. 2014 May;11(5):572-8.

doi: 10.1038/nmeth.2888. Epub 2014 Mar 16.

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

Jun Chu¹, Russell D Haynes², Stéphane Y Corbel², Pengpeng Li³, Emilio González-González⁴, John S Burg⁵, Niloufar J Ataie⁶, Amy J Lam¹, Paula J Cranfill⁷, Michelle A Baird⁷, Michael W Davidson⁷, Ho-Leung Ng⁸, K Christopher Garcia⁹, Christopher H Contag⁴, Kang Shen¹⁰, Helen M Blau², Michael Z Lin¹¹

Affiliations

¹ 1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.
² 1] Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA. [2] Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.
³ Department of Biological Sciences, Stanford University, Stanford, California, USA.
⁴ 1] Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA. [2] Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California, USA.
⁵ 1] Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA. [2] Department of Structural Biology, Stanford University School of Medicine, Stanford, California, USA.
⁶ Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
⁷ 1] Department of Biological Science, Florida State University, Tallahassee, Florida, USA. [2] National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA.
⁸ 1] Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA. [2] University of Hawaii Cancer Center, Honolulu, Hawaii, USA.
⁹ 1] Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA. [2] Department of Structural Biology, Stanford University School of Medicine, Stanford, California, USA. [3] Howard Hughes Medical Institute, Stanford University, Stanford, California, USA.
¹⁰ 1] Department of Biological Sciences, Stanford University, Stanford, California, USA. [2] Howard Hughes Medical Institute, Stanford University, Stanford, California, USA.
¹¹ 1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA. [3] Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California, USA.

PMID: 24633408
PMCID: PMC4008650
DOI: 10.1038/nmeth.2888

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

Jun Chu et al. Nat Methods. 2014 May.

. 2014 May;11(5):572-8.

doi: 10.1038/nmeth.2888. Epub 2014 Mar 16.

Authors

Affiliations

¹ 1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.
² 1] Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA. [2] Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.
³ Department of Biological Sciences, Stanford University, Stanford, California, USA.
⁴ 1] Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA. [2] Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California, USA.
⁵ 1] Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA. [2] Department of Structural Biology, Stanford University School of Medicine, Stanford, California, USA.
⁶ Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
⁷ 1] Department of Biological Science, Florida State University, Tallahassee, Florida, USA. [2] National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA.
⁸ 1] Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA. [2] University of Hawaii Cancer Center, Honolulu, Hawaii, USA.
⁹ 1] Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, USA. [2] Department of Structural Biology, Stanford University School of Medicine, Stanford, California, USA. [3] Howard Hughes Medical Institute, Stanford University, Stanford, California, USA.
¹⁰ 1] Department of Biological Sciences, Stanford University, Stanford, California, USA. [2] Howard Hughes Medical Institute, Stanford University, Stanford, California, USA.
¹¹ 1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA. [3] Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California, USA.

PMID: 24633408
PMCID: PMC4008650
DOI: 10.1038/nmeth.2888

Abstract

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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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

**Figure 1**
Spectral characteristics of new far-red FPs. **(a)** Absorbance spectra of oxygenated hemoglobin (oxyHb), deoxygenated hemoglobin (deoxyHb), and monomeric far-red FPs. Myoglobin spectra are similar to hemoglobin spectra. **(b)** Normalized excitation (left) and emission (right) spectra of monomeric far-red FPs. **(c)** Transmittance of mKate2, mNeptune1, mNeptune2, mNeptune2.5, mCardinal, and TagRFP657 at 1 mg/mL of purified mature protein. Blue transmission of mCardinal is due to efficient absorbance of green and red light.

**Figure 2**
Structural basis of red-shifting in mCardinal. **(a)** Structure of Neptune showing the water molecule hydrogen-bonded to the acylimine oxygen of the chromophore. The fluorescent protein is viewed down the axis of the beta-barrel from the direction of the N- and C-termini. Beta-strands and alpha-helices are depicted in cartoon form. The chromophore, the Met63 side chain attached to the chromophore, and the water are depicted in stick representation with carbon colored lavender, nitrogen blue, and oxygen red. Numbers indicate distances between the water oxygen atom and hydrogen-bonding partners. Cyan labels indicate positions mutated to amino acids with side chains capable of donating hydrogen bonds to the acylimine oxygen: Met11, Leu13, Ser28 and Gly41. **(b,c)** Hydrogen bond interactions between Thr28, Gln41, and the chromophore acylimine in mCardinal **(b)** or mCardinal-V218E **(c)**. Rendering is of chain A. Distances shown are averages of measurements from chains with temperature factors for Gln41 amide atoms lower than those for the hydrogen-bonding water molecule in the Neptune structure, specifically chains A and B for mCardinal and chains A and C for mCardinal-V218E. Mesh depicts electron density in the Fo-Fc OMIT map contoured to 3.0 sigma within 2.0 Å of the atoms displayed in stick form.

