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4 Janelia Publications

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    08/13/19 | Bright and photostable chemigenetic indicators for extended in vivo voltage imaging.
    Abdelfattah AS, Kawashima T, Singh A, Novak O, Liu H, Shuai Y, Huang Y, Campagnola L, Seeman SC, Yu J, Zheng J, Grimm JB, Patel R, Friedrich J, Mensh BD, Paninski L, Macklin JJ, Murphy GJ, Podgorski K, Lin B, Chen T, Turner GC, Liu Z, Koyama M, Svoboda K, Ahrens MB, Lavis LD, Schreiter ER
    Science. 2019 Aug 13;365(6454):699-704. doi: 10.1126/science.aav6416

    Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, "Voltron", that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 min of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

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    07/29/19 | Kilohertz frame-rate two-photon tomography.
    Kazemipour A, Novak O, Flickinger D, Marvin JS, Abdelfattah AS, King J, Borden P, Kim J, Al-Abdullatif S, Deal P, Miller E, Schreiter E, Druckmann S, Svoboda K, Looger L, Podgorski K
    Nature Methods. 2019 Jul 29;16(8):778-86. doi: 10.1101/357269

    Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits imaging speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and uses prior information to recover high-resolution images at over 1.4 billion voxels per second. Using a structural image as a prior for recording neural activity, we imaged visually-evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 microns and frame-rates over 1 kHz. Dendritic glutamate transients in anaesthetized mice are synchronized within spatially-contiguous domains spanning tens of microns at frequencies ranging from 1-100 Hz. We demonstrate high-speed recording of acetylcholine and calcium sensors, 3D single-particle tracking, and imaging in densely-labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.

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    06/17/19 | High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.
    Dana H, Sun Y, Mohar B, Hulse BK, Kerlin AM, Hasseman JP, Tsegaye G, Tsang A, Wong A, Patel R, Macklin JJ, Chen Y, Konnerth A, Jayaraman V, Looger LL, Schreiter ER, Svoboda K, Kim DS
    Nature Methods. 2019 Jun 17;16(7):649-57. doi: 10.1038/s41592-019-0435-6

    Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

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    01/21/19 | A genetically encoded near-infrared fluorescent calcium ion indicator.
    Qian Y, Piatkevich KD, Mc Larney B, Abdelfattah AS, Mehta S, Murdock MH, Gottschalk S, Molina RS, Zhang W, Chen Y, Wu J, Drobizhev M, Hughes TE, Zhang J, Schreiter ER, Shoham S, Razansky D, Boyden ES, Campbell RE
    Nature Methods. 2019 Jan 21;16(2):171-4. doi: 10.1038/s41592-018-0294-6

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

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