3D reconstruction of a gastrulating human embryo.

Progress in understanding early human development has been impeded by the scarcity of reference datasets from natural embryos, particularly those with spatial information during crucial stages like gastrulation. We conducted high-resolution spatial transcriptomics profiling on 38,562 spots from 62 transverse sections of an intact Carnegie stage (CS) 8 human embryo. From this spatial transcriptomic dataset, we constructed a 3D model of the CS8 embryo, in which a range of cell subtypes are identified, based on gene expression patterns and positional register, along the anterior-posterior, medial-lateral, and dorsal-ventral axis in the embryo. We further characterized the lineage trajectories of embryonic and extra-embryonic tissues and associated regulons and the regionalization of signaling centers and signaling activities that underpin lineage progression and tissue patterning during gastrulation. Collectively, the findings of this study provide insights into gastrulation and post-gastrulation development of the human embryo.

Reconstruction of neocortex: Organelles, compartments, cells, circuits, and activity.

We assembled a semi-automated reconstruction of L2/3 mouse primary visual cortex from ∼250 × 140 × 90 µm3 of electron microscopic images, including pyramidal and non-pyramidal neurons, astrocytes, microglia, oligodendrocytes and precursors, pericytes, vasculature, nuclei, mitochondria, and synapses. Visual responses of a subset of pyramidal cells are included. The data are publicly available, along with tools for programmatic and three-dimensional interactive access. Brief vignettes illustrate the breadth of potential applications relating structure to function in cortical circuits and neuronal cell biology. Mitochondria and synapse organization are characterized as a function of path length from the soma. Pyramidal connectivity motif frequencies are predicted accurately using a configuration model of random graphs. Pyramidal cells receiving more connections from nearby cells exhibit stronger and more reliable visual responses. Sample code shows data access and analysis.

Digital Museum of Retinal Ganglion Cells with Dense Anatomy and Physiology.

When 3D electron microscopy and calcium imaging are used to investigate the structure and function of neural circuits, the resulting datasets pose new challenges of visualization and interpretation. Here, we present a new kind of digital resource that encompasses almost 400 ganglion cells from a single patch of mouse retina. An online "museum" provides a 3D interactive view of each cell's anatomy, as well as graphs of its visual responses. The resource reveals two aspects of the retina's inner plexiform layer: an arbor segregation principle governing structure along the light axis and a density conservation principle governing structure in the tangential plane. Structure is related to visual function; ganglion cells with arbors near the layer of ganglion cell somas are more sustained in their visual responses on average. Our methods are potentially applicable to dense maps of neuronal anatomy and physiology in other parts of the nervous system.

Cryogenic electron microscopy and single-particle analysis.

About 20 years ago, the first three-dimensional (3D) reconstructions at subnanometer (<10-Å) resolution of an icosahedral virus assembly were obtained by cryogenic electron microscopy (cryo-EM) and single-particle analysis. Since then, thousands of structures have been determined to resolutions ranging from 30 Å to near atomic (<4 Å). Almost overnight, the recent development of direct electron detectors and the attendant improvement in analysis software have advanced the technology considerably. Near-atomic-resolution reconstructions can now be obtained, not only for megadalton macromolecular complexes or highly symmetrical assemblies but also for proteins of only a few hundred kilodaltons. We discuss the developments that led to this breakthrough in high-resolution structure determination by cryo-EM and point to challenges that lie ahead.

Cardiomyocyte orientation recovery at micrometer scale reveals long-axis fiber continuum in heart walls.

Coordinated cardiomyocyte contraction drives the mammalian heart to beat and circulate blood. No consensus model of cardiomyocyte geometrical arrangement exists, due to the limited spatial resolution of whole heart imaging methods and the piecemeal nature of studies based on histological sections. By combining microscopy and computer vision, we produced the first-ever three-dimensional cardiomyocyte orientation reconstruction across mouse ventricular walls at the micrometer scale, representing a gain of three orders of magnitude in spatial resolution. We recovered a cardiomyocyte arrangement aligned to the long-axis direction of the outer ventricular walls. This cellular network lies in a thin shell and forms a continuum with longitudinally arranged cardiomyocytes in the inner walls, with a complex geometry at the apex. Our reconstruction methods can be applied at fine spatial scales to further understanding of heart wall electrical function and mechanics, and set the stage for the study of micron-scale fiber remodeling in heart disease.

Ultrastructural analysis of whole glomerulus using array tomography.