**Figure 3**
Comparison of far-red FPs for deep-tissue imaging. **(a,b,c)** Left, representative fluorescence images of equal amounts of purified far-red FPs placed within the esophagus of euthanized mice. Images were acquired with 605/30 nm excitation in an IVIS Spectrum **(a)**, 640/30 nm excitation in an IVIS spectrum **(b)**, or with 620/20 nm excitation on a SZX-12 fluorescence stereomicroscope **(c)**. Pseudocolor scale represents signal-to-background ratio, calculated as (F_FP − F_PBS)/F_PBS, where F_FP and F_PBS were total intensities measured in a common region of interest encompassing all fluorescence signal in images of FP and PBS, respectively. Scale bars, 1 cm. Right, quantification of signal-to-background ratio and signal intensity subtracted by background in units of 10⁸ photons per second per steradian (p s⁻¹ sr⁻¹ μW⁻¹ × 10⁸) presented as mean ± standard error of the mean (SEM), n = 4 for **(a,b)** and 5 for **(c)**. Differences are statistically significant by one-way ANOVA (P < 0.0001). Asterisks indicate significant differences by Dunnett’s multiple comparison test versus mNeptune1 (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

**Figure 4**
Non-invasive longitudinal visualization of muscle regeneration in living mice. **(a)** Tibialis anterior (TA) muscles injected with 1 million myoblasts expressing mCardinal, imaged with a fluorescence stereoscope with 620/20 nm excitation. All images are normalized to the same intensity scale. Series is representative of 5 repeats. **(b)** Images from 3, 7, and 14 days post-injection (d.p.i.) are shown with intensity scaling tighter than in **(a)** by a factor of 10, 10, and 5 respectively. **(c)** Magnified view of the muscle at 7 d.p.i., showing an early regenerating fiber (arrow). The image at right is deliberately enlarged until pixelated to show that this fiber appears just a few pixels wide. **(d)** Fluorescence signal from TA muscles injected with 1000 muscle stem cells expressing mCardinal. **(e)** Magnified view of the muscle at 44 d.p.i., showing multiple regenerating fibers (arrows). **(f)** Bioluminescence imaging at 42 d.p.i. of stem cells shown in **(d)**. A single 8-min bioluminescence image was acquired at the highest possible resolution with no binning. The resulting sampling resolution (21 μm/pixel) is similar to that (15 μm/pixel) of the fluorescence images in **(e). (g)** Enlargement of the luminescence image in **(f)** shows the absence of structures resembling myofibers. The right panel is enlarged to show the presence of noise at the level of individual pixels. In a 40-pixel × 30-pixel region containing the brightest pixels, standard deviation was 33 while mean intensity was 290 counts over that of a background region, indicating presence of substantial shot and read noise relative to signal.

**Figure 5**
Comparison of mCardinal with mNeptune1, iRFP and Clover GFP for non-invasive visualization of muscle regeneration in living mice. **(a)** Representative fluorescence images of tibialis anterior (TA) muscles injected with 1 million myoblasts expressing iRFP and mCardinal or iRFP and mNeptune1. Images were taken in the absence of exogenous biliverdin. The bar graph shows contrast over background (mean ± SEM, n = 6 and n = 8 for mNeptune1 and mCardinal, respectively). Asterisks indicate significant differences by unpaired two-tailed Student t test (* P < 0.05 and *** P < 0.001). **(b)** Representative fluorescence images of TA muscles expressing mCardinal and iRFP in the absence (top panel) and presence (middle panel) of 250 nM biliverdin. The leg injected with untransfected myoblasts (bottom panel) was used as a negative control. **(c)** Representative fluorescence images of TA muscles injected with 5000 muscle stem cells expressing Clover and mCardinal at 29 d.p.i. The white arrow indicates mCardinal-positive muscle fibers. Shadows of blood vessels (highlighted in the inset) are expected from absorption of green autofluorescence by blood.

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

Affiliations

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

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Substances

Associated data

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Research Materials

Miscellaneous