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Whole-brain serial-section electron microscopy in larval zebrafish

Abstract

High-resolution serial-section electron microscopy (ssEM) makes it possible to investigate the dense meshwork of axons, dendrites, and synapses that form neuronal circuits1. However, the imaging scale required to comprehensively reconstruct these structures is more than ten orders of magnitude smaller than the spatial extents occupied by networks of interconnected neurons2, some of which span nearly the entire brain. Difficulties in generating and handling data for large volumes at nanoscale resolution have thus restricted vertebrate studies to fragments of circuits. These efforts were recently transformed by advances in computing, sample handling, and imaging techniques1, but high-resolution examination of entire brains remains a challenge. Here, we present ssEM data for the complete brain of a larval zebrafish (Danio rerio) at 5.5 days post-fertilization. Our approach utilizes multiple rounds of targeted imaging at different scales to reduce acquisition time and data management requirements. The resulting dataset can be analysed to reconstruct neuronal processes, permitting us to survey all myelinated axons (the projectome). These reconstructions enable precise investigations of neuronal morphology, which reveal remarkable bilateral symmetry in myelinated reticulospinal and lateral line afferent axons. We further set the stage for whole-brain structure–function comparisons by co-registering functional reference atlases and in vivo two-photon fluorescence microscopy data from the same specimen. All obtained images and reconstructions are provided as an open-access resource.

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Figure 1: Targeted, multi-scale ssEM of a larval zebrafish brain.
Figure 2: Neuron reconstructions capturing sensory input and motor output.
Figure 3: Reconstruction of a larval zebrafish projectome.
Figure 4: Bilateral symmetry in myelinated reticulospinal axon reconstructions.

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Acknowledgements

We thank D. D. Bock and K.-H. Huang for preliminary studies; L.-H. Ma, M. B. Ahrens, and D. Schoppik for dissection help; E. Raviola, H. S. Kim, J. A. Buchanan, E. J. Benecchi, and S. Ito for histology guidance; K. J. Hayworth, J. L. Morgan, N. Kasthuri, and R. Schalek for ssEM advice; T. Kazimiers and J. A. Bogovic for software assistance at the Harvard CBS Neuroengineering Core; B. L. Shanny, A. M. Roberson, M. A. Afifi, F. Gao, A. D. Wong, F. Camacho Garcia, C. S. Elkhill, T. J. Cawley, R. J. Plummer, K. M. Runci, A. Haddad, P. E. Lewis, I. Odstrcil, A. H. Cohen, and P. I. Petkova for reconstructions; and R. C. Reid, R. I. Wilson, and J. R. Sanes for valuable input. Support was provided by the NIH through NINDS and NIMH to F.E. (DP1NS082121, RC2NS069407, U01NS090449) and W.-C.A.L. (R21NS085320) and the Harvard CBS Neuroengineering Core (P30NS062685), through NIGMS to MMBioS via the Pittsburgh Supercomputing Center (P41GM103712); by the DARPA SIMPLEX program through SPAWAR to R.B. and J.T.V. (N66001-15-C-4041); by the Korea NRF through MSIP (NRF-2015M3A9A7029725) and MOE (NRF-2014R1A1A2058773) to W.-K.J.; and by the NIH (T32MH20017, T32HL007901) and the NSF (IIA-EAPSI-1317014) to D.G.C.H.

Author information

Authors and Affiliations

Authors

Contributions

F.E. and D.G.C.H. designed experiments. R.P., D.G.C.H., and I.H.B. conducted light microscopy. D.G.C.H. prepared and sectioned samples. D.G.C.H. and G.S.P. completed ssEM. A.W.W., S.S., and D.G.C.H. aligned ssEM. D.G.C.H. and O.R. registered light and ssEM. R.M.T., B.J.G., and D.G.C.H. processed reconstructions. M.C. and D.G.C.H. analysed symmetry. W.C., T.M.Q., J.M., D.G.C.H., and W.-K.J. made visualizations. A.S.C. modified annotation software. A.D.B., K.L., R.B., and J.T.V. provided hosting. F.E., J.W.L., W.-C.A.L., and A.F.S. supplied resources. D.G.C.H., F.E., and J.W.L. wrote the manuscript.

