Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.
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Most of the datasets generated for characterizing the new sensors are included in the published article (and its Supplementary Information files). Additional data that support the findings of this study are available from the corresponding authors upon reasonable request. Correspondence and requests for flies should be addressed to V.J. and L.L.L. for jGCaMP7 protein structure information and constructs and K.S. for neuronal culture screen information and mice.
DNA constructs and AAV plasmids for the jGCaMP7 variants were deposited for distribution at Addgene (http://www.addgene.org, plasmid numbers 104463, 104483–84, 104487–89, 104491–93, 104495–97, 105321–23). Drosophila stocks were deposited at the Bloomington Drosophila Stock Center (http://flystocks.bio.indiana.edu). Gene sequences were deposited at NCBI GenBank (https://www.ncbi.nlm.nih.gov/nuccore/, accession codes: jGCaMP7s MK749391, jGCaMP7f MK749392, jGCaMP7b MK749393, jGCaMP7c MK749394).
AAnalysis codes that support the findings of this study are available from the corresponding authors upon reasonable request.
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We thank D. Flickinger for design of the microscope used for spine imaging, M. Reiser, M. Isaacson, J. Chen, J. Liu and A. Chiu for the G4.0 panel display system used in the fly-on-ball imaging experiments and D. Walpita and J. Hagemeier for neuronal culture (all from Janelia). This work is part of the GENIE Project at the Howard Hughes Medical Institute, Janelia Research Campus. A.K. is supported by the Hertie Foundation.
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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Measurement of excitation and emission spectra for GCaMP6 and jGCaMP7 sensors. Emission spectra were calculated with 495 nm excitation light, excitation spectra were calculated with 515 nm emission light (data from a single measurement per sensor).
Measurement of the two-photon excitation spectra for GCaMP6 and jGCaMP7 sensors (averaged data from n = 2 independent measurements per sensor).
Measurement of jGCaMP7 properties in purified protein solutions: a. Molecular brightness (average of n = 2 independent measurements). b. Two-photon excitation cross-section (average of n = 2 independent measurements). c. Bleaching time constant in calcium-saturated solution (n = 3 independent measurements, blue dots). d. Bleaching time constant in calcium-free solution (bleaching was measured with 6.5 mW of 488 nm illumination, light intensity was 4.5 W/cm2, protein concentration was 4 μM, n=3 independent measurements, blue dots, errorbars show mean ± s.d.).
The response amplitude was averaged and normalized over 980 neurons expressing jGCaMP7s and plotted vs. trial number. No stimulus adaptation was evident (mean ± s.e.m.).
Supplementary Figure 5 Comparison of orientation tuning in V1 neurons measured with different sensors.
a. Distribution of orientation selectivity index (OSI) for visually responsive cells measured using different sensors (n = 625 cells, jGCaMP7s; 614, jGCaMP7f; 844, jGCaMP7b; 345, jGCaMP7c; 337, GCaMP6s; 221, GCaMP6f). The left-shifted distributions of jGCaMP7s and GCaMP6s are probably due to high sensitivity and affinity compared to the other sensors, which enables detection of the weaker responses to non-preferred grating directions and reduces the OSI value. b. Distribution of OSI values across sensors (same data as in a). Red lines correspond to medians, each box shows the 25th to 75th percentile range, whisker length is the shorter of 1.5 times the 25th to 75th range or until the extreme data point. c. Averaged ΔF/F0 traces of all cells detected as responsive from a single mouse (gray traces, left: n = 2 mice, 384 neurons, for jGCaMP7s; right: n = 3 mice, 321 neurons, for jGCaMP7f) and their mean (red and green lines), shifted that the preferred response for each cell appears first. jGCaMP7s shows a small increase in its response to the drifting grating moving to the orthogonal direction to the preferred stimulus, compared to jGCaMP7f, which is likely to contribute to the left shift of its OSI distribution shown in a.
a. Expression schema for targeted single-neuron labeling. b. Experimental setup, V1 imaging. c. Mean images of example dendritic imaging session for GCaMP6s (top, one example cell out of 5 different recorded neurons) and jGCaMP7b (bottom, one example cell out of 5 different recorded neurons). d. GCaMP7b labelled neurons had more detectable spines (medians [95% CI]: 0.23 [0.17–0.32] vs. 0.3 [0.23–0.37] spines/μm for 6s and 7b respectively; n = 35 dendritic segments (5 neurons from 4 animals) and 37 dendritic segments (5 neurons from 4 animals) for 6s and 7b respectively, p = 0.01, Wilcoxon rank-sum test). e. GCaMP7b labelled neurons had a higher number of spontaneously active spines (medians [CI]: 0.17 [0.12–0.24] vs. 0.23 [0.21–0.33] spines/μm for 6s and 7b respectively; n = 35 dendritic segments (5 neurons from 4 animals) and 37 dendritic segments (5 neurons from 4 animals) for 6s and 7b respectively, p = 0.001, two-sided Wilcoxon rank-sum test. Orange lines correspond to medians, each box shows the 25th to 75th percentile range, whisker length is the shorter of 1.5 times the 25th to 75th range or until the extreme data point.
Supplementary Figs. 1–6 and Supplementary Tables 1–3
Comprehensive neuronal culture screening results for variants of GCaMP6s
Recording of jGCaMP7s fluorescence signal of layer 2/3 neurons in the mouse visual cortex in vivo: this video shows 83 s of continuous fluorescence recording from one FOV of jGCaMP7s-expressing neurons (shown also in Fig. 5a) in the mouse visual cortex, in response to drifting grating stimulation (6 s of blank display, followed by 4 s of gratings moving in one of eight directions, 0.05 cycles per degree, 1 Hz temporal frequency). The times when the grating appears and its movement direction are indicated by the arrow in the upper left corner of the respective frames. FOV size 250 μm, 50 FOVs were recorded from n = 4 mice expressing jGCaMP7s with similar results, as summarized in Fig. 5.
Recording of jGCaMP7f fluorescence signal of layer 2/3 neurons in the mouse visual cortex in vivo: this video shows 83 s of continuous fluorescence recording from one FOV of jGCaMP7f-expressing neurons in the mouse visual cortex, in response to drifting grating stimulation (4 s of blank display, followed by 4 sec of gratings moving in one of eight directions, 0.05 cycles per degree, 1 Hz temporal frequency). The times when the grating appears and its movement direction are indicated by the arrow in the upper left corner of the respective frames. FOV size 250 μm, 55 FOVs were recorded from n = 5 mice expressing jGCaMP7f with similar results, as summarized in Fig. 5.
Recording of jGCaMP7c fluorescence signal of layer 2/3 neurons in the mouse visual cortex in vivo: this video shows 83 s of continuous fluorescence recording from one FOV of jGCaMP7c-expressing neurons in the mouse visual cortex, in response to drifting grating stimulation (6 s of blank display, followed by 4 s of gratings moving in one of eight directions, 0.05 cycles per degree, 1 Hz temporal frequency). The times when the grating appears and its movement direction are indicated by the arrow in the upper left corner of the respective frames. FOV size 250 μm, 38 FOVs were recorded from n = 4 mice expressing jGCaMP7c with similar results, as summarized in Fig. 5.
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