Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy

Journal name:
Nature Methods
Volume:
11,
Pages:
727–730
Year published:
DOI:
doi:10.1038/nmeth.2964
Received
Accepted
Published online

High-speed, large-scale three-dimensional (3D) imaging of neuronal activity poses a major challenge in neuroscience. Here we demonstrate simultaneous functional imaging of neuronal activity at single-neuron resolution in an entire Caenorhabditis elegans and in larval zebrafish brain. Our technique captures the dynamics of spiking neurons in volumes of ~700 μm × 700 μm × 200 μm at 20 Hz. Its simplicity makes it an attractive tool for high-speed volumetric calcium imaging.

At a glance

Figures

  1. Light-field deconvolution microscopy.
    Figure 1: Light-field deconvolution microscopy.

    (a) A microlens array was appended to the camera port of a wide-field microscope and placed in the primary image plane of the fluorescence microscope. The array itself was imaged with a 1:1 relay lens system onto the chip of a scientific complementary metal-oxide semiconductor (sCMOS) camera (Online Methods). Inset, close-up of the microlens array. (b) Point spread function (PSF) of a subdiffraction-sized bead located at z = 7.5 μm off the focal plane, as seen through the microlens array. (c) Axial (xz) PSF at z = 7.5 μm, reconstructed using the LFDM, and corresponding x and z profiles showing lateral and axial resolution, respectively. a.u., arbitrary units. (d) Maximum-intensity projection (MIP) of a deconvolved wide-field focal stack taken without microlenses. The sample consists of 6-μm-sized fluorescent beads in agarose. (e) Red box in d; xz- and yz-section MIPs are shown. (f,g) Corresponding volume of the same beads in d,e, 4–28 μm off the focal plane, reconstructed via 15 iterations of the light-field deconvolution algorithm. Scale bars, 150 μm (a,b), 3 μm (c) and 10 μm (dg).

  2. Whole-animal Ca2+ imaging of C. elegans using LFDM.
    Figure 2: Whole-animal Ca2+ imaging of C. elegans using LFDM.

    (a) Wide-field image of the worm inside a microfluidic poly(dimethylsiloxane) (PDMS) device used for immobilization. The head is at the bottom right. (b) Maximum-intensity projection (MIP) of a light-field deconvolved image (15 iterations) containing 14 distinct z planes. Arrows and numbers indicate individual neurons in the head ganglia and ventral cord. (c) Ca2+ intensity traces (ΔF/F0) of NLS-GCaMP5K fluorescence of selected neurons as marked in b and imaged volumetrically at 5 Hz for 200 s (Supplementary Video 1). (d) Close-up of the brain region, with the MIP of the xy plane as well as xz and yz cross-sections indicated by the dashed lines (Supplementary Video 2). (e) Individual z planes of a typical recording of the worm's brain, reconstructed from a single camera exposure (see Supplementary Fig. 2 for neuron IDs). In this recording, the worm's center along the lateral (left–right) (z) axis was placed at the focal plane of the objective. (f) Activity of all 74 neurons identified in e (Supplementary Video 4). Each row shows a time-series heat map of an individual neuron. Color indicates percent fluorescence changes (ΔF/F0); scaling is indicated by the color bar on the right. Scale bars, 50 μm (b,e), 10 μm (d).

  3. Whole-brain Ca2+ imaging of larval zebrafish in vivo.
    Figure 3: Whole-brain Ca2+ imaging of larval zebrafish in vivo.

    (a) Axial point spread function (PSF) of a 0.5-μm-sized bead located at z = 28 μm off the focal plane for the 20×/0.5–numerical aperture (NA) lens, and corresponding x and z profiles. a.u., arbitrary units. (b) Maximum-intensity projection (MIP) of a light-field deconvolved volume (eight iterations) containing 51 z planes, captured at an exposure time of 50 ms per frame and spaced 4 μm apart, showing the xy plane and xz and yz cross-sections. Spatial filters, each representing individual cells, identified using principal-component and independent-component analysis20 are shown. In total, 5,379 filters were automatically identified, most of which correspond to individual neurons. (c) Extracted Ca2+ intensity signal (ΔF/F0) of GCaMP5 fluorescence using spatial filters shown in b. Each row shows a time-series heat map. Color bars denote encircled regions in b, which include the olfactory epithelium, olfactory bulb and telencephalon. The arrow at ~15 s denotes the addition of an aversive odor. A close-up of the dashed box is shown (right, lower panel); neurons with subtle differences in response onset are highlighted by colored arrows. The location of these neurons in the MIP is also shown (right, upper panel). (d) Overlay of the MIP with randomly selected spatial filters (colored dots and arrows). (e) Ca2+ intensity traces of selected cells shown in d. Neurons were manually selected from the olfactory system, midbrain and hindbrain. Trace color matches spatial-filter color in d. Also see Supplementary Video 6. Scale bars, 10 μm (a) and 100 μm bd.

