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      A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

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          Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

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          Most cited references 47

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          Optimization of a GCaMP calcium indicator for neural activity imaging.

          Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
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            Improving FRET dynamic range with bright green and red fluorescent proteins

            A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of cyan and yellow fluorescent proteins (CFP and YFP), but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light, and complex photokinetic events such as reversible photobleaching and photoconversion. Here, we engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date, and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP in reporters of kinase activity, small GTPase activity, and transmembrane voltage significantly improves photostability, FRET dynamic range, and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action potential firing and RhoA activation in growth cones.
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              Main-chain bond lengths and bond angles in protein structures.

              The main-chain bond lengths and bond angles of protein structures are analysed as a function of resolution. Neither the means nor standard deviations of these parameters show any correlation with resolution over the resolution range investigated. This is as might be expected as bond lengths and bond angles are likely to be heavily influenced by the geometrical restraints applied during structure refinement. The size of this influence is then investigated by performing an analysis of variance on the mean values across the five most commonly used refinement methods. The differences in means are found to be highly statistically significant, suggesting that the different target values used by the different methods leave their imprint on the structures they refine. This has implications concerning the actual target values used during refinement and stresses the importance of the values being not only accurate but also consistent from one refinement method to another.

                Author and article information

                Nat Biotechnol
                Nat. Biotechnol.
                Nature biotechnology
                30 March 2016
                30 May 2016
                July 2016
                30 November 2016
                : 34
                : 7
                : 760-767
                [1 ]Department of Bioengineering, Stanford University, Stanford, California, USA
                [2 ]Department of Pediatrics, Stanford University, Stanford, California, USA
                [3 ]Department of Neurobiology, Stanford University, Stanford, California, USA
                [4 ]Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA
                [5 ]Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA
                [6 ]Max Planck Florida Institute, Jupiter, Florida, USA
                [7 ]Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
                [8 ]Department of Genetics, Stanford University, Stanford, California, USA
                [9 ]Department of Biological Science, Florida State University, Tallahassee, Florida, USA
                [10 ]National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA
                [11 ]University of Hawaii Cancer Center, Honolulu, Hawaii, USA
                Author notes
                Correspondence should be addressed to M.Z.L. mzlin@ 123456stanford.edu

                Current address: Research Lab for Biomedical Optics and Molecular Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.


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