Sensitive red protein calcium indicators for imaging neural activity
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- Weizmann Institute of Science, Israel
- Howard Hughes Medical Institute, The Rockefeller University, United States
Figures
Figure 1
Mutagenesis and screening of jRCaMP1 and jRGECO1 in dissociated neurons.
(a) RCaMP1h and R-GECO1 structure and mutations introduced in jRCaMP1a, jRCaMP1b, and jRGECO1a. M13 peptide (yellow), linker 1 (gray), cpmRuby or cpmApple (red), linker 2 (gray), CaM (blue). …
Figure 2 with 4 supplements
jRGECO1 and jRCaMP1 performance in dissociated neurons.
(a) Average responses in response to one action potential (AP) for RCaMP1h (9479 neurons, 605 wells), R-GECO1 (8988 neurons, 539 wells), R-CaMP2 (265 neurons, 22 wells), jRGECO1a (383 neurons, 26 …
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Figure 2—source data 1
Biophysical properties of purified jRGECO1 and jRCaMP1 sensors.
Summary of red GECI biophysical properties, mean ± s.d., where indicated, for independently purified protein samples (Materials and methods).
- https://doi.org/10.7554/eLife.12727.005
Figure 2—figure supplement 1
Absorption and emission spectra of red GECIs.
(a) One-photon excitation (dashed lines) and emission (solid lines) spectra for jRGECO1a (left panel), jRCaMP1a (middle), and jRCaMP1b (right) in Ca-free (blue lines) and Ca-saturated (red lines) …
Figure 2—figure supplement 2
Biophysical properties.
(a) Fluorescence quantum yield of red GECIs and red FPs (all at 1070 nm), and GCaMP6s (940 nm). (b) Peak two-photon molecular brightness (Ca-bound state for GECIs) of red GECIs, red FPs, and GCaMP6s …
Figure 2—figure supplement 3
Photoswitching in purified protein assay.
Fluorescence traces of purified Ca2+-free protein droplets (red traces) of jRCaMP1a (left panel), jRGECO1a (middle), and R-CaMP2 (right) constantly illuminated with 561 nm excitation light (40 mW/mm2…
Figure 2—figure supplement 4
jRCaMP1a is more compatible than jRGECO1a for simultaneous use with ChR2.
(a) Fluorescence signal from cultured rat hippocampal neurons transfected with either jRGECO1a alone (gray) or jRGECO1a+ChR2-Venus (blue) following stimulation with blue light (33 mW, 10 ms pulses, …
Figure 3 with 2 supplements
jRGECO1a and jRCaMP1a and jRCaMP1b performance in the mouse primary visual cortex.
(a) Top, schematic of the experiment. Bottom, image of V1 L2/3 cells expressing jRGECO1a (left), and the same field of view color-coded according to the neurons’ preferred orientation (hue) and …
Figure 3—figure supplement 1
Comparison of orientation tuning in V1 neurons measured with different red GECIs.
Distribution of orientation selectivity index (OSI, 3 upper rows) for all cells detected as responsive, measured using different GECIs. Bottom panel, mean ± s.d for all constructs show similar …
Figure 3—figure supplement 2
Long-term expression of red GECIs in mouse V1.
(a) Example images of V1 L2/3 neurons after long term expression of red GECIs. (b) Comparison of the proportion of responsive cells, orientation-tuned cells, and the ratio between them. Each time …
Figure 4 with 3 supplements
Combined imaging and electrophysiology in the mouse visual cortex.
(a) Simultaneous fluorescence dynamics and spikes measured from jRGECO1a (top, blue) and jRCaMP1a (bottom, black) expressing neurons. The number of spikes for each burst is indicated below the trace …
Figure 4—figure supplement 1
jRGECO1a accumulates in lysosomes.
(a) Red GECI expression (top, red) and LAMP-1 immunostaining (bottom, green) of fixed tissue sections from mouse V1. (b) Distribution of the fraction of somatic area covered by protein aggregates …
Figure 4—figure supplement 2
Red GECIs lack functional response at 900nm excitation.
Functional responses of three example cells (jRGECO1a, expressed in L4 V1 neurons) at 1040 nm excitation (left) and 900 nm (25 vs. 1 cells were detected as responsive out of 50 cells in FOV; …
Figure 4—figure supplement 3
Complex spectral and functional characteristics of the red GECIs after long-term expression in vivo.
