively. H.-L.N., K.C.G., M.W.D., C.H.C., K.S. and H.M.B. provided supervision. M.Z.L. performed protein mutagenesis and characterization, analyzed data, cowrote the paper, provided supervision and directed the project.

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Correspondence to Michael Z Lin.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–16 and Supplementary Table 1 (PDF 11836 kb)

Time-lapse laser-scanning confocal fluorescence microscopy imaging of a mCardinal-18aa-actin fusion protein targeting actin filaments in a fox lung fibroblast

Frames were acquired 18 s apart with a 633 nm laser and a 60× objective. Playback was encoded at 15 frames per s. (MOV 4127 kb)

Time-lapse laser-scanning confocal fluorescence microscopy imaging of a PDHA1-10-aa-mCardinal fusion protein targeting mitochondria in an NIH3T3 fibroblast

Frames were acquired 15 s apart with a 633 nm laser and a 60× objective. Playback was encoded at 15 frames per s. (MOV 1805 kb)

Fast time-lapse laser-scanning confocal fluorescence microscopy imaging of freely moving C. elegans with mCardinal-labeled pharynx

Frames were acquired 125 ms apart with a 635 nm laser and a 20× objective (NA = 0.75). Power measured at the specimen was 100 μW, corresponding to an irradiance of 0.030 J/cm2 per frame. Playback was encoded at 7 frames per s. (MOV 13440 kb)

Fast time-lapse laser-scanning confocal fluorescence microscopy imaging of partially immobilized C. elegans with mCardinal-labeled pharynx. Worms were partially immobilized by dotting cyanoacrylate glue on their tails

1 mg/mL serotonin in M9 was added to keep the sample hydrated during recordings and to induce pharyngeal pumping. Frames were acquired 125 ms apart with a 635 nm laser and a 20× objective (NA = 0.75). Power measured at the specimen was 100 μW, corresponding to an irradiance of 0.030 J/cm2 per frame. Playback was encoded at 7 frames per s. (MOV 11800 kb)

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Chu, J., Haynes, R., Corbel, S. et al. Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein. Nat Methods 11, 572–578 (2014).

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