Abstract
Understanding information processing in the brain requires monitoring neuronal activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope empowered by all-optical laser scanning, we imaged neuronal activity in vivo at up to 3,000 frames per second and submicrometer spatial resolution. This imaging method enabled monitoring of both supra- and subthreshold electrical activity down to 345 μm below the brain surface in head-fixed awake mice.
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Data availability
Raw image sequences used to generate all spontaneous activity traces and representative sensory-evoked activity traces in Fig. 2 are deposited at https://figshare.com/articles/Image_sequences_for_Fig_2/11492712. The raw image sequences for the other data presented in Fig. 2 are not available at a public repository due to their large size (>1 TB) but are available upon reasonable request to the correspondence authors.
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Acknowledgements
We thank C. Rodriguez for help with the laser system; E. Carroll for help with electronics; R. Lu for help with visual stimulation experiments; G. Cao (Janelia Research Campus) for providing cultured neuron samples; J. Marvin and L. Looger (Janelia Research Campus) for providing glutamate sensors; and the Janelia JET team for designing and assembling the dispersion compensation unit. This work was supported by the Howard Hughes Medical Institute (J.W., Y.L., C.-L.H., N.J.) and an American Epilepsy Society predoctoral fellowship (M.C.); the China Scholarship Council Joint PhD Training Program (D.S.); Stanford Neuroscience PhD Program training grant (no. 5T32MH020016) and the Post-9/11 GI Bill (S.W.E.); the Research Grants Council of the Hong Kong Special Administrative Region of China (grant nos. 17209017, 17259316, 17207715 to J.W. and K.K.T.); and NIH BRAIN Initiative grants (nos. 1U01NS103464 and 1RF1MH114105 to M.Z.L. and 1UF1NS107696 to J.W., N.J. and K.K.T.).
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Contributions
N.J. conceived the project. M.Z.L., K.K.T. and N.J. supervised research. J.W., K.K.T. and N.J. designed the FACED module. Y.L., S.C. and C.-L.H. prepared samples. M.C. created ASAP3. M.C., S.W.E. and D.S. characterized ASAP3 and ASAP3-expressing viruses. J.W. collected and analyzed the data. J.W. and N.J. wrote the manuscript with input from all authors.
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K.K.T. and the University of Hong Kong have filed a US patent application (14/733,454) that relates to the all-optical laser-scanning imaging methods.
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Integrated supplementary information
Supplementary Figure 1 Optical layout of the FACED two-photon fluorescence microscope.
2X: 2-fold beam expander; λ/2: Half-wave plate; PBS: polarizing beam splitter; λ/4: quarter-wave plate; α: misalignment angle of the mirror pair; PMT: photomultiplier tube. The FACED module scans the foci along the X galvo direction. Note that the X galvo is deactivated for high-speed functional imaging, but can be used to tile the images to cover a larger field of view.
Supplementary Figure 2 Raw images taken at 1,000 fps for different indicators.
These images are representative of 1000 or 6000 frames captured continuously for each FOV. They are provided here to demonstrate the quality and signal-to-noise ratio of raw images captured at 1,000 Hz. Calcium indicators: GCaMP6s/6f; Glutamate indicators: A184V and S72A variants of SF-iGluSnFR; voltage indicator: ASAP3-Kv. Scale bar: 20 μm.
Supplementary Figure 3 1 kHz imaging of GCaMP6s-expressing cultured neurons.
(a) Morphological image. (b) Calcium transients of 3 cells in (a) evoked by field electrode stimulation and recorded at 1,000 fps. Darker lines were 100-point boxcar averages of the raw traces to guide the eye. Scale bar: 20 µm.
Supplementary Figure 4 1 kHz imaging of a GCaMP6f-expressing neuron in an acute brain slice.
(a) Morphological image. (b) Calcium transient of the cell in (a) evoked by field electrode stimulation and recorded at 1,000 fps. Darker line was 50-point boxcar average of the raw trace to guide the eye. Two independent stimulations were repeated with similar results. Scale bar: 20 μm.
Supplementary Figure 5 1 kHz imaging of spontaneous calcium increases in neurites of GCaMP6s-expressing cultured neurons.
(a) Mean intensity projection of 2,000 frames. (b) Temporal color coding of the 2,000 frames highlights the sites where calcium increases were initially observed (white arrowheads). (c) Calcium transients at the color masked positions in (a), indicating a calcium propagating speed of ~25 μm/s. Darker lines were 50-point boxcar averages of the raw traces to guide the eye. 5 datasets were recorded independently with similar results. Scale bar: 10 μm.
Supplementary Figure 6 1 kHz imaging of genetically encoded glutamate indicator iGluSnFR variants in cultured neurons and in V1 of awake mice in vivo.
