Serial Section Images Research Articles

Rapid full-color serial sectioning tomography with speckle illumination and ultraviolet excitation

Three-dimensional (3D) high-resolution large-volume imaging has remained a challenge. Translational rapid ultraviolet-excited sectioning tomography (TRUST) achieves rapid and cost-effective whole-organ subcellular imaging through iterative optical scanning and mechanical sectioning. However, the axial resolution is limited by the mechanical sectioning thickness or the UV light penetration depth in tissue. Here, assisted with high-and-low-frequency (HiLo) microscopy (HiLoTRUST), the optical sectioning thickness has been reduced from tens of micrometers to ~5.8 µm. In addition, HiLoTRUST has attained a finer mechanical sectioning thickness (10–15 µm) compared to TRUST (50 µm). For high-content imaging as in TRUST, we employed two additional UV light-emitting diodes (LEDs) specifically for uniform illumination. The full-color imaging ability and improved axial resolution of HiLoTRUST have been validated by two-dimensional (2D)/3D imaging of various mouse organs and human lung cancer specimens. HiLoTRUST offers a cost-effective, full-color, and high-resolution 3D imaging approach, showing its great potential in 3D histology applications.

Combining array tomography with electron tomography provides insights into leakiness of the blood-brain barrier in mouse cortex

Like other volume electron microscopy approaches, automated tape-collecting ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-stable thin and fragile films, thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed ‘ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood-brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlative ATUM-Tomo. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures.

Combining array tomography with electron tomography provides insights into leakiness of the blood-brain barrier in mouse cortex.

Like other volume electron microscopy approaches, automated tape-collecting ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-stable thin and fragile films, thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed 'ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood-brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlative ATUM-Tomo. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures.

Three-Dimensional Ultrastructure of Flower-Spray Nerve Endings in the Rat Carotid Sinus.

The flower-spray nerve endings are afferent nerve terminals in the carotid sinus that arise from carotid sinus nerve of glossopharyngeal nerve. However, the three-dimensional ultrastructural characteristics of flower-spray nerve endings and spatial relationships between the terminal parts and other cellular elements have not been fully understood. To elucidate their detailed relationship, backscattered electron imaging of serial sections was performed with a scanning electron microscope to produce a three-dimensional reconstruction of the flower-spray endings. The terminal parts of flower-spray endings were distributed horizontally approximately 5µm outside the external elastic membrane in the tunica adventitia of the internal carotid artery. The three-dimensional reconstruction showed that the terminal parts of flower-spray endings were flat with irregular contours and were partially covered by the thin cytoplasmic processes of Schwann cells. The complex consisting of the nerve terminals and associated Schwann cells was surrounded by a multilayered basement membrane. The terminal parts of the endings were also surrounded by fibroblasts with elastic fibers and collagen fibrils. Secretory vesicles without an electron-dense core were observed in the terminal parts of the endings. The accumulation of vesicles just below the axonal membrane was observed in terminal parts not covered by Schwann cell cytoplasmic processes on both the luminal and basal sides. Swollen mitochondria, concentric membranous structures, and glycogen granule-like electron-dense materials were often noted in some of the terminal parts of the endings and the parent axon. Collectively, the present results suggest that flower-spray endings are baroreceptors because their morphology was similar to other mechanoreceptors. Furthermore, flower-spray endings may be affected by glutamate secreted in an autocrine manner.

A deep learning-based strategy for producing dense 3D segmentations from sparsely annotated 2D images.

Producing dense 3D reconstructions from biological imaging data is a challenging instance segmentation task that requires significant ground-truth training data for effective and accurate deep learning-based models. Generating training data requires intense human effort to annotate each instance of an object across serial section images. Our focus is on the especially complicated brain neuropil, comprising an extensive interdigitation of dendritic, axonal, and glial processes visualized through serial section electron microscopy. We developed a novel deep learning-based method to generate dense 3D segmentations rapidly from sparse 2D annotations of a few objects on single sections. Models trained on the rapidly generated segmentations achieved similar accuracy as those trained on expert dense ground-truth annotations. Human time to generate annotations was reduced by three orders of magnitude and could be produced by non-expert annotators. This capability will democratize generation of training data for large image volumes needed to achieve brain circuits and measures of circuit strengths.

