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      A genetically encoded near-infrared fluorescent calcium ion indicator

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          Abstract

          We report an intensiometric, near-infrared (NIR) fluorescent, genetically encoded calcium ion (Ca 2+) indicator (GECI) with excitation and emission maxima at 678 nm and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca 2+ transients in cultured mammalian cells and brain tissue with sensitivity comparable to currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca 2+ imaging in combination with other optogenetic indicators and actuators.

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          Most cited references19

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          The green fluorescent protein.

          R Tsien (1998)
          In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.
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            Gene splicing and mutagenesis by PCR-driven overlap extension.

            Extension of overlapping gene segments by PCR is a simple, versatile technique for site-directed mutagenesis and gene splicing. Initial PCRs generate overlapping gene segments that are then used as template DNA for another PCR to create a full-length product. Internal primers generate overlapping, complementary 3' ends on the intermediate segments and introduce nucleotide substitutions, insertions or deletions for site-directed mutagenesis, or for gene splicing, encode the nucleotides found at the junction of adjoining gene segments. Overlapping strands of these intermediate products hybridize at this 3' region in a subsequent PCR and are extended to generate the full-length product amplified by flanking primers that can include restriction enzyme sites for inserting the product into an expression vector for cloning purposes. The highly efficient generation of mutant or chimeric genes by this method can easily be accomplished with standard laboratory reagents in approximately 1 week.
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              Is Open Access

              Sensitive red protein calcium indicators for imaging neural activity

              Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging. DOI: http://dx.doi.org/10.7554/eLife.12727.001
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                Author and article information

                Journal
                101215604
                32338
                Nat Methods
                Nat. Methods
                Nature methods
                1548-7091
                1548-7105
                5 December 2018
                21 January 2019
                February 2019
                21 July 2019
                : 16
                : 2
                : 171-174
                Affiliations
                [1 ]Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada.
                [2 ]Media Lab and McGovern Institute for Brain Research, MIT, Cambridge, Massachusetts, 02139, United States.
                [3 ]Institute for Biological and Medical Imaging (IBMI), Helmholtz Center Munich, Neuherberg, Germany.
                [4 ]Faculty of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675 Munich, Germany.
                [5 ]Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, 20147, United States.
                [6 ]Department of Pharmacology, University of California San Diego, La Jolla, California, USA.
                [7 ]Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana, 59717, United States.
                [8 ]Departments of Ophthalmology and of Neuroscience and Physiology, New York University Langone Health, New York City, New York, 10010, United States.
                [9 ]Faculty of Medicine and Institute of Pharmacology and Toxicology, University of Zurich, Switzerland.
                [10 ]Department of Information Technology and Electrical Engineering and Institute for Biomedical Engineering, ETH Zurich, Switzerland
                [11 ]Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan
                [12 ]These authors contributed equally to this work.
                Author notes
                [* ]Correspondence should be addressed to R.E.C. ( robert.e.campbell@ 123456ualberta.ca ).

                Author Contributions

                Y.Q. developed NIR-GECO1 and performed in vitro characterization. Y.Q., K.D.P., A.S.A and M.H.M performed characterization in hippocampal neurons. K.D.P. and M.H.M. characterized NIR-GECO1 in intact brain slice. B.M. and S.G. performed in vivo mesoscale imaging. S.M. performed live-cell imaging in MIN6 β-cells. R.S.M. and M.D. measured two-photon spectra. W.Z. built the pcDuEx2 vector. Y.C. and J.W. worked on development of the smURFP-based GECI. M.D., T.E.H., J.Z., E.R.S., S.S., D.R., E.S.B., and R.E.C. supervised research. All authors were involved in data analysis. Y.Q., K.D.P., and R.E.C. wrote the manuscript.

                Article
                NIHMS1515854
                10.1038/s41592-018-0294-6
                6393164
                30664778
                f141ecb7-d205-4e08-bd0b-18fe702000b3

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