Abstract
Ratiometric genetically encoded calcium indicators (GECIs) record neural activity with high brightness while mitigating motion-induced artifacts. Recently developed ratiometric GECIs primarily employ cyan and yellow-fluorescent fluorescence resonance energy transfer pairs, and thus fall short in some applications that require deep tissue penetration and resistance to photobleaching. We engineered a set of green-red ratiometric calcium sensors that fused two fluorescent proteins and calcium sensing domain within an alternate configuration. The best performing elements of this palette of sensors, Twitch-GR and Twitch-NR, inherited the superior photophysical properties of their constituent fluorescent proteins. These properties enabled our sensors to outperform existing ratiometric calcium sensors in brightness and photobleaching metrics. In turn, the shot-noise limited signal fidelity of our sensors when reporting action potentials in cultured neurons and in the awake behaving mice was higher than the fidelity of existing sensors. Our sensor enabled a regime of imaging that simultaneously captured neural structure and function down to the deep layers of the mouse cortex.
Highlights
Ratiometric genetically encoded calcium indicators (GECIs) record neural activity with high brightness while mitigating motion-induced artifacts
GECIs largely fall into two categories: the single fluorescence channel category of GECIs includes families of sensors such as the GCaMPs5–7 and RCaMPs8–10; the two fluorescence channel category of GECIs includes families of sensors such as the yellow cameleons (YCs)[11,12,13,14] and Twitches[15,16,17]
The sensing domain responds to changes in calcium concentration by modulating the relative positioning and fluorescence resonance energy transfer (FRET) efficiency between the two fluorescent proteins, and modulating the fluorescence intensities of the two fluorescent proteins[13,17]
Summary
Ratiometric genetically encoded calcium indicators (GECIs) record neural activity with high brightness while mitigating motion-induced artifacts. The best performing elements of this palette of sensors, Twitch-GR and Twitch-NR, inherited the superior photophysical properties of their constituent fluorescent proteins These properties enabled our sensors to outperform existing ratiometric calcium sensors in brightness and photobleaching metrics. 1234567890():,; Genetically encoded calcium indicators (GECIs) are powerful tools for reporting neural activity and elucidating neural function[1,2,3] Neural activity such as action potentials modulate calcium fluxes across the neuron membrane through voltage-gated calcium channels, and drive intracellular calcium concentration changes. Two-channel ratiometric sensors can potentially overcome issues of brightness by combining a calcium sensing domain with two fluorescent proteins to report calcium activity. The bright ratiometric sensors provide structural information, such as sub-cellular axonal or dendritic compartments, in conjunction with dynamic information during experiments This ability would allow experimentalists to chronically and repeatably locate target structures for imaging
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