Unveiling tumor invasiveness: enhancing cancer diagnosis with phase-contrast microtomography for 3D virtual histology

Malignant diseases are characterized by a critical trait known as invasiveness, where tumor cells tend to spread from the primary tissue layer into surrounding healthy tissues and distant organs. Presently, histopathology offers essential insights for diagnosing, classifying, predicting outcomes, and guiding patient-specific treatments. However, histology offers two-dimensional data from chosen cutting planes. Although 3D histological volumes can be generated through serial sectioning or whole slide imaging, this method is laborious, may introduce processing artefacts, and lacks isotropic spatial resolution. These limitations pose a considerable challenge to accurate diagnoses, particularly when dealing with micro-infiltrating carcinomas. These lesions, characterized by minute infiltrations, demand a three-dimensional representation for comprehensive visualization, essential for precise identification and assessment. Emerging X-ray-based virtual histology technology offers three-dimensional visualization of soft-tissue specimens, enabling virtual slicing in any direction or at any point. This approach can assist in guiding tissue sectioning for optimal representation of tumor cross sections during histological analysis. Micro-infiltrating carcinomas from the breast, cervix, and thyroid were imaged using X-ray phase-contrast microtomography (PhC-μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT) at the Elettra synchrotron facility in Trieste, Italy. Comparative assessment of histological and CT slices by pathologists revealed that PhC-μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mu$$\\end{document}CT aids in classifying lesions by highlighting distinct tissue components and, notably, identifying tissue invasion. Reviewing a volume image allows pathologists to trace the entire lesion, identifying invasion sites that might be overlooked in individual or serial histological sections. Consequently, this proposed method could complement pathologists’ tools, potentially enhancing diagnoses by minimizing under-staging and reducing false negative results.

Volume X-Ray Micro-Computed Tomography Analysis of the Early Cephalized Central Nervous System in a Marine Flatworm, Stylochoplana pusilla.

Platyhelminthes are a phylum of simple bilaterian invertebrates with prototypic body systems. Compared with non-bilaterians such as cnidarians, the bilaterians are likely to exhibit integrated free-moving behaviors, which require a concentrated nervous system "brain" rather than the distributed nervous system of radiatans. Marine flatworms have an early cephalized 'central' nervous system compared not only with non-bilaterians but also with parasitic flatworms or freshwater planarians. In this study, we used the marine flatworm Stylochoplana pusilla as an excellent model organism in Platyhelminthes because of the early cephalized central nervous system. Here, we investigated the three-dimensional structures of the flatworm central nervous system by the use of X-ray micro-computed tomography (micro-CT) in a synchrotron radiation facility. We found that the obtained tomographic images were sufficient to discriminate some characteristic structures of the nervous system, including nerve cords around the cephalic ganglion, mushroom body-like structures, and putative optic nerves forming an optic commissure-like structure. Through the micro-CT imaging, we could obtain undistorted serial section images, permitting us to visualize precise spatial relationships of neuronal subpopulations and nerve tracts. 3-D micro-CT is very effective in the volume analysis of the nervous system at the cellular level; the methodology is straightforward and could be applied to many other non-model organisms.

Array tomography of in vivo labeled synaptic receptors.

Array tomography (AT) allows one to localize sub-cellular components within the structural context of cells in 3D through the imaging of serial sections. Using this technique, the z-resolution can be improved physically by cutting ultra-thin sections. Nevertheless, conventional immunofluorescence staining of those sections is time consuming and requires relatively large amounts of costly antibody solutions. Moreover, epitopes are only readily accessible at the section's surface, leaving the volume of the serial sections unlabeled. Localization of receptors at neuronal synapses in 3D in their native cellular ultrastructural context is important for understanding signaling processes. Here, we present in vivo labeling of receptors via fluorophore-coupled tags in combination with super-resolution AT. We present two workflows where we label receptors at the plasma membrane: first, in vivo labeling via microinjection with a setup consisting of readily available components and self-manufactured microscope table equipment and second, live receptor labeling by using a cell-permeable tag. To take advantage of a near-to-native preservation of tissues for subsequent scanning electron microscopy (SEM), we also apply high-pressure freezing and freeze substitution. The advantages and disadvantages of our workflows are discussed.

The efferent connections of the orbitofrontal, posterior parietal, and insular cortex of the rat brain

The orbitofrontal, posterior parietal, and insular cortices are sites of higher-order cognitive processing implicated in a wide range of behaviours, including working memory, attention guiding, decision making, and spatial navigation. To better understand how these regions contribute to such functions, we need detailed knowledge about the underlying structural connectivity. Several tract-tracing studies have investigated specific aspects of orbitofrontal, posterior parietal and insular connectivity, but a digital resource for studying the cortical and subcortical projections from these areas in detail is not available. We here present a comprehensive collection of brightfield and fluorescence microscopic images of serial coronal sections from 49 rat brain tract-tracing experiments, in which discrete injections of the anterograde tracers biotinylated dextran amine and/or Phaseolus vulgaris leucoagglutinin were placed in the orbitofrontal, parietal, or insular cortex. The images are spatially registered to the Waxholm Space Rat brain atlas. The image collection, with corresponding reference atlas maps, is suitable as a reference framework for investigating the brain-wide efferent connectivity of these cortical association areas.