Corresponding authors

Correspondence to David Grant Colburn Hildebrand or Florian Engert.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks A. Cardona and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Preparing larval zebrafish brain tissue for ssEM.

a, Immersion of intact specimens into tissue processing solutions resulted in poor preservation of brain ultrastructure due to membranes (arrowheads). b, c, Dissecting away skin and membranes (b) allowed solutions to diffuse into the brain, resulting in improved preservation (c). To minimize damage, dissections were initiated by puncturing the thin epithelial layer over the rhombencephalic ventricle dorsal to the hindbrain with a sharpened tungsten needle (red cross). Small anterior-directed incisions along the midline were then made as close to the surface as possible until the brain was exposed up to the anterior optic tectum (red dashed line). df, Following dissection and aldehyde fixation (d), samples were post-fixed with a reduced osmium solution (e), and stained with uranyl acetate (f). g, h, Processed specimens were then dehydrated with acetonitrile, infiltrated with a low-viscosity resin, mounted in a micromachined pre-cast resin mould to orient the sample for transverse sectioning (g), and surrounded by support tissue that stabilized sectioning (h). i, Representative ultrastructure acquired as a transmission electron micrograph from a section through the optic tectum of an early dissection test specimen. Scale bars: a, c, i, 1 μm; b, 100 μm; df, 500 μm; g, h, 1 mm.

Extended Data Figure 2 Serial sectioning and ultrathin section library assembly.

a, Serial sections of resin-embedded samples were picked up with an automated tape-collecting ultramicrotome modified for compatibility with larger reels containing enough tape to accommodate tens of thousands of sections. b, c, Direct-to-tape sectioning resulted in consistent section spacing and orientation. Just as a section left the diamond knife, it was caught by the tape. d, After sectioning, the tape was divided onto silicon wafers that functioned as a stage in a scanning electron microscope and formed an ultrathin section library. For a series containing all of a 5.5 dpf larval zebrafish brain, 68 m of tape was divided onto 80 wafers (typically 227 sections per wafer). e, Wafer images were used as a coarse guide for targeting electron microscopic imaging. Fiducial markers (copper circles) provided a reference for a per-wafer coordinate system, enabling storage of the position associated with each section for multiple rounds of re-imaging at varying resolutions as needed. f, Overview micrographs (758.8 × 758.8 × 60 nm3 per voxel) were acquired for each section to ascertain sectioning reliability and determine the extents of the ultrathin section library. Embedding the larval zebrafish (green dashed circle) in support tissue stabilized sectioning. Scale boxes: a, 5 × 5 × 5 cm3; b, 1 × 1 × 1 cm3; c, 1 × 1 × 1 mm3. Scale bars: e, 1 cm; f, 250 μm.

Extended Data Figure 3 Serial sectioning through the anterior quarter of a 5.5 dpf larval zebrafish.

a, Overview micrographs from a collection of 17,963 approximately 60-nm-thick transverse serial sections that span 1.09 mm through a 5.5 dpf larval zebrafish. Embedding the larval zebrafish (green dashed circle) in support tissue stabilized sectioning. Straight dashed lines indicate cropping of the section overview. b, Volume rendering of aligned overview micrographs. Magenta and yellow planes correspond to reslice planes in c. Green plane corresponds to section outlined in a. c, Reslice planes through the aligned overview image volume reveal structures contained within the series and illustrate the sectioning plane relative to the horizontal (upper) and sagittal (lower) body planes. This series spans from myotome 7 through the anterior larval zebrafish, encompassing part of the spinal cord and the entire brain. Dashed lines indicate where reslice planes intersect. d, Histograms of lost, partial (missing any larval zebrafish tissue), or adjacent (lost–partial or partial–partial) events per bin of 50 sections. In total, 244 (1.34%) sections were lost and 283 (1.55%) were partial for this series. No two adjacent sections were lost. Inset histograms expand the shaded regions to provide a detailed view of sectioning reliability with bin sizes of five sections. Dashed lines indicate the number of lost sections if uniformly distributed throughout the series. Scale box: b, 250 × 250 × 250 μm3. Scale bars: a, c, 250 μm.

Extended Data Figure 4 Serial sectioning through most of the brain of a 7 dpf larval zebrafish.

a, Overview micrographs from a collection of 15,046 approximately 50-nm-thick transverse serial sections that span 0.75 mm through a 7 dpf larval zebrafish. Surrounding part of the larval zebrafish (green dashed circle) with support tissue stabilized sectioning. Straight dashed lines indicate cropping of the section overview. b, Volume rendering of aligned overview micrographs. Magenta and yellow planes correspond to reslice planes in c. Green plane corresponds to section outlined in a. c, Reslice planes through the aligned overview image volume reveal structures contained within the series and illustrate the sectioning plane relative to the horizontal (upper) and sagittal (lower) body planes. This series spans from the posterior hindbrain through the anterior larval zebrafish, encompassing most of the brain. Dashed lines indicate where reslice planes intersect. d, Histograms depicting the number of lost, partial (missing any larval zebrafish tissue), or adjacent (lost–partial or partial–partial) events per bin of 50 sections throughout the series. In total, 6 (0.04%) sections were lost and 25 (0.17%) were partial for this series. No two adjacent sections were lost. Scale box: b, 250 × 250 × 250 μm3. Scale bars: a, c, 250 μm.

