Svoboda Karel

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Svoboda
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Karel
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  • Article
    Optimization of a GCaMP calcium indicator for neural activity imaging
    (Society for Neuroscience, 2012-10-03) Akerboom, Jasper ; Chen, Tsai-Wen ; Wardill, Trevor J. ; Tian, Lin ; Marvin, Jonathan S. ; Mutlu, Sevinc ; Calderon, Nicole Carreras ; Esposti, Federico ; Borghuis, Bart G. ; Sun, Xiaonan Richard ; Gordus, Andrew ; Orger, Michael B. ; Portugues, Ruben ; Engert, Florian ; Macklin, John J. ; Filosa, Alessandro ; Aggarwal, Aman ; Kerr, Rex A. ; Takagi, Ryousuke ; Kracun, Sebastian ; Shigetomi, Eiji ; Khakh, Baljit S. ; Baier, Herwig ; Lagnado, Leon ; Wang, Samuel S.-H. ; Bargmann, Cornelia I. ; Kimmel, Bruce E. ; Jayaraman, Vivek ; Svoboda, Karel ; Kim, Douglas S. ; Schreiter, Eric R. ; Looger, Loren L.
    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.
  • Article
    High throughput instrument to screen fluorescent proteins under two-photon excitation
    (Optical Society of America, 2020-12-01) Molina, Rosana S. ; King, Jonathan ; Franklin, Jacob ; Clack, Nathan ; McRaven, Christopher ; Goncharov, Vasily ; Flickinger, Daniel ; Svoboda, Karel ; Drobizhev, Mikhail ; Hughes, Thomas E.
    Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.