Extended Data Fig. 8: Performance of NEMO sensors in rodent neurons.

(a) Signal-to-noise ratio (SNR) of GECIs in cultured neurons (unpaired Student’s t-test, two-tailed; Left panel, NEMOs vs NCaMP7 at 20 Hz, ^*, p = 0.0182; 60 Hz, ^**, p = 0.0035; 100 Hz, ^****, p = 2.98E-8; 180 Hz, ^****, p = 1.50E-6; right panel, NEMOf vs GCaMP6f at 20 Hz, ^***, p = 1.05E-4; 60 Hz, ^****, p = 2.24E-7, 100 Hz, ^****, p = 2.41E-9; 180 Hz, ^****, p = 1.24E-6) (For stimulation at varied frequencies, NCaMP7, n = 12, 12, 12, 14, 12 cells. The ‘n’ values of other sensors were equal to Fig. 3. Each GECI measurement set was analyzed from multiple dendrites of neurons in three different primary hippocampal neuron cultures. (b) Fluorescence responses of NEMO variants in neurons of acute mouse brain slices. Left, cartoon showing the set up of two-photon fluorescence imaging under a whole-cell patch clamp configuration. Middle, statistics showing the peak SBR-frequency of action potentials (AP) (NEMOf vs GCaMP6s at 50 Hz, p = 3.5E-4; at 100 Hz, p = 1.4E-5; unpaired Student’s t-test, two-tailed) (GCaMP6f, n = 57 cells from 3 mice; GCaMP6s, n = 51 cells from 2 mice; NEMOs, n = 30 cells from 4 mice; NEMOm, n = 45 cells from 3 mice; NEMOf, n = 54 cells, from 3 mice); right, half decay time of NEMO variants under different intensities of stimuli (For stimulation from 5-100 AP, GCaMP6f, n = 12,14,18,18,18 cells from 3 mice; GCaMP6s, n = 17 cells from 2 mice; NEMOm, n = 2,4,14,16,16 cells from 3 mice; NEMOs, n = 8,9,10,10,9 cells from 4 mice; NEMOf, n = 5,9,15,18,17 cells from 3 mice; for stimulation at 1 AP, GCaMP6s, n = 10 cells from 2 mice; GCaMP6f, n = 3 cells from 3 mice). (c-f) In vivo two-photon imaging of visual cortex neurons in response to drift gratings. (C) Typical responses; (D-F) Statistics of results shown in Fig. 4a–c; (D) Cumulative distribution of orientation (left, ^****, p = 0.000102) or direction (right, for NEMOf vs GCaMP6f and NEMOs vs GCaMP6s, ^****, p = 0.0002; ^***, p = 0.005) selectivity (Kolmogorov–Smirnov test, two-tailed). (E) Statistics of half decay time (GCaMP6f, n = 46 cells from 2 mice; GCaMP6s, n = 157 cells from 3 mice; NEMOs, n = 223 cells from 4 mice; NEMOm, n = 105 cells from 3 mice; NEMOf, n = 40 cells from 3 mice). (F) Statistics showing fraction of responsive cells (NEMOf vs GCaMP6f, p = 0.1356; NEMOs vs GCaMP6s, p = 0.9805, unpaired Student’s t-test, two-tailed) (GCaMP6f, n = 21 cells from 2 mice; GCaMP6s, n = 23 cells from 3 mice; NEMOs, n = 35 cells from 4 mice; NEMOm, n = 24 cells, from 3 mice; NEMOf, n = 30 cells from 3 mice). (g) Cumulative distribution of SNR (NEMOf vs GCaMP6f and NEMOs vs GCaMP6s, p = 0.25; ^****, p = 1.69E-6, Kolmogorov-Smirnov test, two-tailed). At least n = 3 independent biological replicates. Data in A, B, E and F panels were shown as mean ± s.e.m.

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