title: Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein.
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.
authors.affiliation: 1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Chu
authors.affiliation: 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.
authors.forename: Russell D
authors.initials: RD
authors.lastname: Haynes
authors.affiliation: 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.
authors.forename: Stéphane Y
authors.initials: SY
authors.lastname: Corbel
authors.affiliation: Department of Biological Sciences, Stanford University, Stanford, California, USA.
authors.forename: Pengpeng
authors.initials: P
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Emilio
authors.initials: E
authors.lastname: González-González
authors.affiliation: 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.
authors.forename: John S
authors.initials: JS
authors.lastname: Burg
authors.affiliation: Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
authors.forename: Niloufar J
authors.initials: NJ
authors.lastname: Ataie
authors.affiliation: 1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Lam
authors.affiliation: 1] Department of Biological Science, Florida State University, Tallahassee, Florida, USA. [2] National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA.
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Cranfill
authors.affiliation: 1] Department of Biological Science, Florida State University, Tallahassee, Florida, USA. [2] National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Baird
authors.affiliation: 1] Department of Biological Science, Florida State University, Tallahassee, Florida, USA. [2] National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Davidson
authors.affiliation: 1] Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA. [2] University of Hawaii Cancer Center, Honolulu, Hawaii, USA.
authors.forename: Ho-Leung
authors.initials: HL
authors.lastname: Ng
authors.affiliation: 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.
authors.forename: K Christopher
authors.initials: KC
authors.lastname: Garcia
authors.affiliation: 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.
authors.forename: Christopher H
authors.identifier: 0000000210118278
authors.initials: CH
authors.lastname: Contag
authors.affiliation: 1] Department of Biological Sciences, Stanford University, Stanford, California, USA. [2] Howard Hughes Medical Institute, Stanford University, Stanford, California, USA.
authors.forename: Kang
authors.initials: K
authors.lastname: Shen
authors.affiliation: 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.
authors.forename: Helen M
authors.initials: HM
authors.lastname: Blau
authors.affiliation: 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.
authors.forename: Michael Z
authors.initials: MZ
authors.lastname: Lin
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D018360:Crystallography, X-Ray; D003952:Diagnostic Imaging / Q000379:methods*; D015723:Gene Library; D006367:HeLa Cells; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D008164:Luminescent Proteins / Q000378:metabolism*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D032342:Muscle Cells / Q000378:metabolism; D018482:Muscle, Skeletal / Q000473:pathology; D009132:Muscles / Q000473:pathology; D016296:Mutagenesis; D032446:Myoblasts / Q000378:metabolism; D009211:Myoglobin / Q000737:chemistry; D041681:NIH 3T3 Cells; D012038:Regeneration; D013234:Stem Cells / Q000166:cytology; D000097573:Red Fluorescent Protein
chemical_list: D006454:Hemoglobins; D008164:Luminescent Proteins; D009211:Myoglobin; D000097573:Red Fluorescent Protein