(a) (Left) mean intensity projections of cultured neurons expressing either the A184V or the S72A variants of SF-iGluSnFR; (Right) transients associated with glutamate release triggered by extracellular electric stimulation (green dashed line) for structures labeled on the left. 10 experiments were repeated independently with similar results. (b) (Left) Representative images of layer 2/3 neurons (depth: 150–250 µm) expressing either A184V or S72A in V1 of awake mice; (Right) Transients associated with spontaneous glutamate releases at sites indicated by arrowheads on the left. Here, darker lines were 20-point boxcar averages of the raw traces to guide the eye. Green arrowheads on the traces highlight the fast rising edge of the glutamate transients. 4 experiments were repeated independently with similar results. Scale bars: 20 μm. Post-objective power: 25–30 mW for cultured neurons, 40 mW in vivo.
Supplementary Figure 7 Tracking rapid movement of a fluorescent particle in the awake mouse brain in vivo.
(a) Morphological image of layer-2/3 neurons expressing the A184V variant of the glutamate sensor iGluSnFR in V1 of an awake mouse. (b) Time traces at the three pixels indicated by the three asterisks in (a). (c) Top panel shows the maximum intensity projection of the 10 image frames collected at 1 kHz of the boxed region in (a); bottom panel shows the ten individual frames imaged at 1-ms intervals. We observed similar rapid movements of fluorescent particles in 3 independent experiments. Scale bars: 20 μm.
Supplementary Figure 8 Representative ASAP3-Kv fluorescence photobleaching during 6-s continuous 1KHz FACED 2PFM imaging.
The mean fluoresce intensity for the field of view (FOV) in Fig. 2g during a 6-sec recording. Imaging depth: 75 μm; FOV: 50 × 250 μm2; post-objective power: 35 mW.
Supplementary Figure 9 Representative bleaching and recovery dynamics of ASAP3-Kv fluorescence during intermittent 1-s duration 1KHz FACED 2PFM imaging.
Data obtained from ROI 2 in Fig. 2b. (a) Fluorescence signal change for eight 1-s recordings that were collected with a 2.5-s gap in between. (b) Box-and-whisker and scatter plots of photobleaching and recovery from 80 1-s 1,000-fps recordings. Bleaching is quantified by the ratio between the mean fluorescence signal of the last 50 frames and first 50 frames within a sequence of 1,000 frames (0.84 ± 0.04, mean ± s.d., n = 80 1-s 1,000-fps recordings). Recovery is quantified by the ratio of mean fluorescence signal between the n + 1th 1,000 frames and nth 1,000 frames (0.98 ± 0.05, mean ± s.d., n = 70 1-s 1,000-fps recordings). Gray filled circles are outliers in the box-and-whisker plots. Imaging depth: 125 μm; FOV: 50 × 50 μm2; post-objective power: 35 mW.
Supplementary Figure 10 Data processing for in vivo voltage traces.
(a) Baseline fluorescence F (red) was obtained by low-pass filtering (500-ms rolling percentile (50%) filter) of the raw signal trace (black). (b) Raw SNR trace (ΔF/√F, fluorescence change-to-noise ratio). (c) Smoothened SNR trace obtained by applying a 250-Hz low-pass Butterworth filter to (b). Optical spikes (green ticks) were identified as local SNR maxima above a spiking threshold (e.g., 7.5) at least 3 ms apart. (d) Raw ΔF/F. (e) Smoothened trace representing the subthreshold response obtained by applying a 50-Hz low-pass Butterworth filter to (d). All traces in Fig. 2 are raw traces as in (b) and (d).
Supplementary Figure 11 1 kHz imaging of voltage responses in V1 of awake mice at a 50 × 250 μm2 FOV.
(a) Dashed red box indicates the FOV for 1 kHz FACED imaging; within this FOV, 15 ASAP3-expressing cells were imaged simultaneously. (b) Voltage traces for the 7 cells labeled in (a). During a 6-s recording, a burst of two spikes were detected for ROI 1. Note the absence of these spikes in ROIs 2-5, which were in close proximity with ROI 1. Image depth: 155 μm; post-objective power: 75 mW. 7 experiments were repeated independently with similar results. Scale bar: 50 μm.
Supplementary Figure 12 Imaging the subthreshold oscillations in V1 of awake mice.
(a) Representative raw ΔF/F traces and (b) their corresponding power spectra from individual neurons. (c) Histogram of the peak oscillation frequencies (192 traces from 120 cells in three mice).
Supplementary Figure 13 Orientation and direction tuning of neurons in V1 of awake mice.