The renal glomerulus produces primary urine from blood plasma by ultrafiltration. The ultrastructure of the glomerulus is closely related to filtration function and disease development. The ultrastructure of the glomeruli has mainly been evaluated using transmission electron microscopy. However, the volume that can be observed using transmission electron microscopy is extremely limited relative to the total volume of the glomerulus. Consequently, observing structures that exist in only one location in each glomerulus, such as the vascular pole, and evaluating low-density or localized lesions are challenging tasks. Array tomography (AT) is a technique used to analyze the ultrastructure of tissues and cells via scanning electron microscopy of serial sections. In this study, we propose an AT workflow optimized for observing complete serial sections of the whole glomerulus, shared several analytical examples using the optimized AT workflow, and demonstrate the usefulness of this approach. Overall, this AT workflow can be a powerful tool for structural and pathological evaluation of the glomerulus. This workflow is also expected to provide new insights into the ultrastructure of the glomerulus and its constituent cells.

Soft, malleable double diamond twin.

A twin boundary (TB) is a common low energy planar defect in crystals including those with the atomic diamond structure (C, Si, Ge, etc.). We study twins in a self-assembled soft matter block copolymer (BCP) supramolecular crystal having the double diamond (DD) structure, consisting of two translationally shifted, interpenetrating diamond networks of the minority polydimethyl siloxane block embedded in a polystyrene block matrix. The coherent, low energy, mirror-symmetric double tubular network twin has one minority block network with its nodes offset from the (222) TB plane, while nodes of the second network lie in the plane of the boundary. The offset network, although at a scale about a factor of 103 larger, has precisely the same geometry and symmetry as a (111) twin in atomic single diamond where the tetrahedral units spanning the TB retain nearly the same strut (bond) lengths and strut (bond) angles as in the normal unit cell. In DD, the second network undergoes a dramatic restructuring-the tetrahedral nodes transform into two new types of mirror-symmetric nodes (pentahedral and trihedral) which alternate and link to form a hexagonal mesh in the plane of the TB. The collective reorganization of the supramolecular packing highlights the hierarchical structure of ordered BCP phases and emphasizes the remarkable malleability of soft matter.

A semiparametric Gaussian mixture model for chest CT-based 3D blood vessel reconstruction.

Computed tomography (CT) has been a powerful diagnostic tool since its emergence in the 1970s. Using CT data, 3D structures of human internal organs and tissues, such as blood vessels, can be reconstructed using professional software. This 3D reconstruction is crucial for surgical operations and can serve as a vivid medical teaching example. However, traditional 3D reconstruction heavily relies on manual operations, which are time-consuming, subjective, and require substantial experience. To address this problem, we develop a novel semiparametric Gaussian mixture model tailored for the 3D reconstruction of blood vessels. This model extends the classical Gaussian mixture model by enabling nonparametric variations in the component-wise parameters of interest according to voxel positions. We develop a kernel-based expectation-maximization algorithm for estimating the model parameters, accompanied by a supporting asymptotic theory. Furthermore, we propose a novel regression method for optimal bandwidth selection. Compared to the conventional cross-validation-based (CV) method, the regression method outperforms the CV method in terms of computational and statistical efficiency. In application, this methodology facilitates the fully automated reconstruction of 3D blood vessel structures with remarkable accuracy.

Ablation of Sam50 is associated with fragmentation and alterations in metabolism in murine and human myotubes.

The sorting and assembly machinery (SAM) Complex is responsible for assembling ß-barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing system complex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity. While the role of Sam50 in mitochondrial structure and metabolism in skeletal muscle remains unclear, this study aims to investigate its impact. Serial block-face-scanning electron microscopy and computer-assisted 3D renderings were employed to compare mitochondrial structure and networking in Sam50-deficient myotubes from mice and humans with wild-type (WT) myotubes. Furthermore, autophagosome 3D structure was assessed in human myotubes. Mitochondrial metabolic phenotypes were assessed using Gas Chromatography-Mass Spectrometry-based metabolomics to explore differential changes in WT and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-deficient myotubes compared to controls. Metabolomic analysis indicated elevated metabolism of propanoate and several amino acids, including ß-Alanine, phenylalanine, and tyrosine, along with increased amino acid and fatty acid metabolism in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation in both murine and human myotubes, as measured with the XF24 Seahorse Analyzer. Collectively, these findings support the critical role of Sam50 in establishing and maintaining mitochondrial integrity, cristae structure, and mitochondrial metabolism. By elucidating the impact of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle.

3-Dimensional Reconstruction From Histopathological Sections: A Systematic Review.

Although pathological tissue analysis is typically performed on single 2-dimensional (2D) histologic reference slides, 3-dimensional (3D) reconstruction from a sequence of histologic sections could provide novel opportunities for spatial analysis of the extracted tissue. In this review, we analyze recent works published after 2018 and report information on the extracted tissue types, the section thickness, and the number of sections used for reconstruction. By analyzing the technological requirements for 3D reconstruction, we observe that software tools exist, both free and commercial, which include the functionality to perform 3D reconstruction from a sequence of histologic images. Through the analysis of the most recent works, we provide an overview of the workflows and tools that are currently used for 3D reconstruction from histologic sections and address points for future work, such as a missing common file format or computer-aided analysis of the reconstructed model.