title: Colorful protein-based fluorescent probes for collagen imaging.
abstract: Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, MB Eindhoven, The Netherlands.
authors.forename: Stijn J A
authors.initials: SJ
authors.lastname: Aper
authors.affiliation: Soft Tissue Biomechanics and Engineering, Department of Biomedical Engineering, Eindhoven University of Technology, MB Eindhoven, The Netherlands.
authors.forename: Ariane C C
authors.initials: AC
authors.lastname: van Spreeuwel
authors.affiliation: Soft Tissue Biomechanics and Engineering, Department of Biomedical Engineering, Eindhoven University of Technology, MB Eindhoven, The Netherlands.
authors.forename: Mark C
authors.initials: MC
authors.lastname: van Turnhout
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, MB Eindhoven, The Netherlands.
authors.forename: Ardjan J
authors.initials: AJ
authors.lastname: van der Linden
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, MB Eindhoven, The Netherlands.
authors.forename: Pascal A
authors.initials: PA
authors.lastname: Pieters
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, MB Eindhoven, The Netherlands.
authors.forename: Nick L L
authors.initials: NL
authors.lastname: van der Zon
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, MB Eindhoven, The Netherlands.
authors.forename: Sander L
authors.initials: SL
authors.lastname: de la Rambelje
authors.affiliation: Soft Tissue Biomechanics and Engineering, Department of Biomedical Engineering, Eindhoven University of Technology, MB Eindhoven, The Netherlands.
authors.forename: Carlijn V C
authors.initials: CV
authors.lastname: Bouten
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, MB Eindhoven, The Netherlands.
authors.forename: Maarten
authors.initials: M
authors.lastname: Merkx
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001426:Bacterial Proteins / Q000378:metabolism*; D002264:Carboxylic Acids / Q000378:metabolism*; D002478:Cells, Cultured; D003094:Collagen / Q000032:analysis* / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D010496:Pericardium / Q000166:cytology / Q000378:metabolism; D017785:Photons; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012867:Skin / Q000166:cytology / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D002264:Carboxylic Acids; D005456:Fluorescent Dyes; C436864:Oregon Green 488 carboxylic acid; D011993:Recombinant Fusion Proteins; D003094:Collagen