Videos

  1. Whole animal Ca2+-imaging of C. elegans
    Video 1: Whole animal Ca2+-imaging of C. elegans
    Maximum intensity projection of 14 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 seconds of recording of basal activity at 5 Hz volume rate (1000 frames in total). Video frame rate is 100 frames per second, which equates to 20 seconds in real time in the video (i.e. playback speed 20x). See also Fig. 2a-c.
  2. Whole animal Ca2+-imaging of C. elegans
    Video 2: Whole animal Ca2+-imaging of C. elegans
    Maximum intensity projection of 14 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 seconds of recording of basal activity at 5 Hz volume rate. Video frame rate is 100 frames per second, which equates to 20 seconds in real time in the video (i.e. playback speed 20x). See also Fig. 2d.
  3. High-speed Ca2+-imaging of unrestrained C. elegans at 50 Hz
    Video 3: High-speed Ca2+-imaging of unrestrained C. elegans at 50 Hz
    Maximum intensity projection of 12 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 15 seconds of recording of basal activity of freely-moving worms at 50 Hz volume rate. Exposure time for individual volumes was 20 ms. Video frame rate is 50 frames per second, which equates to 50 volumes per second in the video (i.e. playback speed 1x – real time).
  4. Brain-wide Ca2+-imaging of C. elegans
    Video 4: Brain-wide Ca2+-imaging of C. elegans
    Maximum intensity projection of 15 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 seconds of recording of basal activity in the head ganglia at 5 Hz volume rate. Video frame rate is 100 frames per second, which equates to 20 seconds in real time in the video (i.e. playback speed 20x). See also Fig. 2e-f.
  5. Brain-wide Ca2+-imaging of C. elegans during chemosensory stimulation
    Video 5: Brain-wide Ca2+-imaging of C. elegans during chemosensory stimulation
    Maximum intensity projection of 7 z-planes at 2 μm distance of a of a Punc-31::NLS-GCaMP5K worm during chemosensory stimulation. Shown are 240 seconds of recording of basal activity at 5 Hz volume rate. Video frame rate is 50 frames per second, which equates to 24 seconds in real time in the video (i.e. playback speed 10x). Also see Supplementary Fig. 3.
  6. Brain-wide Ca2+-imaging of zebrafish larvae
    Video 6: Brain-wide Ca2+-imaging of zebrafish larvae
    Maximum intensity projection of 51 z-planes at 4 μm distance of a 5 dpf - HuC:GCaMP5G zebrafish larvae. Shown are 240 seconds of recording of activity at 20 Hz volume rate. Playback speed of video is 10x. At ~15 sec (~1.5 sec in the video), decomposed fish water is supplied manually to the fish chamber in order to evoke activity in the olfactory system. Also see Fig. 3 in the main manuscript.

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Author information

  1. These authors contributed equally to this work.

    • Robert Prevedel &
    • Young-Gyu Yoon

Affiliations

  1. Research Institute of Molecular Pathology, Vienna, Austria.

    • Robert Prevedel,
    • Maximilian Hoffmann,
    • Saul Kato,
    • Tina Schrödel,
    • Manuel Zimmer &
    • Alipasha Vaziri
  2. Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.

    • Robert Prevedel,
    • Maximilian Hoffmann &
    • Alipasha Vaziri
  3. Research Platform Quantum Phenomena & Nanoscale Biological Systems (QuNaBioS), University of Vienna, Vienna, Austria.

    • Robert Prevedel,
    • Maximilian Hoffmann &
    • Alipasha Vaziri
  4. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.

