Supplementary Figure 1 In vitro screening of recombinant R-CaMP2 variants. Baseline fluorescence compared to R-CaMP1.07 at nominally zero calcium plotted versus dynamic range ( F/F) for 150 recombinant candidate sensors (gray dots). As a reference, the values for R-GECO1 (orange), R-CaMP1.07 (blue), R-CaMP2 (black), and R- GECO2L (red) are indicated.
Supplementary Figure 2 Fluorescence spectra of R-CaMP2 and R-GECO2L. (a,b) Normalized fluorescence excitation (dashed lines) and emission (solid lines) spectra of Ca 2+ -free (bottom traces) and Ca 2+ - saturated (upper traces) R-CaMP2 (a) and R-GECO2L (b). The fluorescence intensity of each indicator was normalized to the peaks of the Ca 2+ -saturated spectra. (c) Two-photon cross-section (δ) spectra of R-CaMP2 in the Ca 2+ -free (bottom dotted traces) or Ca 2+ -saturated (upper solid traces) state. Error bar, s.e.m. (n = 3).
Supplementary Figure 3 ph titration of normalized fluorescence intensity and dynamic change ( F/F) of red GECIs. (a-c) ph titration of normalized fluorescence intensity of Ca 2+ -free (black) and Ca 2+ -saturated (red) states. The fluorescence intensity of each indicator was normalized to the peak of the calcium-saturated fluorescence intensity. Gray line indicates the dynamic range ( F/F). Experimental pk a values are summarized in Table 1. (a) R-GECO1 (b) R-CaMP1.07 (c) R-CaMP2
Supplementary Figure 4 Stopped-flow kinetic characterization of R-CaMP1.07 and R-CaMP2. Observed relaxation rate constants (K obs) plotted as a function of Ca 2+ concentrations. The association and dissociation rate constants and the Hill coefficients (K on, K off and n, respectively) were determined by fitting to the equation K obs = K on [Ca 2+ ] n + K off; these values are shown in Supplementary Table 2.
Supplementary Figure 5 Comparison of R-CaMP sensors during 1AP-triggered Ca 2+ imaging of synaptic boutons. (a) Representative image of synaptic boutons in cultured hippocampal neuron expressing R-CaMP2 (top) and R-CaMP1.07 (bottom), 50 msec after one field stimulus evoked one action potential. Left: EGFP (in monochrome). Right: Pseudocolor image of R-CaMP2 and R-CaMP1.07. Scale bar, 5 µm. (b) Single trials (gray) and trial-averaged responses of R-CaMP2 (black), R-GECO2L (red), and R-CaMP1.07 (blue) in response to a single AP. (c) Amplitude, SNR, rise times, and decay time constants for a single AP. Error bars, s.e.m. (n = 13, 8, 17 dishes sampled from 81, 52, 108 synaptic boutons for R-CaMP2, R-GECO2L, R-CaMP1.07, respectively, *P < 0.05, **P < 0.01, ***P < 0.001 in Tukey s post-hoc test following one-way ANOVA. Individual P values: Amplitude, 9.9 10 5 (R-CaMP2/R-GECO2L), 4.7 10 14 (R-CaMP2/R- CaMP1.07); SNR, 1.8 10 8 (R-CaMP2/R-GECO2L), 3.1 10-13 (R-CaMP2/R-CaMP1.07); Rise time, 1.1 10-2 (R-CaMP2/R- CaMP1.07).
Supplementary Figure 6 Comparison of R-CaMP2 and R-CaMP1.07 during a single Glu photolysis-induced somatic AP-triggered Ca 2+ imaging at the soma in cultured hippocampal neurons. (a) Single trials (n = 9, gray) and trial-averaged responses of R-CaMP2 (left, black) and R-CaMP1.07 (right, blue) in response to a single pulse of MNI-glutamate uncaging. (b) Amplitude, SNR, rise times, and decay time constants for a single pulse of MNI-glutamate uncaging. Error bars, s.e.m. (n = 9 cells each, *P < 0.05, ***P < 0.001 in Welch s t-test. Individual P values: Amplitude, 8.0 10 4 ; SNR, 4.8 10 2 ; Rise time, 2.6 10-2 ; Decay time, 2.7 10 7.
Supplementary Figure 7 Comparison of R-CaMP2 and R-CaMP1.07 during a 1AP-evoked Ca 2+ transient in acute cortical slices. (a) Amplitude, (b) SNR, (c) rise times, and (d) decay time constants of somatic Ca 2+ transients induced by a single AP. Error bars, s.e.m. (n = 10 cells each, ***P < 0.001 in Welch s t-test. Individual P values: Amplitude, 2.0 10 4 ; SNR, 4.3 10 6 ; Rise time, 1.3 10-7 ; Decay time, 3.0 10 11.
Supplementary Figure 8 Trial-averaged fluorescence transients of R-CaMP2 and R-CaMP1.07 to five spike trains delivered at different frequencies. (a,b) Trial-averaged data from experiments in Fig. 3d,e. Gray lines indicate individual sweeps (n = 10). Black and blue thick lines indicate average traces of R-CaMP2 (a) and R-CaMP1.07 (b). Right side of both figures: responses to 10, 20, and 40 Hz spike trains are shown in an expanded time scale. Stimulus time stamps are indicated by the vertical bars and dotted lines.