A Three-Dimensional Technique for The Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a three-dimensional (3D) perspective, is critical for mechanistic studies. Electron microscopy (EM) offers good imaging depth and allows for the reconstruction of high-resolution image stacks to investigate the ultrastructural morphology of cellular organelles even at the nanometer scale; therefore, 3D reconstruction is gaining importance due to its incomparable advantages. Scanning electron microscopy (SEM) provides a high-throughput image acquisition technology that allows for reconstructing large structures in 3D from the same region of interest in consecutive slices. Therefore, the application of SEM in large-scale 3D reconstruction to restore the true 3D ultrastructure of organelles is becoming increasingly common. In this protocol, we suggest a combination of serial ultrathin section and 3D reconstruction techniques to study mitochondrial cristae in pancreatic cancer cells. The details of how these techniques are performed are described in this protocol in a step-by-step manner, including the osmium-thiocarbohydrazide-osmium (OTO) method, the serial ultrathin section imaging, and the visualization display.

Three-dimensional investigation of fragment distribution in Al – 7wt.% Si solidified in microgravity

Three-dimensional (3D) reconstruction of the microstructure of Al – 7 wt.% Si samples solidified in microgravity conditions on board of the International Space Station was obtained from serial-sectioning images. The main objective was to quantify the number of dendrite fragments issued from the solidification process in absence of buoyancy force and with motionless dendrite fragments on contrary to ground-based experiments. The results show that the number of fragments strongly depends on the dendrite network configuration. More fragments are observed in the sample containing several dendrites with different orientations compared to a sample with well-aligned dendrites. Moreover, it has been found that the highest number of fragments is mainly located in the region corresponding to an incipient grain boundary where dendrites with different orientations compete. Indeed, in this region solute accumulates and the microstructure is finer which is more prone to fragmentation of dendritic arms growing in this region. Thus, a mechanism that leads to the formation of dendrite fragmentation is identified: dendrite misorientation during solidification.

An integrative web-based software tool for multi-dimensional pathology whole-slide image analytics

Objective. In the era of precision medicine, human tumor atlas-oriented studies have been significantly facilitated by high-resolution, multi-modal tissue based microscopic pathology image analytics. To better support such tissue-based investigations, we have developed Digital Pathology Laboratory (DPLab), a publicly available web-based platform, to assist biomedical research groups, non-technical end users, and clinicians for pathology whole-slide image visualization, annotation, analysis, and sharing via web browsers. Approach. A major advancement of this work is the easy-to-follow methods to reconstruct three-dimension (3D) tissue image volumes by registering two-dimension (2D) whole-slide pathology images of serial tissue sections stained by hematoxylin and eosin (H&E), and immunohistochemistry (IHC). The integration of these serial slides stained by different methods provides cellular phenotype and pathophysiologic states in the context of a 3D tissue micro-environment. DPLab is hosted on a publicly accessible server and connected to a backend computational cluster for intensive image analysis computations, with results visualized, downloaded, and shared via a web interface. Main results. Equipped with an analysis toolbox of numerous image processing algorithms, DPLab supports continued integration of community-contributed algorithms and presents an effective solution to improve the accessibility and dissemination of image analysis algorithms by research communities. Significance. DPLab represents the first step in making next generation tissue investigation tools widely available to the research community, enabling and facilitating discovery of clinically relevant disease mechanisms in a digital 3D tissue space.

Anatomical study of the adductor canal: three-dimensional micro-computed tomography, histological, and immunofluorescence findings relevant to neural blockade.

A precise anatomical understanding of the adductor canal (AC) and its neural components is essential for discerning the action mechanism of the AC block. We therefore aimed to clarify the detailed anatomy of the AC using micro-computed tomography (micro-CT), histological evaluation, and immunofluorescence (IF) assays. Gross dissections of 39 thighs provided morphometric data relevant to injection landmarks. Serial sectional images of the AC were defined using micro-CT and ultrasonography. The fascial and neural structures of the AC proper were histologically evaluated using Masson's trichrome and Verhoeff-Van Gieson staining, and double IF staining using choline acetyltransferase (ChAT) and neurofilament 200 antibodies. The posteromedial branch insertion of the nerve to vastus medialis (NVM) into the lateral border of the AC proper was lower (14.5 ± 2.4 cm [mean ± SD] above the base of the patella) than the origin of the proximal AC. The AC consists of a thin subsartorial fascia in the proximal region and a thick aponeurosis-like vastoadductor membrane in the distal region. In the proximal AC, the posteromedial branch of the NVM (pmNVM) consistently contained both sensory and motor fibers, and more ChAT-positive fibers were observed than in the saphenous nerve (27.5 ± 11.2 / 104 vs. 4.2 ± 2.6 / 104 [counts/µm2], P < 0.001). Anatomical differences in fascial structures between the proximal and distal AC and a mixed neural component of the neighboring pmNVM have been visualized using micro-CT images, histological evaluation, and IF assays.

