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

MPG-Autoren
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Thestrup,  Thomas
Research Group: Cellular Dynamics / Griesbeck, MPI of Neurobiology, Max Planck Society;

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Litzlbauer,  Julia
Research Group: Cellular Dynamics / Griesbeck, MPI of Neurobiology, Max Planck Society;

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Bartholomäus,  Ingo
Emeritus Group: Neuroimmunology / Wekerle, MPI of Neurobiology, Max Planck Society;

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Mues,  Marsilius
Emeritus Group: Neuroimmunology / Wekerle, MPI of Neurobiology, Max Planck Society;

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Geiger,  Anselm
Research Group: Cellular Dynamics / Griesbeck, MPI of Neurobiology, Max Planck Society;

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Griesbeck,  Oliver
Research Group: Cellular Dynamics / Griesbeck, MPI of Neurobiology, Max Planck Society;

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Zitation

Thestrup, T., Litzlbauer, J., Bartholomäus, I., Mues, M., Russo, L., Dana, H., et al. (2014). Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes. NATURE METHODS, 11(2), 175-182. doi:10.1038/NMETH.2773.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0017-BB51-E
Zusammenfassung
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.