Supplementary Figure 9 Persistent R-CaMP2 expression does not affect passive membrane properties or Ca 2+ transient kinetics. (a) Representative traces of membrane potential deflections induced by current injection. Control (left, untransfected contralateral hemisphere), CAG-R-CaMP2 expressing neuron (right) (b) Resting membrane potential (left), membrane capacitance (middle), membrane input resistance (right) were unchanged between groups. Error bars, s.e.m. (n = 18, 17 cells for control (untransfected contralateral hemisphere) neuron, and CAG-R-CaMP2 expressing neuron, respectively, P = 0.824, 0.365, 0.588 respectively, ns (not significant), Welch s t-test). (c) Representative traces of fluorescence changes ( F/F) in response to a single AP in neurons expressing either a TRE or a CAG promoter-driven R-CaMP2 in acute slices. Ten-trial average response of a single sweep each (gray) and averages of 10 sweeps (black) are overlaid (n = 10, 8 cells for TRE-R-CaMP2, CAG-R-CaMP2, respectively). (d) Either TRE or CAG promoter-driven R-CaMP2-expressing neurons showed undistinguishable single AP-evoked Ca 2+ transient kinetics.
Supplementary Figure 10 Ectopic R-CaMP2 expression has little effect on radial migration and does not cause toxic cell death. Mouse embryo were electroporated at E14 with a plasmid expressing control GCaMP6f (a,d: control) or R-CaMP2 (b,e: R-CaMP2), and sacrificed at E18 (a-c) or P18 (d-f). Coronal brain slices were imaged for baseline fluorescence (white) and immunostained with activated-caspase-3 (red). White arrowheads indicate labeled activated-caspase-3 cell. Scale bars, 100 µm (a,b) and 200 µm (d,e) respectively. (c,f) Quantification of the transfected cells that are activated-caspase-3 positive (Cas3+). Error bars, s.e.m. (n = 9182, 8242 cells for control (GCaMP6f), R-CaMP2 from 7 pairs of littermate animals (E18) for each condition, P = 0.736 (c), ns (not significant), Welch s t- test). (n = 5171, 6302 cells for control (GCaMP6f), R-CaMP2 from 6 pairs of littermate animals (P18) for each condition, P = 0.978 (f), ns (not significant), Welch s t-test).
Supplementary Figure 11 Simultaneous Ca 2+ imaging and electrophysiology in neocortical layer 2/3 neurons in vivo. (a) Representative traces of simultaneous recordings of calcium transients (top) and APs (bottom) in an R-CaMP2-expressing layer 2/3 neuron in the barrel cortex. The number of spikes for each burst is indicated below the trace. Scale bar, 5 µm. (b) Amplitude, SNR, and integral of calcium transients induced by number of APs in a 200 msec bin (n = 254, 115, 45, 26, 13 events for 1, 2, 3, 4, 5 action potentials, n = 9 cells in 7 mice). Error bars, s.e.m. (c) Median fluorescence change in response to a single action potential in vivo. Shading indicates s.e.m. (n = 9 cells). (d) The rise time and decay time constants of calcium transients in vivo induced by a single AP measured with cell-attached recording. Error bars, s.e.m.
Supplementary Figure 12 All-optical interrogation of an avoidance circuit using R-CaMP2 in freely moving C. elegans. (a) Schematic diagram of the ASH neuron-mediated avoidance circuit. (b) Representative R-CaMP2 response images in a freely moving transgenic animal jqex467, coexpressing R-CaMP2 (red) and EGFP (green) in AVA neurons (AVA::R-CaMP2), and expressing ChR2 in ASH neurons (ASH::ChR2). R-CaMP2 and EGFP signals were also weakly detected in a few other cells including AVE and AVD neurons. Arrowhead indicates AVA neuron. Bar 20 µm. (c) Behavioral responses of the jqex467 worms with or without all-trans-retinal (ATR). The observable backward response during the blue light illumination was counted. ***P < 0.001 in Tukey s post-hoc test following one-way ANOVA. Individual P values: 1.2 10 4 (Wild type without ATR/ASH::ChR2; AVA::R-CaMP2 with ATR), 5.3 10 5 (with/without ATR in ATR/ASH::ChR2; AVA::R-CaMP2 background). (d) Causal relation between blue light-activated ASH neuronal activity (ChR2 on, light blue shade), AVA activity (R-CaMP2, blue trace) and backward movement (behavioral response). R-CaMP2 traces and backward responses of a jqex467 worm. Gray line shows a raw data of R-CaMP2/EGFP ratio image ( R/R) and black line shows a moving average of across 1 sec window.
Supplementary Table 1. Optical characteristics of R-CaMP1.07 and R-CaMP2. GECI Ca 2+ l abs a (ε b ) R-CaMP1.07 R-CaMP2 - + - + 446 (22900) 577 (5320) 445 (7370) 563 (43800) 445 (28200) 576 (14200) 445 (16600) 563 (48100) l em c (Φ d ) Brightness e 592 (0.09) 490 583 (0.20) 8910 591 (0.11) 1590 583 (0.23) 11200 a Absorbance peak (nm). b Extinction coefficient (M -1 cm -1 ). c Emission peak (nm). d Quantum yield. e Product of ε and Φ.
Supplementary Table 2. Kinetic characterization of R-CaMP1.07 and R-CaMP2 by stopped-flow photometry. GECI K on (M -n s -1 ) K off (s -1 ) n K d kinetic (nm) a K d static (nm) b R-CaMP1.07 2.38 10 9 0.86 1.4 226 192 R-CaMP2 1.46 10 8 1.00 1.1 56 69 a K d kinetic = (K off /K on ) 1/n. b From Figure 1c.