Three-Dimensional Reconstruction of Printed Circuit Boards: Comparative Study between 3D Femtosecond Laser Serial Sectioning and Optical Imaging versus 3D X-Ray Computed Tomography

Three-Dimensional Reconstruction of Printed Circuit Boards: Comparative Study between 3D Femtosecond Laser Serial Sectioning and Optical Imaging versus 3D X-Ray Computed Tomography - Volume 28 Issue S1

Stereotaxic Atlas of the Human Lumbar-Sacral Spinal Cord

A Stereotaxic Atlas of the Human Lumbar-Sacral Spinal Cord has been created to provide an anatomical basis for radiologic and ultrasonic imaging and electrophysiological examination, which are used to target the placement of lumbar-sacral epidural stimulating electrodes and cellular transplantation in order to restore movement in individuals with sustained spinal cord injury or a degenerative disorder of the spinal cord. Through the availability of an atlas that exhibits axial images of the cytoarchitecture of each cord segment with a stereotaxic millimeter grid of dorsal-ventral depth from the midline dorsal surface of the cord and right-left distances from the midline of the cord, neuromodulation, and cellular therapy would undoubtedly be made not only more precise but also safer for patients. The atlas is based upon dimension measurements and subsequent serial sectioning, staining and high-resolution digital imaging of the lumbar-sacral enlargement of 20 adult human spinal cords. Nissl stained cross-sections from cord segments L1-S3 illustrate the cytoarchitecture and stereotactic coordinates. The atlas provides an anatomical basis for radiologic and physiologic confirmation of target localization in the lumbar-sacral spinal cord.

Virtual liver needle biopsy from reconstructed three-dimensional histopathological images: Quantification of sampling error

Introduction:Prevalently considered as the “gold-standard” for diagnosis of hepatic fibrosis and cirrhosis, the clinical liver needle biopsy is known to be subject to inadequate sampling and a high mis-sampling rate. However, quantifying such sampling bias has been difficult as generating a large number of needle biopsies from the same living patient is practically infeasible. We construct a three-dimension (3D) virtual liver tissue volume by spatially registered high resolution Whole Slide Images (WSIs) of serial liver tissue sections with a novel dynamic registration method. We further develop a Virtual Needle Biopsy Sampling (VNBS) method that mimics the needle biopsy sampling process. We apply the VNBS method to the reconstructed digital liver volume at different tissue locations and angles. Additionally, we quantify Collagen Proportionate Area (CPA) in all resulting virtual needle biopsies in 2D and 3D. Results:The staging score of the center 2D longitudinal image plane from each 3D biopsy is used as the biopsy staging score, and the highest staging score of all sampled needle biopsies is the diagnostic staging score. The Mean Absolute Difference (MAD) in reference to the Scheuer and Ishak diagnostic staging scores are 0.22 and 1.00, respectively. The absolute Scheuer staging score difference in 22.22% of sampled biopsies is 1. By the Ishak staging method, 55.56% and 22.22% of sampled biopsies present score difference 1 and 2, respectively. There are 4 (Scheuer) and 6 (Ishak) out of 18 3D virtual needle biopsies with intra-needle variations. Additionally, we find a positive correlation between CPA and fibrosis stages by Scheuer but not Ishak method. Overall, CPA measures suffer large intra- and inter- needle variations. Conclusions:The developed virtual liver needle biopsy sampling pipeline provides a computational avenue for investigating needle biopsy sampling bias with 3D virtual tissue volumes. This method can be applied to other tissue-based disease diagnoses where the needle biopsy sampling bias substantially affects the diagnostic results.

