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


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.

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Figure 1: Minimizing the calcium-binding domain in Twitch sensors.
Figure 2: Structural characterization of Twitch calcium sensors.
Figure 3: Functional screening of large FRET calcium-sensor libraries.
Figure 4: Use of Twitch-3 and Twitch-2B for high-resolution in vivo imaging in the mouse brain.
Figure 5: In vivo calcium imaging of T lymphocytes with Twitch-1 and Twitch-2B.

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This work was funded by the Max Planck Society, the Howard Hughes Medical Institute, Deutsche Forschungsgemeinschaft (DFG) grant SFB 870 (to O. Griesbeck), DFG grant GRK1721 (to G.W.), EU FP7 EuroV1sion grant (to O. Griesbeck), US National Science Foundation grant IBN-9985315 (to T.A.), US National Science Foundation grant IOS 1145981 (to L.C.R.) and the DFG Center for Integrative Neuroscience (to O. Garaschuk).

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T.T. characterized the minimal domain, cloned constructs and performed protein purifications and in vitro spectroscopic characterizations; J.L. established the bacterial colony screen and performed colony screening and further protein purifications; M.M. and I.B. performed in vivo imaging of T lymphocytes; L.R., S.B., Y. Laukat and C.G. performed NMR structure determination and interpreted results; T.A. and L.C.R. cloned toadfish TnC; A.G. and T.T. collected SAXS data; G.W. calculated SAXS models; H.D. performed in vivo characterization in mouse visual cortex; Y.K., Y. Liang, G.K. and O. Garaschuk planned, performed and interpreted characterization of the sensors in cortical slices in situ and mouse olfactory bulb in vivo; T.T., T.W.C., H.D. and D.S.K. planned, performed and interpreted neuronal screening results. O. Griesbeck designed experiments, supervised sensor engineering and screening and integrated results from the collaborators. T.T., H.D., C.G., O. Garaschuk and O. Griesbeck wrote the manuscript.

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Correspondence to Oliver Griesbeck.

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Thestrup, T., Litzlbauer, J., Bartholomäus, I. et al. Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes. Nat Methods 11, 175–182 (2014). https://doi.org/10.1038/nmeth.2773

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