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      Colorful Protein-Based Fluorescent Probes for Collagen Imaging

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          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 Ni 2+-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.

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          The recent explosion in the diversity of available fluorescent proteins (FPs) promises a wide variety of new tools for biological imaging. With no unified standard for assessing these tools, however, a researcher is faced with difficult questions. Which FPs are best for general use? Which are the brightest? What additional factors determine which are best for a given experiment? Although in many cases, a trial-and-error approach may still be necessary in determining the answers to these questions, a unified characterization of the best available FPs provides a useful guide in narrowing down the options.
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            Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation.

            Multicolor nonlinear microscopy of living tissue using two- and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermolecular structures produces images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provides crucial information for physiology and pathology. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimer's disease and cancer.
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              Improving the photostability of bright monomeric orange and red fluorescent proteins.

              All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

                Author and article information

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                9 December 2014
                : 9
                : 12
                [1 ]Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, MB Eindhoven, The Netherlands
                [2 ]Soft Tissue Biomechanics and Engineering, Department of Biomedical Engineering, Eindhoven University of Technology, MB Eindhoven, The Netherlands
                Institute of Dentistry, Barts & The London School of Medicine and Dentistry, Queen Mary University of London, United Kingdom
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: SJAA ACCVS CVCB MM. Performed the experiments: SJAA ACCVS MCVT AJVDL PAP NLLVDZ SLDLR. Analyzed the data: SJAA ACCVS MCVT AJVDL PAP NLLVDZ SLDLR CVCB MM. Wrote the paper: SJAA ACCVS CVCB MM.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 21
                This work was supported by an ERC starting grant (ERC-2011-StG 280255) and a grant from the Dutch government to the Netherlands Institute for Regenerative Medicine (NIRM, grant no. FES0908). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Biology and Life Sciences
                Extracellular Matrix Proteins
                Luminescent Proteins
                Tissue Mechanics
                Synthetic Bioengineering
                Macromolecular Engineering
                Protein Engineering
                Tissue Engineering
                Cell Biology
                Cellular Structures and Organelles
                Extracellular Matrix
                Engineering and Technology
                Medicine and Health Sciences
                Connective Tissue Diseases
                Collagen Diseases
                Research and Analysis Methods
                Imaging Techniques
                Fluorescence Imaging
                Light Microscopy
                Fluorescence Microscopy
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.



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