Extended Data Figure 5 Description and categorization of partial sections.

Collected sections were deemed partial if any larval zebrafish tissue appeared to be missing. In total, 283 sections of 18,207 attempted were classified as partial for the 5.5 dpf larval zebrafish series. Partial sections imaged at 56.4 × 56.4 × 60 nm3 per voxel were further categorized into minor, moderate, or severe subclasses. In minor cases, only tissue outside the brain was absent. Moderate cases lacked less than half of the brain. Severe cases were missing half of the brain or more. Note that it is possible that apparently missing tissue is contained in a slightly thicker adjacent section, in which case it is not entirely lost and may be accessible with different imaging strategies. a–c, Posterior examples of partial sections from each category. Line and arrow indicate the orientation and direction of sectioning. d–f, Expanded views of brain tissue from the sections depicted in a–c. Red dashed contours define the brain outline expected from an adjacent section. g–i, Anterior examples of partial sections from each category. j, Number of sections in each category for the 208 partial sections contained within the 16,000 imaged at 56.4 × 56.4 × 60 nm3 per voxel resolution. Scale bars: ai, 50 μm.

Extended Data Figure 6 Software modifications for co-registered ssEM datasets and reference atlas overlays.

The reconstruction of neuronal structures across multi-resolution ssEM image volumes acquired from the same specimen profits from the ability to simultaneously access and view separate but co-registered datasets. Without this ability, some of the time benefits of our imaging approach would be offset by needing to register and track structures across volumes that span both low-resolution, large fields of view and high-resolution, specific regions of interest. With this in mind, we added a feature to the CATMAID neuronal circuit mapping software to overlay and combine image stacks acquired with varying resolutions in a single viewer. This feature is now available in the main open-source release. a, Images from two co-registered ssEM datasets acquired at different resolutions from the same section. The combined view (left) overlays 4.0 × 4.0 × 60 nm3 per voxel data (right) onto 56.4 × 56.4 × 60 nm3 per voxel data (middle). b, Integrated view of co-registered ssEM datasets overlaid with manual reconstructions (coloured dots) and the spinal backfill label (red) from the Z-Brain atlas. As expected, spinal backfill fluorescence is visible directly over a Mauthner cell body (arrowhead).

Extended Data Figure 7 Neuron identity correspondence across whole-brain in vivo light and post hoc ssEM datasets.

Co-registration of in vivo light microscopy and post hoc ssEM datasets can be accomplished with thin-plate spline coordinate transformations guided by manually identified landmarks. a, Volume renderings of the 5.5 dpf larval zebrafish ssEM dataset (top), warped in vivo two-photon imaging of elavl3:GCaMP5G fluorescence from the same specimen (middle), and a merge (bottom). Reslice planes shown in b are indicated by magenta planes. b, Near-horizontal reslice planes from the ssEM volume (upper) and the warped in vivo light microscopy image volume (lower) show gross correspondence throughout the brain. c, d, Magnified views reveal single-neuron matches in the optic tectum (c) and telencephalon (d). Arrowheads indicate the same structures as observed in each modality. Elongated structures are blood vessels. e–g, This exercise revealed the imaging conditions, labelling density, and structural tissue features necessary for reliable matching across imaging modalities. This process was difficult in regions (enclosed by dotted contours) where the fluorescence signal was low (e), where many cells were packed closely together (f), or where new neurons were likely to have been added between light microscopy and preparation for ssEM (g). Improving the light-level data with specific labelling of all nuclei and faster light-sheet or other imaging approaches should improve the ease and accuracy of matching. This ability to assign neuron identity across imaging modalities demonstrates proof-of-principle for the integration of rich neuronal activity maps with subsequent whole-brain structural examination of functionally characterized neurons and their networks. Scale box: a, 100 × 100 × 100 μm3. Scale bars: b, 100 μm; cg, 10 μm.

Extended Data Figure 8 Registration of functional reference atlases to the ssEM dataset.