レーザ粉末床溶融結合法によるAl-10%Si-0.4%Mg合金積層造形体の特異的な熱伝導率のイメージベース有限要素解析

Laser powder bed fused Al-10Si-0.4Mg (mass%) alloy exhibits low and anisotropic thermal conductivity. To elucidate dominant microstructural features generating the characteristic thermal conductivity, thermal conduction analysis was performed by an image-based finite element method (FEM) using scanning electron microscope (SEM) images. The serial sectioning images of as-fabricated and 300ºC/2 h heat treated Al-10Si-0.4Mg alloys were obtained by focused ion beam SEM (FIB-SEM) and were used for constructing three-dimensional models. The homogenization method was applied to calculate average thermal conductivity of these models. When the thermal conductivity of α-Al phase was set at the thermal conductivity of pure aluminum, calculated thermal conductivity of as-fabricated model was much higher than experimentally measured thermal conductivity of as-fabricated Al-10Si-0.4Mg alloy, indicating that α-Al phase in the as-fabricated Al-10Si-0.4Mg alloy had much lower thermal conductivity than pure aluminum. In addition, the anisotropy of thermal conductivity appeared only in the as-fabricated model in which the α-Al/Si interfacial thermal resistance was taken into account. These results were used for discussing dominant contributors of low and anisotropic thermal conductivity of laser additive manufactured Al-10Si-0.4Mg alloy.

Neuronal Morphological Model-Driven Image Registration for Serial Electron Microscopy Sections.

Registration of a series of the two-dimensional electron microscope (EM) images of the brain tissue into volumetric form is an important technique that can be used for neuronal circuit reconstruction. However, complex appearance changes of neuronal morphology in adjacent sections bring difficulty in finding correct correspondences, making serial section neural image registration challenging. To solve this problem, we consider whether there are such stable "markers" in the neural images to alleviate registration difficulty. In this paper, we employ the spherical deformation model to simulate the local neuron structure and analyze the relationship between registration accuracy and neuronal structure shapes in two adjacent sections. The relevant analysis proves that regular circular structures in the section images are instrumental in seeking robust corresponding relationships. Then, we design a new serial section image registration framework driven by this neuronal morphological model, fully utilizing the characteristics of the anatomical structure of nerve tissue and obtaining more reasonable corresponding relationships. Specifically, we leverage a deep membrane segmentation network and neural morphological physical selection model to select the stable rounded regions in neural images. Then, we combine feature extraction and global optimization of correspondence position to obtain the deformation field of multiple images. Experiments on real and synthetic serial EM section neural image datasets have demonstrated that our proposed method could achieve more reasonable and reliable registration results, outperforming the state-of-the-art approaches in qualitative and quantitative analysis.

Axonal and Dendritic Morphology of Excitatory Neurons in Layer 2/3 Mouse Barrel Cortex Imaged Through Whole-Brain Two-Photon Tomography and Registered to a Digital Brain Atlas.

Communication between cortical areas contributes importantly to sensory perception and cognition. On the millisecond time scale, information is signaled from one brain area to another by action potentials propagating across long-range axonal arborizations. Here, we develop and test methodology for imaging and annotating the brain-wide axonal arborizations of individual excitatory layer 2/3 neurons in mouse barrel cortex through single-cell electroporation and two-photon serial section tomography followed by registration to a digital brain atlas. Each neuron had an extensive local axon within the barrel cortex. In addition, individual neurons innervated subsets of secondary somatosensory cortex; primary somatosensory cortex for upper limb, trunk, and lower limb; primary and secondary motor cortex; visual and auditory cortical regions; dorsolateral striatum; and various fiber bundles. In the future, it will be important to assess if the diversity of axonal projections across individual layer 2/3 mouse barrel cortex neurons is accompanied by functional differences in their activity patterns.

Comparative morphometric analysis of age-related changes in the pyramidal neurons of the human prefrontal and posterior associative cortex from birth to 7 years

Aim: This article is devoted to the study of age-related changes in the body volume of pyramidal neurons (VP) of the human prefrontal (PFC) and posterior associative cortex (PAC) in children from birth to 7 years of age. Methods: The material consisted of left cerebral hemispheres obtained from 60 male individuals. We studied the areas 8, 45 and 10 in the PFC and area 37 in subareas 37ac, 37a, 37d in the PAC. For morphometry of pyramidal neurons in cortical sublayer III3, we used virtual images of serial frontal paraffin sections of cortex 10 thick stained by Nissl. We determined the mean value of VP (mean VP), the standard error of the mean, and the confidence interval for each age group. Results: The most significant changes in the mean VP in all studied cortical areas occurred during the first 6 months of life. The greatest increase in the VP was detected in the PFC from 3.5 to 7 years, and in the PAC from 1.5 to 3 years of age. There are close positive correlations between agerelated changes in the size of neurons in the PFC and in the PAC. Conclusion: In children, the development of pyramidal neurons of the external pyramidal plate in the PAC occurs earlier and at a more intensive pace compared to the PFC. We suggest that VP is an important quantitative indicator that allows us to assess the timing and rate of age-related structural changes in the cerebral cortex.