A petascale automated imaging pipeline for mapping neuronal circuits with high-throughput transmission electron microscopy

Yin, Wenjing; Brittain, Derrick; Borseth, Jay; Scott, Marie E.; Williams, Derric; Perkins, Jedediah; Own, Christopher S.; Murfitt, Matthew F.; Torres, Russel; Kapner, Daniel; Mahalingam, Gayathri; Bleckert, Adam; Castelli, Daniel; Reid, David; Lee, Wei-Chung Allen; Graham, Brett J.; Takeno, Marc; Bumbarger, Daniel J.; Farrell, Colin; Reid, R. Clay; Costa, Nuno Maçarico da

doi:10.1038/s41467-020-18659-3

Cited by 117 publications

(180 citation statements)

References 35 publications

(47 reference statements)

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“…This improvement was in large part due to two noteworthy advances: fast imaging owing to multibeam scanning electron microscopy (Eberle et al 2015) and the profound effect of AI on image processing and analysis (Januszewski et al 2018). The rapid improvements over the past few years (Briggman, Helmstaedter, and Denk 2011;Bock et al 2011;Helmstaedter et al 2013;Takemura et al 2013;Lee et al 2016;Motta et al 2019;Scheffer et al 2020;Dorkenwald et al 2020;Yin et al 2020;Gour et al 2021) argues that analyzing volumes that are even three orders of magnitude larger, such as an exascale whole mouse brain connectome, will likely be in reach within a decade (Abbott et al 2020).…”

Section: Discussionmentioning

confidence: 99%

A connectomic study of a petascale fragment of human cerebral cortex

Shapson-Coe

Januszewski

Berger

et al. 2021

Preprint

212

262

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We acquired a rapidly preserved human surgical sample from the temporal lobe of the cerebral cortex. We stained a 1 mm3 volume with heavy metals, embedded it in resin, cut more than 5000 slices at ~30 nm and imaged these sections using a high-speed multibeam scanning electron microscope. We used computational methods to render the three-dimensional structure of 50,000 cells, hundreds of millions of neurites and 130 million synaptic connections. The 1.3 petabyte electron microscopy volume, the segmented cells, cell parts, blood vessels, myelin, inhibitory and excitatory synapses, and 100 manually proofread cells are available to peruse online. Despite the incompleteness of the automated segmentation caused by split and merge errors, many interesting features were evident. Glia outnumbered neurons 2:1 and oligodendrocytes were the most common cell type in the volume. The E:I balance of neurons was 69:31%, as was the ratio of excitatory versus inhibitory synapses in the volume. The E:I ratio of synapses was significantly higher on pyramidal neurons than inhibitory interneurons. We found that deep layer excitatory cell types can be classified into subsets based on structural and connectivity differences, that chandelier interneurons not only innervate excitatory neuron initial segments as previously described, but also each others initial segments, and that among the thousands of weak connections established on each neuron, there exist rarer highly powerful axonal inputs that establish multi-synaptic contacts (up to ~20 synapses) with target neurons. Our analysis indicates that these strong inputs are specific, and allow small numbers of axons to have an outsized role in the activity of some of their postsynaptic partners.

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Section: Discussionmentioning

confidence: 99%

A connectomic study of a petascale fragment of human cerebral cortex

Shapson-Coe

Januszewski

Berger

et al. 2021

Preprint

212

262

View full text Add to dashboard Cite

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“…As with any magnification process, the electron density at the phosphor drops off as the column is extended. To mitigate the impact of reduced electron density on image quality (shot noise), a highsensitivity sCMOS camera was selected and the scintillator composition tuned in order to generate high quality EM images within exposure times of 90 -200 ms 63 .…”

Section: Electron Microscopy Imagingmentioning

confidence: 99%

Oligodendrocyte precursor cells prune axons in the mouse neocortex

Buchanan

Elabbady

Collman

et al. 2021

Preprint

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Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This transformation is facilitated by the engulfment and degradation of excess axonal branches and inappropriate synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia has made it difficult to determine the contribution of these and other glial cell types to this process. Here, we used large scale, serial electron microscopy (ssEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded terminal axon branches and included numerous phagolysosomes (PLs) containing fragments of axons and presynaptic terminals. Single- nucleus RNA sequencing indicated that cortical OPCs express key phagocytic genes, as well as neuronal transcripts, consistent with active axonal engulfment. PLs were ten times more abundant in OPCs than in microglia in P36 mice, and declined with age and lineage progression, suggesting that OPCs contribute very substantially to refinement of neuronal circuits during later phases of cortical development.

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“…However, upscaling these results to whole mouse brain imaging is challenging; one estimate for diamond-cut blockface scanning electron microscopy is that it would take eight years to image a volume of (1 mm) 3 at 16 nm voxel resolution (Xu et al, 2017), while another estimate is that it would take 12 years for the same volume at 10 Â 10 Â 25 nm resolution (Titze & Genoud, 2016). The time for imaging (1 mm) 3 could conceivably drop to less than one year using multi-beam scanning electron microscopy (Eberle & Zeidler, 2018), and recently a highly automated pipeline has been used to image 1 mm 3 in less than six months using six transmission electron microscopes (Yin et al, 2020). However, serial sectioning is still accompanied by inherently anisotropic resolution (Kreshuk et al, 2011;Kornfeld & Denk, 2018) and unavoidable knife-cutting artifacts (Khalilian-Gourtani et al, 2019), which can complicate faithful 3D characterization of the connectome.…”

Section: Introductionmentioning

confidence: 99%

Upscaling X-ray nanoimaging to macroscopic specimens

Du¹,

Zw²,

Gürsoy³

et al. 2021

J Appl Crystallogr

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Upscaling X-ray nanoimaging to macroscopic specimens has the potential for providing insights across multiple length scales, but its feasibility has long been an open question. By combining the imaging requirements and existing proof-of-principle examples in large-specimen preparation, data acquisition and reconstruction algorithms, the authors provide imaging time estimates for howX-ray nanoimaging can be scaled to macroscopic specimens. To arrive at this estimate, a phase contrast imaging model that includes plural scattering effects is used to calculate the required exposure and corresponding radiation dose. The coherent X-ray flux anticipated from upcoming diffraction-limited light sources is then considered. This imaging time estimation is in particular applied to the case of the connectomes of whole mouse brains. To image the connectome of the whole mouse brain, electron microscopy connectomics might require years, whereas optimized X-ray microscopy connectomics could reduce this to one week. Furthermore, this analysis points to challenges that need to be overcome (such as increased X-ray detector frame rate) and opportunities that advances in artificial-intelligence-based `smart' scanning might provide. While the technical advances required are daunting, it is shown that X-ray microscopy is indeed potentially applicable to nanoimaging of millimetre- or even centimetre-size specimens.

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A petascale automated imaging pipeline for mapping neuronal circuits with high-throughput transmission electron microscopy

Cited by 117 publications

References 35 publications

A connectomic study of a petascale fragment of human cerebral cortex

A connectomic study of a petascale fragment of human cerebral cortex

Oligodendrocyte precursor cells prune axons in the mouse neocortex

Upscaling X-ray nanoimaging to macroscopic specimens

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