Left panels: FACED 2PFM images of the neurons. Middle panels: Raster plots showing optical spikes detected from each neuron (blue arrowheads) during the presentation of grating stimuli (10 trials for each of the 8 stimuli). Right panels: Polar plots showing mean spike rate (black) and average subthreshold ΔF/F (red) for (a) an orientation-selective and (b,c) two direction-selective neurons. Shaded areas: standard deviation. Representative results from 8 neurons. Image depth and post-objective power for a, b, c: 140, 170, 130 μm; 35, 30, 30 mW. Scale bar: 20 μm.
Supplementary Figure 14 3 kHz neural activity imaging in V1 of awake mouse in vivo.
(Left) Mean intensity projection of 9,000 frames recorded in 3 s; (Right) Top panel: a spontaneous calcium transient from the soma of a cell (red dashed ROI); bottom panel: fluorescence traces within two ROIs (orange and green ROIs) show calcium propagation along dendrites. Gray lines: raw data traces; colored lines: 100-point boxcar averaging of the raw traces. 4 experiments were repeated independently with similar results. Indicator: GCaMP6s; imaging depth: 125 μm; FOV: 50 × 100 μm2; post-objective power: 90 mW; excitation wavelength: 1035 nm. Scale bar: 10 µm.
Supplementary information
Supplementary Information
Supplementary Figs. 1–14 and Table 1
Supplementary Video 1
Imaging calcium transients at 1,000 fps in GCaMP6s-expressing cultured neurons evoked by extracellular electric stimulation. Same data as in Supplementary Fig. 3. The raw image sequence was binned every 50 frames, saved at 20 binned fps and compressed for video output.
Supplementary Video 2
Imaging calcium transients at 1,000 fps in GCaMP6f-expressing neurons in acute mouse brain slices evoked by extracellular electric stimulation. Same data as in Supplementary Fig. 4. The raw image sequence was binned every 50 frames, saved at 20 binned fps and compressed for video output.
Supplementary Video 3
Imaging spontaneous calcium release events at 1,000 fps in neurites of GCaMP6s-expressing cultured neurons. Same data as in Supplementary Fig. 5. The raw image sequence was binned every 20 frames, saved at 20 binned fps and compressed for video output.
Supplementary Video 4
Imaging glutamate transients at 1,000 fps in cultured neurons expressing A184V variant of iGluSnFR evoked by extracellular electric stimulation. Same A184V data as in Supplementary Fig. 6a. The raw image sequence was binned every 50 frames, saved at 20 binned fps and compressed for video output.
Supplementary Video 5
Imaging glutamate transients at 1,000 fps in cultured neurons expressing A184V variant of iGluSnFR evoked by extracellular electric stimulation. Same A184V data as in Supplementary Fig. 6a. The raw image sequence was binned every 50 frames, saved at 20 binned fps and compressed for video output.
Supplementary Video 6
Imaging glutamate transients at 1,000 fps in cultured neurons expressing S72A variant of iGluSnFR evoked by extracellular electric stimulation. Same S72A data as in Supplementary Fig. 6a. The raw image sequence was binned every 50 frames, saved at 20 binned fps and compressed for video output.
Supplementary Video 7
Imaging glutamate transients at 1,000 fps in cultured neurons expressing S72A variant of iGluSnFR evoked by extracellular electric stimulation. Same S72A data as in Supplementary Fig. 6a. The raw image sequence was binned every 50 frames, saved at 20 binned fps and compressed for video output.
Supplementary Video 8
Imaging glutamate transients of layer 2/3 neurons expressing A184V variant of iGluSnFR in V1 of awake mice at 1,000 fps. The white arrow points to the glutamate releasing site. Same A184V data as in Supplementary Fig. 6b. The raw image sequence was binned every 20 frames, saved at 20 binned fps and compressed for video output.
Supplementary Video 9
Imaging glutamate transients of layer 2/3 neurons expressing S72A variant of iGluSnFR in V1 of awake mice at 1,000 fps. The white arrow points to the glutamate releasing site. Same S72A data as in Supplementary Fig. 6b. The raw image sequence was binned every 20 frames, saved at 20 binned fps and compressed for video output.
Supplementary Video 10
Imaging rapid movement of a fluorescent particle at 1,000 fps in the layer 2/3 of awake mouse brain in vivo. Same data as in Supplementary Fig. 7. A sequence of 50 raw images recorded at 1,000 fps was saved at 20 fps and compressed for video output.
Supplementary Video 11
Imaging spontaneous calcium transients of layer 2/3 neurons expressing GCaMP6s in V1 of awake mice at 3,000 fps. Same data as in Supplementary Fig. 14. The raw image sequence was binned every 60 frames, saved at 20 binned fps and compressed for video output.
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Wu, J., Liang, Y., Chen, S. et al. Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo. Nat Methods 17, 287–290 (2020). https://doi.org/10.1038/s41592-020-0762-7
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DOI: https://doi.org/10.1038/s41592-020-0762-7
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