    • Young-Gyu Yoon
  5. MIT Media Lab, MIT, Cambridge, Massachusetts, USA.

    • Young-Gyu Yoon,
    • Nikita Pak,
    • Gordon Wetzstein,
    • Ramesh Raskar &
    • Edward S Boyden
  6. Department of Mechanical Engineering, MIT, Cambridge, Massachusetts, USA.

    • Nikita Pak
  7. Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA.

    • Edward S Boyden
  8. Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA.

    • Edward S Boyden
  9. McGovern Institute, MIT, Cambridge, Massachusetts, USA.

    • Edward S Boyden

Contributions

R.P. designed microlenses, built the imaging system and performed experiments together with M.H. Y.-G.Y. designed and wrote 3D-deconvolution software with contributions from G.W. under the guidance of R.R. R.P. and M.H. refined and rebuilt the imaging system and analyzed data together with Y.-G.Y. N.P. implemented and tested the LFDM prototype. T.S. generated transgenic animals, provided microfluidic devices and performed cell identifications under the guidance of M.Z. S.K. wrote analysis software. E.S.B. and A.V. conceived of and led project. R.P., Y.-G.Y. and A.V. wrote the manuscript, with input from all authors.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Author details

Supplementary information

Video

  1. Video 1: Whole animal Ca2+-imaging of C. elegans (7.14 MB, Download)
    Maximum intensity projection of 14 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 seconds of recording of basal activity at 5 Hz volume rate (1000 frames in total). Video frame rate is 100 frames per second, which equates to 20 seconds in real time in the video (i.e. playback speed 20x). See also Fig. 2a-c.
  2. Video 2: Whole animal Ca2+-imaging of C. elegans (9.19 MB, Download)
    Maximum intensity projection of 14 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 seconds of recording of basal activity at 5 Hz volume rate. Video frame rate is 100 frames per second, which equates to 20 seconds in real time in the video (i.e. playback speed 20x). See also Fig. 2d.
  3. Video 3: High-speed Ca2+-imaging of unrestrained C. elegans at 50 Hz (9.31 MB, Download)
    Maximum intensity projection of 12 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 15 seconds of recording of basal activity of freely-moving worms at 50 Hz volume rate. Exposure time for individual volumes was 20 ms. Video frame rate is 50 frames per second, which equates to 50 volumes per second in the video (i.e. playback speed 1x – real time).
  4. Video 4: Brain-wide Ca2+-imaging of C. elegans (9.44 MB, Download)
    Maximum intensity projection of 15 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 seconds of recording of basal activity in the head ganglia at 5 Hz volume rate. Video frame rate is 100 frames per second, which equates to 20 seconds in real time in the video (i.e. playback speed 20x). See also Fig. 2e-f.
  5. Video 5: Brain-wide Ca2+-imaging of C. elegans during chemosensory stimulation (10.8 MB, Download)
    Maximum intensity projection of 7 z-planes at 2 μm distance of a of a Punc-31::NLS-GCaMP5K worm during chemosensory stimulation. Shown are 240 seconds of recording of basal activity at 5 Hz volume rate. Video frame rate is 50 frames per second, which equates to 24 seconds in real time in the video (i.e. playback speed 10x). Also see Supplementary Fig. 3.
  6. Video 6: Brain-wide Ca2+-imaging of zebrafish larvae (3.66 MB, Download)
    Maximum intensity projection of 51 z-planes at 4 μm distance of a 5 dpf - HuC:GCaMP5G zebrafish larvae. Shown are 240 seconds of recording of activity at 20 Hz volume rate. Playback speed of video is 10x. At ~15 sec (~1.5 sec in the video), decomposed fish water is supplied manually to the fish chamber in order to evoke activity in the olfactory system. Also see Fig. 3 in the main manuscript.

PDF files

  1. Supplementary Text and Figures (6,474 KB)

    Supplementary Figures 1–4 and Supplementary Notes 1 and 2

Zip files

  1. Supplementary Software (7,217 KB)

    Software for 3D volume reconstruction from light-field images
    The software for 3D volume reconstruction from light-field images is available as Supplementary Software. It is written in MATLAB and requires a workstation with MATLAB2013a or later version and at least 32GB of RAM. A user manual for the software (LFM_Software_readme.pdf) is included in the .zip file.

Additional data