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Nat Methods. 2014 Feb;11(2):175-82. doi: 10.1038/nmeth.2773. Epub 2014 Jan 5.

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

Author information

1
1] Max Planck Institute of Neurobiology, Martinsried, Germany. [2] Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
2
Max Planck Institute of Neurobiology, Martinsried, Germany.
3
Max Planck Institute for Biophysical Chemistry, NMR-based Structural Biology, Göttingen, Germany.
4
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
5
Institute of Physiology II, University of Tübingen, Tübingen, Germany.
6
Gene Center, Department of Biochemistry, University of Munich, Munich, Germany.
7
Oberlin College, Oberlin, Ohio, USA.
8
1] Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA. [2] The Whitman Center, Marine Biological Lab, Woods Hole, Massachusetts, USA.

Abstract

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.

Comment in

PMID:
24390440
DOI:
10.1038/nmeth.2773
[Indexed for MEDLINE]

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