Main Menu (Mobile)- Block

Main Menu - Block

custom | custom

Search Results

general_search_page-panel_pane_1 | views_panes

12 Janelia Publications

Showing 1-10 of 12 results
Your Criteria:
    07/10/20 | A general approach to engineer positive-going eFRET voltage indicators
    Abdelfattah AS, Valenti R, Zheng J, Wong A, Podgorski K, Koyama M, Kim DS, Schreiter ER
    Nature Communications. 2020 Jul 10;11(1):

    We engineered electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with “positive-going” fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transformed the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further applied this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.

    View Publication Page
    07/08/20 | Bright and high-performance genetically encoded Ca indicator based on mNeonGreen fluorescent protein.
    Zarowny L, Aggarwal A, Rutten VM, Kolb I, GENIE Project , Patel R, Huang H, Chang Y, Phan T, Kanyo R, Ahrens MB, Allison WT, Podgorski K, Campbell RE
    ACS Sensors. 2020 Jul 08:. doi: 10.1021/acssensors.0c00279

    Genetically encodable calcium ion (Ca) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost 2 decades of steady improvements in the GFP-based GCaMP series of GECIs, the performance of the most recent generation (i.e., jGCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression toward ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.

    View Publication Page
    06/16/20 | Comprehensive imaging of sensory-evoked activity of entire neurons within the awake developing brain using ultrafast AOD-based random-access two-photon microscopy.
    Sakaki KD, Podgorski K, Dellazizzo Toth TA, Coleman P, Haas K
    Frontiers in Neural Circuits. 2020 Jun 16;14:33. doi: 10.3389/fncir.2020.00033

    Determining how neurons transform synaptic input and encode information in action potential (AP) firing output is required for understanding dendritic integration, neural transforms and encoding. Limitations in the speed of imaging 3D volumes of brain encompassing complex dendritic arbors using conventional galvanometer mirror-based laser-scanning microscopy has hampered fully capturing fluorescent sensors of activity throughout an individual neuron's entire complement of synaptic inputs and somatic APs. To address this problem, we have developed a two-photon microscope that achieves high-speed scanning by employing inertia-free acousto-optic deflectors (AODs) for laser beam positioning, enabling random-access sampling of hundreds to thousands of points-of-interest restricted to a predetermined neuronal structure, avoiding wasted scanning of surrounding extracellular tissue. This system is capable of comprehensive imaging of the activity of single neurons within the intact and awake vertebrate brain. Here, we demonstrate imaging of tectal neurons within the brains of albino tadpoles labeled using single-cell electroporation for expression of a red space-filling fluorophore to determine dendritic arbor morphology, and either the calcium sensor jGCaMP7s or the glutamate sensor iGluSnFR as indicators of neural activity. Using discrete, point-of-interest scanning we achieve sampling rates of 3 Hz for saturation sampling of entire arbors at 2 μm resolution, 6 Hz for sequentially sampling 3 volumes encompassing the dendritic arbor and soma, and 200-250 Hz for scanning individual planes through the dendritic arbor. This system allows investigations of sensory-evoked information input-output relationships of neurons within the intact and awake brain.

    View Publication Page
    05/25/20 | jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths.
    Mohr MA, Bushey D, Aggarwal A, Marvin JS, Kim JJ, Marquez EJ, Liang Y, Patel R, Macklin JJ, Lee C, Tsang A, Tsegaye G, Ahrens AM, Chen JL, Kim DS, Wong AM, Looger LL, Schreiter ER, Podgorski K
    Nature Methods. 2020 May 25;17(1):694-97. doi: 10.1038/s41592-020-0835-7

    Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

    View Publication Page
    02/08/20 | A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies.
    Borden P, Zhang P, Shivange AV, Marvin JS, Cichon J, Dan C, Podgorski K, Figueiredo A, Novak O, Tanimoto M, Shigetomi E, Lobas MA, Kim H, Zhu P, Zhang Y, Zheng WS, Fan C, Wang G, Xiang B, Gan L, Zhang G, Guo K, Lin L, Cai Y, Yee AG, Aggarwal A, Ford CP, Rees DC, Dietrich D, Khakh BS, Dittman JS, Gan W, Koyama M, Jayaraman V, Cheer JF, Lester HA, Zhu JJ, Looger LL
    bioRxiv. 2020 Feb 8:. doi: https://doi.org/10.1101/2020.02.07.939504

    Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.

    View Publication Page
    11/25/19 | Two-photon imaging with silicon photomultipliers.
    Modi MN, Daie K, Turner GC, Podgorski K
    Optics Express. 2019 Nov 25;27(24):35830-35841. doi: 10.1364/OE.27.035830

    We compared performance of recently developed silicon photomultipliers (SiPMs) to GaAsP photomultiplier tubes (PMTs) for two-photon imaging of neural activity. Despite higher dark counts, SiPMs match or exceed the signal-to-noise ratio of PMTs at photon rates encountered in typical calcium imaging experiments due to their low pulse height variability. At higher photon rates encountered during high-speed voltage imaging, SiPMs substantially outperform PMTs.

    View Publication Page
    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.

    View Publication Page
    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.

    View Publication Page
    07/15/19 | A genetically encoded fluorescent sensor for in vivo imaging of GABA.
    Marvin JS, Shimoda Y, Magloire V, Leite M, Kawashima T, Jensen TP, Kolb I, Knott EL, Novak O, Podgorski K, Leidenheimer NJ, Rusakov DA, Ahrens MB, Kullmann DM, Looger LL
    Nature Methods. 2019 Jul 15;16(8):763-770. doi: 10.1038/s41592-019-0471-2

    Current techniques for monitoring GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in vertebrates, cannot follow transients in intact neural circuits. To develop a GABA sensor, we applied the design principles used to create the fluorescent glutamate receptor iGluSnFR. We used a protein derived from a previously unsequenced Pseudomonas fluorescens strain and performed structure-guided mutagenesis and library screening to obtain intensity-based GABA sensing fluorescence reporter (iGABASnFR) variants. iGABASnFR is genetically encoded, detects GABA release evoked by electric stimulation of afferent fibers in acute brain slices and produces readily detectable fluorescence increases in vivo in mice and zebrafish. We applied iGABASnFR to track mitochondrial GABA content and its modulation by an anticonvulsant, swimming-evoked, GABA-mediated transmission in zebrafish cerebellum, GABA release events during interictal spikes and seizures in awake mice, and found that GABA-mediated tone decreases during isoflurane anesthesia.

    View Publication Page
    11/01/18 | Stability, affinity and chromatic variants of the glutamate sensor iGluSnFR.
    Marvin JS, Scholl B, Wilson DE, Podgorski K, Kazemipour A, Mueller JA, Schoch-McGovern S, Wang SS, Quiroz FJ, Rebola N, Bao H, Little JP, Tkachuk AN, Hantman AW, Chapman ER, Dietrich D, DiGregorio DA, Fitzpatrick D, Looger LL
    Nature Methods. 2018 Nov;15(11):9386-9. doi: 10.1038/s41592-018-0171-3

    Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

    View Publication Page