Abstract

Calcium imaging with cellular resolution typically requires an animal to be tethered under a microscope, which substantially restricts the range of behaviors that can be studied. To expand the behavioral repertoire amenable to imaging, we have developed a tracking microscope that enables whole-brain calcium imaging with cellular resolution in freely swimming larval zebrafish. This microscope uses infrared imaging to track a target animal in a behavior arena. On the basis of the predicted trajectory of the animal, we applied optimal control theory to a motorized stage system to cancel brain motion in three dimensions. We combined this motion-cancellation system with differential illumination focal filtering, a variant of HiLo microscopy, which enabled us to image the brain of a freely swimming larval zebrafish for more than an hour. This work expands the repertoire of natural behaviors that can be studied with cellular-resolution calcium imaging to potentially include spatial navigation, social behavior, feeding and reward.

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Acknowledgements

We thank M. Burns, K. Taute, H. Berg, L. Stern and D. Schaack for feedback on the manuscript. We thank M. Burns and C. Friend for helpful advice and discussions throughout the project. We thank W. Hill for custom electronic circuit design, and C. Stokes for engineering assistance. We thank D.N. Congreve (Rowland Institute at Harvard, Cambridge, Massachusetts, USA) for providing submicrometer fluorescent sheets. We thank H.C. Park (Korea University, Seoul, South Korea) for Tg(elavl3:Kaede) zebrafish. We thank J. Todd for help with software development, and E. Kane for motivating the project. This work was financially supported by the Rowland Institute at Harvard.

Author information

Author notes

    • David G C Hildebrand

    Present address: Laboratory of Neural Systems, Rockefeller University, New York, New York, USA.

Affiliations

  1. Rowland Institute at Harvard University, Cambridge, Massachusetts, USA.

    • Dal Hyung Kim
    • , Jungsoo Kim
    • , João C Marques
    • , Wenchao Gu
    • , Jennifer M Li
    •  & Drew N Robson
  2. Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, Massachusetts, USA.

    • Abhinav Grama
    •  & David G C Hildebrand
  3. Program in Neuroscience, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA.

    • David G C Hildebrand

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Contributions

D.N.R. and J.M.L. conceived and designed the project, instrumentation, and algorithms for DIFF microscopy, MPC-based tracking and image registration; D.H.K. integrated the real-time MPC-based tracking system and DIFF imaging, implemented online and offline image registration, and performed all tracking experiments, with guidance from D.N.R. and J.M.L.; J.K. contributed to the design and implementation of the global shutter imaging system and DIFF microscopy; J.C.M. performed kinematic analyses of fish movement and regression analysis between behavior and neural activity; A.G. and D.G.C.H. generated Tg(elavl3:GCaMP6s)a13203 zebrafish; W.G. contributed to fish genetics and sample preparation; and J.M.L., D.N.R., D.H.K., J.K. and J.C.M. analyzed the data and wrote the manuscript.

Competing interests

US Patent application 62/487,793 has been filed, with D.N.R., D.K. and J.M.L. named as inventors.

Corresponding authors

Correspondence to Jennifer M Li or Drew N Robson.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15 and Supplementary Notes 1–3

  2. 2.

    Life Sciences Reporting Summary

Zip files

  1. 1.

    Supplementary Software

    Software for operating the tracking microscope and image registration, as well as license information, implementation, and usage notes.

Videos

  1. 1.

    Brain-wide cellular-resolution imaging of neural activity in a swimming larval zebrafish by DIFF microscopy.

    Left, trajectory of an elavl3:GCaMP6s larval zebrafish navigating a linear thermal gradient. During this behavioral epoch, the animal is in exploration mode. Right, DIFF-sectioned images acquired at 2 volumes per second during live tracking. Structured fluorescence image pairs were acquired at 200 Hz and processed by DIFF optical sectioning to obtain DIFF-sectioned images at 100 Hz. A piezo Z stage sweeps the objective axially to record a 100 μm target brain volume with closed loop real-time Z tracking (2 μm axial step per DIFF image, 50 DIFF-sectioned images per sweep, two sweeps per second). Bottom left, the imaging volume (red box) and current sweep location (cyan plane) are shown. For simplicity, the sweep offset applied by real-time live Z tracking is not shown. Dataset was collected from an awake and freely swimming 6 dpf larval zebrafish.

  2. 2.

    Brain-wide cellular-resolution imaging of neural activity during restricted-area search behavior.

    Left, trajectory of an elavl3:GCaMP6s larval zebrafish navigating a linear thermal gradient. During this behavioral epoch, the animal has escaped the hot side of the linear gradient and is displaying restricted-area search behavior to stay localized in the cooler half of the arena. Right, DIFFsectioned images acquired at 2 volumes per second during live tracking. Fluorescence imaging data for this fish was collected with a 100 μm target brain volume generated with a 2 μm axial step per DIFF image, 50 DIFFsectioned images per sweep, and two sweeps per second. Dataset was collected from an awake and freely swimming 6 dpf larval zebrafish.

  3. 3.

    Registered DIFF-sectioned images from multiple focal planes in the brain of a freely swimming larval zebrafish.

