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

      Nature Methods

      Troponin C, Animals, metabolism, cytology, T-Lymphocytes, Rats, Neurons, Molecular Sequence Data, Microscopy, Fluorescence, Mice, Inbred C57BL, Mice, Magnetic Resonance Spectroscopy, Lymphocyte Activation, Luminescent Proteins, Image Processing, Computer-Assisted, Humans, Hippocampus, HEK293 Cells, Fluorescent Dyes, Fluorescence Resonance Energy Transfer, Calcium, methods, Biosensing Techniques, Animals, Newborn

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          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.

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          Author and article information

          Journal
          10.1038/nmeth.2773
          24390440

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