Abstract
Genetically-encoded calcium indicators (GECI) are indispensable tools for real-time monitoring of intracellular calcium signals and cellular activities in living organisms. Current GECIs face the challenge of sub-optimal peak signal-to-baseline-ratio (SBR) with limited resolution for reporting subtle calcium transients. We report herein the development of a suite of calcium sensors, designated NEMO, with fast kinetics and ultra-wide dynamic ranges (>200-fold). NEMO indicators report Ca2+ transients with peak SBRs ∼20-fold larger than the top-of-the-range GCaMP series. NEMO sensors further enable the quantification of absolution calcium concentration with ratiometric or photochromic imaging. Compared to GCaMPs, NEMOs could detect single action potentials in neurons with a peak SBR two times higher and a median peak SBR four times larger in vivo, thereby outperforming most existing state-of-the-art GECIs. Given their ultra-high sensitivity and resolution to report intracellular Ca2+ signals, NEMO sensors may find broad applications in monitoring neuronal activities and other Ca2+-modulated physiological processes in both mammals and plants.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
(1) We performed more two-photon characterization of NEMO sensors as advised by reviewer 1 and 3: i) Obtained in vitro two-photon cross section of NEMOc. ii) Examined the dynamic ranges of NEMOs excited by two-photon light in HEK cells. We found that the dynamic range and relative basal brightness of NEMOs were similar to those observed with one-photon excitation. iii) Carried out in vivo recordings of NEMOf with optimal two-photon excitation, and confirmed that optimally-excited NEMOf signals showed brighter basal fluorescence and greatly improved signal-to-noise ratio (SNR). (2) Measured the photostability of all NEMO sensors as suggested by reviewer 3. (3) We characterized more merits of NEMO sensors arising from its larger dynamic range as advised by reviewer 1: i) Using a photo-activatible Ca2+ channel or store operated Ca2+ entry as readouts, we demonstrated NEMO's capacity to resolve small differences in amplitudes of Ca2+ signals. ii) We performed more recordings on cell lines, cultured neurons, and in vivo. Together with data analysis on previously obtained data, we showed that NEMOm, NEMOs and NEMOf have better SNR than the corresponding GCaMP6 or NCaMP7 indicators with comparable Ca2+ affinities. (4) To better demonstrate that photochromic NEMO sensors can directly show absolute Ca2+ levels, we replaced previous NEMOf responses with those of NEMOs, because NEMOs is more suitable for detecting basal Ca2+ level and small Ca2+ transients.