Cross-modal registration of the Z-Brain atlas and the Zebrafish Brain Browser allows characterization of specific domains within the 5.5 dpf larval zebrafish ssEM dataset defined, for example, by genetically restricted labels (a–f) or retrograde labelling (g, h). a, c, e, g, Dorsal (left) and lateral (right) views through dual-volume renderings of the ssEM data and Z-Brain atlas data from a elavl3:H2B-RFP transgenic line (a), vglut2a:GFP transgenic line (c), hcrt:RFP transgenic line (e), and spinal backfill retrograde labelling (g). b, d, f, h, Z-Brain atlas fluorescence signal for the same labels overlaid onto horizontal reslice planes through the ssEM dataset. As expected, the fluorescence associated with the Mauthner cell and nucMLF neuron positions overlaps with the ssEM data associated with these identifiable neurons in the spinal backfill label case (h). Scale bars: ah, 100 μm.

Extended Data Figure 9 Symmetry analysis descriptions and examples.

ac, Analysis of symmetry in 3D position and shape for one example left–right neuron pair with axons in the MLF. a, In the comparison between the left MiD2cm axon and its right homologue, the left side was first reflected across the plane of symmetry (dotted line). b, The comparison cost value representing the similarity in position and shape of the two axons was then computed using a dynamic time warping (DTW) sequence matching approach. c, Each cost value was calculated as the sum of the Euclidian distances between points matched by the DTW algorithm, normalized by the number of matches, and finally multiplied by a penalty factor proportional to the unmatched sequence lengths (total length divided by matched length; not illustrated). d, In a globally optimal pairwise assignment for a selection of 22 identified left–right MLF homologues, one pair of myelinated axon reconstructions were not assigned to their contralateral homologues (see Fig. 4b, red asterisks). Upon investigating this unexpected assignment further, it was clear that similar pairwise comparison costs resulted for the assigned non-homologues (left column) and unassigned left–right homologues (right column). However, the combined non-homologue cost was slightly lower (by 174) than the combined left–right homologue cost. Because the global assignment sought to minimize the total cost summed over all pairwise comparisons, this difference is likely to explain why non-homologues were grouped over left–right homologues. eh, Analysis of symmetry in 2D neighbour relations. e, In cross-sectional slices, the vector between each pair of left axons was compared to the reflected vector between the right axons with the same identities. Two metrics were then calculated to relate the original and reflected pairs: the angle difference (measured as the dot product between the vectors) and the distance difference (measured as the difference between the lengths of the vectors). f, For each slice, a difference matrix was constructed from the angle and distance difference values for all pairwise combinations. g, h, Linearizing difference matrices (g) and then concatenating them (h) enabled visualization of changes in relative positional arrangements across slices. ik, Extension of 2D symmetry analysis to the 22 identified left–right MLF pairs. i, Examined myelinated axon reconstructions. j, k, Trend towards mirror-symmetrical relative positional arrangements over long MLF stretches apparent by linearizing angle (j) and distance (k) differences. Neighbour relations for many pairs returned to symmetrical state despite local perturbations, while others showed more variability. Black indicates insufficient data for comparing the given pair. Scale bars: a, d, i, 50 μm; b, 5 μm.

Extended Data Figure 10 Examples of non-neuronal tissue contained within the ssEM dataset.

In addition to capturing the whole brain, the 56.4 × 56.4 × 60 nm3 per voxel image volume contains the anterior quarter of a 5.5 dpf larval zebrafish, thus serving as a high-resolution atlas for several other tissues and structures. Three selected sections (a, h, m) are accompanied by example images (b–g, i–l, n–o) to illustrate the variety of tissues and structures contained within this dataset. Scale bars: a, h, m, 50 μm; bg, 5 μm; il, n, o, 10 μm.

Supplementary information

Survey of sections from the 5.5 dpf larval zebrafish series

Electron micrographs of 17,963 sections acquired at 758.8 × 758.8 × 60 nm3 per voxel to survey sectioning quality and provide a basis for targeting subsequent high-resolution acquisition. Several sections excluded for faster viewing. Scale bar: 250 µm. (MOV 15281 kb)

Section overview volume rendering

Volume rendering of aligned 758.8 × 758.8 × 60 nm3 per voxel overviews from 17,963 sections shows how much of the 5.5 dpf larval zebrafish was captured with this series. Magenta and yellow planes correspond to reslice planes in Extended Data Fig. 3c. Green plane corresponds to section outlined in Extended Data Fig. 3a. Scale box: 250 µm. (MOV 5424 kb)