(a) Images of jRGECO1a (left) and jRCaMP1a (right) L2/3 neurons in vivo excited by 1040 nm (top) and 900 nm light (bottom). Note the punctate fluorescence image in the bottom images. (b) Emission …
Figure 5
Deep tissue imaging using red GECIs.
(a) Left, schematic of the measurement. L5 neuron apical dendrites were imaged at different depths (FOVs 1-n). Right, RCaMP1h fluorescence from an L5 apical dendrite (red dots) as a function of …
Figure 6
Dual color imaging in the mouse visual cortex.
(a) Two-photon action spectra of Ca- saturated GCaMP6s, jRCaMP1a, and jRGECO1a. Measurements were done on purified protein (Materials and methods). (b) Image of L5 apical dendrites (red) and LM …
Figure 7 with 3 supplements
Imaging activity in Drosophila larval NMJ boutouns with red GECIs.
(a) Schematic representation of Drosophila larval neuromuscular junction (NMJ) assay. Segmented motor nerve is electrically stimulated while optically imaging calcium responses in presynaptic …
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Figure 7—source data 1
Summary of results shown in Figure 7—figure supplement 1.
Wilcoxon rank sum test is used for p-value.
- https://doi.org/10.7554/eLife.12727.022
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Figure 7—source data 2
Summary of results shown in Figure 7—figure supplement 2.
Wilcoxon rank sum test is used for p-value.
- https://doi.org/10.7554/eLife.12727.023
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Figure 7—source data 3
Summary of results shown in Figure 7—figure supplement 3.
Wilcoxon rank sum test is used for p-value.
- https://doi.org/10.7554/eLife.12727.024
Figure 7—figure supplement 1
Imaging activity in Drosophila larval NMJ boutons with jRGECO1a.
(a) Schematic of experimental setup. Epifluorescence and high magnification △F/F0 images of Type 1b boutons (green arrowheads) from muscle 13 (segments A3-A5), with image segmentation ROIs …
Figure 7—figure supplement 2
Imaging activity in Drosophila larval NMJ boutons with jRCaMP1 constructs.
(a) Schematic of experimental setup. Epifluorescence and high magnification △F/F0 images of Type 1b boutons (green arrowheads) from muscle 13 (segments A3-A5), with image segmentation ROIs …
Figure 7—figure supplement 3
Comparing red and green GECI activity in Drosophila larval NMJ boutons.
(a–b) Single trial and averaged fluorescence transients after 1 Hz (a) and 5 Hz (b) stimulus for 2 s for jRGECO1a, jRCaMP1a, jRCaMP1b, GCaMP6s and GCaMP6f (jRGECO1a: 12 FOVs in 7 flies, 48 boutons; …
Figure 8
Imaging activity in the zebrafish trigeminal neurons with red GECIs.
(a) Schematic representation of zebrafish trigeminal neurons assay. Zebrafish larvae (3–4 days post fertilization) were paralyzed, embedded in agarose, and stimulated with electrodes (20 ms pulses; …
Figure 9
Imaging activity in the C. elegans ASH and AWC neurons with red GECIs.
(a) Schematic representation of the Caenorhabditis elegans imaging assay. A paralyzed animal is restrained in a microfluidic device with its nose exposed to a fluid channel. Delivery of stimulus (S) …
Author response image 1
Author response image 2
Videos
Video 1
jRGECO1a L2/3 functional imaging in the mouse V1.
The mouse was anesthetized and presented with moving gratings in eight directions to the contralateral eye. Gratings were presented for 4 s (indicated by appearance of an arrowhead in the grating …
Video 2
Dual-color imaging of axons and apical dendrites in L1 of the mouse V1.
jRGECO1a labeled apical L5 dendrites (red), and GCaMP6s labeled axons from LM (green) were imaged in L1 of V1 (left). ΔF/F0 traces from 4 ROIs (Figure 6b) are presented (right). Field of view size …
Additional files
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Supplementary file 1
Comprehensive neuronal culture screening results for RCaMP1h variants.
- https://doi.org/10.7554/eLife.12727.030
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Supplementary file 2
Comprehensive neuronal culture screening results for R-GECO1 variants.
- https://doi.org/10.7554/eLife.12727.031
