Kim Douglas S.

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Kim
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Douglas S.
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Optimization of a GCaMP calcium indicator for neural activity imaging

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.

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Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes

2013-06 , Thestrup, Thomas , Litzlbauer, Julia , Bartholomaus, Ingo , Mues, Marsilius , Russo, Luigi , Dana, Hod , Kovalchuk, Yuri , Liang, Yajie , Kalamakis, Georgios , Lauka, Yvonne , Becker, Stefan , Witte, Gregor , Geiger, Anselm , Allen, Taylor , Rome, Lawrence C. , Chen, Tsai-Wen , Kim, Douglas S. , Garaschuk, Olga , Griesinger, Christian , Griesbeck, Oliver

The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These ‘Twitch’ sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the “Twitch” sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.