Colorful Protein-Based Fluorescent Probes for Collagen Imaging

<div><p>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 <i>in situ</i>, 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 <i>E. coli</i> and purified in one step using Ni²⁺-affinity chromatography. The probes all bind specifically to collagen, both <i>in vitro</i> 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.</p>