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22 Janelia Publications

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    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.

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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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    10/03/18 | High-performance GFP-based calcium indicators for imaging activity in neuronal populations and microcompartments.
    Dana H, Sun Y, Mohar B, Hulse B, 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
    bioRxiv. 2018 Oct 3:. doi: 10.1101/434589

    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 jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and tracking large populations of neurons using 2-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

     

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    08/01/17 | Genetically encoded biosensors.
    Marvin JS, Looger LL, Lee RT, Schreiter ER
    USPTO. 2017 Aug 01;B2:

    The present disclosure provides, inter alia, genetically encoded recombinant peptide biosensors comprising analyte-binding framework portions and signaling portions, wherein the signaling portions are present within the framework portions at sites or amino acid positions that undergo a conformational change upon interaction of the framework portion with an analyte.

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    06/12/17 | Neural signatures of dynamic stimulus selection in Drosophila.
    Sun Y, Nern A, Franconville R, Dana H, Schreiter ER, Looger LL, Svoboda K, Kim DS, Hermundstad AM, Jayaraman V
    Nature Neuroscience. 2017 Jun 12;20(8):1104-13. doi: 10.1038/nn.4581

    Many animals orient using visual cues, but how a single cue is selected from among many is poorly understood. Here we show that Drosophila ring neurons—central brain neurons implicated in navigation—display visual stimulus selection. Using in vivo two-color two-photon imaging with genetically encoded calcium indicators, we demonstrate that individual ring neurons inherit simple-cell-like receptive fields from their upstream partners. Stimuli in the contralateral visual field suppressed responses to ipsilateral stimuli in both populations. Suppression strength depended on when and where the contralateral stimulus was presented, an effect stronger in ring neurons than in their upstream inputs. This history-dependent effect on the temporal structure of visual responses, which was well modeled by a simple biphasic filter, may determine how visual references are selected for the fly's internal compass. Our approach highlights how two-color calcium imaging can help identify and localize the origins of sensory transformations across synaptically connected neural populations.

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    03/01/17 | All-optical functional synaptic connectivity mapping in acute brain slices using CaMPARI.
    Zolnik TA, Sha F, Johenning F, Schreiter ER, Looger LL, Larkum ME, Sachdev RN
    The Journal of Physiology. 2017 Mar 01;595(5):1465-77. doi: 10.1113/JP273116

    The calcium-modulated photoactivatable ratiometric integrator CaMPARI (Fosque et al., 2015) facilitates the study of neural circuits by permanently marking cells active during user-specified temporal windows. Permanent marking enables measurement of signals from large swathes of tissue and easy correlation of activity with other structural or functional labels. One potential application of CaMPARI is labeling neurons postsynaptic to specific populations targeted for optogenetic stimulation, giving rise to all-optical functional connectivity mapping. Here, we characterized the response of CaMPARI to several common types of neuronal calcium signals in mouse acute cortical brain slices. Our experiments show that CaMPARI is effectively converted by both action potentials and sub-threshold synaptic inputs, and that conversion level is correlated to synaptic strength. Importantly, we found that conversion rate can be tuned: it is linearly related to light intensity. At low photoconversion light levels CaMPARI offers a wide dynamic range due to slower conversion rate; at high light levels conversion is more rapid and more sensitive to activity. Finally, we employed CaMPARI and optogenetics for functional circuit mapping in ex vivo acute brain slices, which preserve in vivo-like connectivity of axon terminals. With a single light source, we stimulated channelrhodopsin-2-expressing long-range posteromedial (POm) thalamic axon terminals in cortex and induced CaMPARI conversion in recipient cortical neurons. We found that POm stimulation triggers robust photoconversion of layer 5 cortical neurons and weaker conversion of layer 2/3 neurons. Thus, CaMPARI enables network-wide, tunable, all-optical functional circuit mapping that captures supra- and sub-threshold depolarization. This article is protected by copyright. All rights reserved.

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    03/24/16 | Sensitive red protein calcium indicators for imaging neural activity.
    Dana H, Mohar B, Sun Y, Narayan S, Gordus A, Hasseman JP, Tsegaye G, Holt GT, Hu A, Walpita D, Patel R, Macklin JJ, Bargmann CI, Ahrens MB, Schreiter ER, Jayaraman V, Looger LL, Svoboda K, Kim DS
    eLife. 2016 Mar 24;5:. doi: 10.7554/eLife.12727

    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.

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    10/09/15 | A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store.
    Henderson MJ, Baldwin HA, Werley CA, Boccardo S, Whitaker LR, Yan X, Holt GT, Schreiter ER, Looger LL, Cohen AE, Kim DS, Harvey BK
    PloS one. 2015 Oct 09;10(10):e0139273. doi: 10.1371/journal.pone.0139273

    Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

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    09/18/15 | Green-to-red photoconversion of GCaMP.
    Ai M, Mills H, Kanai M, Lai J, Deng J, Schreiter E, Looger L, Neubert T, Suh G
    PLoS One. 2015 Sep 18;10(9):e0138127. doi: 10.1371/journal.pone.0138127

    Genetically encoded calcium indicators (GECIs) permit imaging intracellular calcium transients. Among GECIs, the GFP-based GCaMPs are the most widely used because of their high sensitivity and rapid response to changes in intracellular calcium concentrations. Here we report that the fluorescence of GCaMPs-including GCaMP3, GCaMP5 and GCaMP6-can be converted from green to red following exposure to blue-green light (450-500 nm). This photoconversion occurs in both insect and mammalian cells and is enhanced in a low oxygen environment. The red fluorescent GCaMPs retained calcium responsiveness, albeit with reduced sensitivity. We identified several amino acid residues in GCaMP important for photoconversion and generated a GCaMP variant with increased photoconversion efficiency in cell culture. This light-induced spectral shift allows the ready labeling of specific, targeted sets of GCaMP-expressing cells for functional imaging in the red channel. Together, these findings indicate the potential for greater utility of existing GCaMP reagents, including transgenic animals.

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