Isotropic ssEM through the anterior quarter of the 5.5 dpf larval zebrafish

Fly-through of aligned 56.4 × 56.4 × 60 nm3 per voxel electron micrographs acquired for 16,000 transverse sections. The resulting image volume contains the anterior quarter and entire brain of a 5.5 dpf larval zebrafish. Several sections excluded for faster viewing. Scale bar: 50 µm. (MOV 15200 kb)

Mauthner cell reconstruction

Fly-through of aligned 56.4 × 56.4 × 60 nm3 per voxel electron micrographs centred on part of a skeleton reconstruction of the left Mauthner cell (magenta dot) from the 5.5 dpf larval zebrafish ssEM dataset. The video begins at the Mauthner lateral dendrite, proceeds through its soma and axon cap, crosses to the contralateral side, and ends in the spinal cord. Extracting vertical line segments from the centre of these micrographs yielded the contour reslices in Fig. 1a−c. Several sections excluded for faster viewing. Scale bar: 5 µm. (MOV 20476 kb)

Correspondence across light and ssEM datasets

Fly-through of matched horizontal reslice planes through the 56.4 × 56.4 × 60 nm3 per voxel ssEM dataset (upper) and co-registered 0.637 × 0.637 × 1 µm3 per voxel in vivo two-photon elavl3:GCaMP5G fluorescence dataset (lower) for the 5.5 dpf larval zebrafish. Scale bar: 100 µm. (MOV 15008 kb)

Lateral line primary sensory afferent reconstruction

Fly-through of the aligned multi-resolution 5.5 dpf larval zebrafish ssEM data centred on part of a skeleton reconstruction of an afferent neuron that innervates a right dorsal neuromast (orange dot). This video contains data from three separate image volumes, starting at 4.0 × 4.0 × 60 nm3 per voxel where the afferent receives input from a neuromast hair cell. The field of view expands to follow the myelinated afferent at 56.4 × 56.4 × 60 nm3 per voxel throughout the periphery. It next magnifies to 18.8 × 18.8 × 60 nm3 per voxel before reaching the soma in the posterior lateral line ganglion and ends in the hindbrain. Scale bar: 1 µm (size changes depending on magnification). (MOV 30703 kb)

Projectome reconstruction

Volume rendering of the projectome reconstruction formed by annotation of all myelinated axons from the multi-resolution 5.5 dpf larval zebrafish ssEM dataset. The reconstructions are presented within a semi-transparent rendering of the aligned ssEM image volume so that it is possible to reference the positions of the myelinated axons. All reconstructions were manually annotated from the ssEM data. Colours assigned randomly. Scale bar: 100 µm. (MOV 15357 kb)

Bilateral symmetry in the lateral line system

Volume rendering of lateral line system reconstructions from the multi-resolution 5.5 dpf larval zebrafish ssEM dataset reveal bilateral symmetry in most neuromasts (blue) and the myelinated axons that innervate them. The myelinated axons innervating identified neuromasts (purple) and members of the posterior lateral line nerve (yellow) that likely innervate neuromasts outside the imaged volume are both displayed. Neuromasts have a bilaterally symmetrical counterpart in all but one case (orange) and the myelinated axons innervating them overlap substantially when viewed from the side. Scale bar: 100 µm. (MOV 4822 kb)

Bilateral symmetry in the reticulospinal system

Volume rendering of the Z-Brain spinal backfill label (red) and reticulospinal axon reconstructions (blue) embedded within semi-transparent 5.5 dpf larval zebrafish ssEM data. Also illustrated is the plane of symmetry (yellow) that was extracted from myelinated axons of identified reticulospinal neurons whose projections formed part of the MLF. Cross-modal registration of the Z-Brain and Zebrafish Brain Browser reference atlases allowed for anatomical characterization of domains within the ssEM dataset and aided in reticulospinal neuron identification. Scale bar: 100 µm. (MOV 15368 kb)

Analysis of symmetry in myelinated axon neighbour relations

Slice-by-slice 2D symmetry analysis of myelinated axon neighbour relations within the MLF. Angle and distance differences (upper right) were calculated from cross-sectional slices (upper left) through a subset of myelinated axon reconstructions (lower), linearized, and aggregated (middle) for display. The video shows how some panels in Fig. 4 and Extended Data Fig. 9 were constructed. (MOV 10172 kb)

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Hildebrand, D., Cicconet, M., Torres, R. et al. Whole-brain serial-section electron microscopy in larval zebrafish. Nature 545, 345–349 (2017). https://doi.org/10.1038/nature22356

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