    Left, trajectory of an elavl3:GCaMP6s larval zebrafish navigating a linear thermal gradient (same timepoints as Supplementary Video 2). Right, DIFF-sectioned fluorescence images from 8 focal planes of a registered fish brain over the same timepoints as the behavior shown on the left. The raw data was acquired at a volume rate of 2 volumes per second during live tracking and then registered offline through a GPU-accelerated registration pipeline. The registration pipeline consists of an initial 6 Degree of Freedom (6-DoF) rigid registration followed by a piecewise affine registration to accommodate non-rigid deformation. The 8 registered focal planes shown span 84 μm along the dorsal-ventral axis. Fluorescence imaging data for this fish was collected with a 100 μm target brain volume generated with a 2 μm axial step per DIFF image, 50 DIFFsectioned images per sweep, and two sweeps per second. Each pixel is presented without spatial filtering. Single pixel shot noise was reduced for presentation by trend filtering each pixel along the time axis (Methods). Dataset was collected from an awake and freely swimming 6 dpf larval zebrafish.

  4. 4.

    Registered DIFF-sectioned images in the brain of a freely swimming larval zebrafish, showing fine sectioning and habenula neurons in the ROI.

    Left, trajectory of an elavl3:GCaMP6s larval zebrafish navigating a linear thermal gradient (same timepoints as in Supplementary Video 2). Middle, DIFF-sectioned fluorescence images from a single focal plane through the registered fish brain over the same timepoints as the behavior shown on the left. Right, a small ROI volume (256 × 128 × 14 μm) in the habenula of the fish is shown as 8 dorsal (top) to ventral (bottom) sections. Each section represents a single optical section through the habenula with no binning along the axial dimension. Adjacent sections are spaced by 2 μm. Fluorescence imaging data for this fish was collected with a 100 μm target brain volume generated with a 2 μm axial step per DIFF image, 50 DIFFsectioned images per sweep, and two sweeps per second. Bottom left, the targeted imaging volume (red box), selected focal plane (cyan), and habenula volume ROI (yellow box) are shown. Each pixel is presented without spatial filtering. Single pixel shot noise was reduced for presentation by trend filtering each pixel along the time axis (Methods). Dataset was collected from an awake and freely swimming 6 dpf larval zebrafish.

  5. 5.

    Neuronal activity (ΔF/F) throughout the brain of a freely swimming larval zebrafish.

    Left, trajectory of an elavl3:GCaMP6s larval zebrafish navigating a linear thermal gradient (same timepoints as in Supplementary Video 2). Middle, neuronal activity (ΔF/F) throughout a single focal plane of the registered fish brain, shown over the same timepoints as the behavior shown on the left. Right, neuronal activity (ΔF/F) in a small ROI volume (256 × 128 × 14 μm) in the habenula of the fish is shown as 8 dorsal (top) to ventral (bottom) sections. Each section represents a single optical section through the habenula with no binning along the axial dimension. Adjacent sections are spaced by 2 μm. Fluorescence imaging data for this fish was collected with a 100 μm target brain volume generated with a 2 μm axial step per DIFF image, 50 DIFF-sectioned images per sweep, and two sweeps per second. Bottom left, the targeted imaging volume (red box), selected focal plane (cyan), and habenula volume ROI (yellow box) are shown. Shot noise was reduced for presentation by sequentially applying a trend filter to each spatial and temporal dimension (Methods). Dataset was collected from an awake and freely swimming 6 dpf larval zebrafish.

  6. 6.

    Registration performance evaluated by temporal projection of 4D imaging volume after registration.

    Anatomical stack obtained by temporal projection of all registered moving images (across a 10 min imaging session) at a given Z plane within the reference volume. Treating each axial sweep as a single timepoint, we project each moving image into a 4-D dataset (XYZT) sharing the same coordinate system as the reference brain. We present both dorsal and sagittal views of this projection stack to show that subcellular features are resolvable throughout the brain after registration. Dataset was collected from an awake and freely swimming 6 dpf elavl3:GCaMP6s larval zebrafish.

  7. 7.

    Event-triggered neuronal activity (ΔF/F) aligned to heat onset in freely swimming elavl3:GCaMP6s and elavl3:Kaede fish.

    We applied periodic heat pulses (5 s duration, 30 s interval) to freely swimming elavl3:GCaMP6s and elavl3:Kaede larval zebrafish. Fluorescence imaging data for each fish was collected with a 100 μm target brain volume generated with a 2 μm axial step per DIFF image, 50 DIFF-sectioned images per sweep, and two sweeps per second. After DIFF-sectioning and registration, we select a single registered focal plane in the brain. DIFF-sectioned images from this focal plane were then temporally aligned to heat onset (-5 s to +25 s) to obtain an event-triggered time series F(t). Fluorescence images collected at 5 s before heat onset were averaged to obtain a baseline fluorescence image Fbaseline. Neuronal activity is defined as ΔF/F = (F(t) - Fbaseline) / Fbaseline, and overlaid as a heat map (ranging from 0.0 to 0.75) over the grayscale Fbaseline image. The heat pulse is indicated by a red square at the upper left corner of the video. Datasets were collected from an awake and freely swimming 6 dpf elavl3:GCaMP6s larval zebrafish and a 7 dpf awake and freely swimming elavl3:Kaede larval zebrafish.

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DOI

https://doi.org/10.1038/nmeth.4429

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