Serial Block-face Scanning Electron Research Articles

Cell Biology Methods to Study Recombinant Proteins in Seeds.

Seeds are an attractive platform for the production of recombinant proteins because of their excellent storage properties and their well-developed endomembrane system, which allows accumulation of the product within specialized storage organelles. Due to the presence of these additional organelles and the resulting complexity of intracellular protein trafficking it is interesting to investigate the transport and storage of a recombinant protein within seed tissues, its interactions with endogenous reserve proteins and its impact on the ultrastructure of the endomembrane system. Possible approaches include sequential extraction procedures, subcellular fractionation and 2D as well as 3D electron microscopy techniques such as electron tomography (ET) and serial block face scanning electron microscopy (SBF-SEM), which are described and discussed in this chapter.

The 3D organisation of mitochondria in primate photoreceptors

Vertebrate photoreceptors contain large numbers of closely-packed mitochondria which sustain the high metabolic demands of these cells. These mitochondria populations are dynamic and undergo fusion and fission events. This activity serves to maintain the population in a healthy state. In the event of mitochondrial damage, sub-domains, or indeed whole mitochondria, can be degraded and population homeostasis achieved. If this process is overwhelmed cell death may result. Death of photoreceptors contributes to loss of vision in aging individuals and is associated with many eye diseases. In this study we used serial block face scanning electron microscopy of adult Macaca fascicularis retinae to examine the 3D structure of mitochondria in rod and cone photoreceptors. We show that healthy-looking photoreceptors contain mitochondria exhibiting a range of shapes which are associated with different regions of the cell. In some photoreceptors we observe mitochondrial swelling and other changes often associated with cellular stress. In rods and cones that appear stressed we identify elongated domains of mitochondria with densely-packed normal cristae associated with photoreceptor ciliary rootlet bundles. We observe mitochondrial fission and mitochondrion fragments localised to these domains. Swollen mitochondria with few intact cristae are located towards the periphery of the photoreceptor inner-segment in rods, whilst they are found throughout the cell in cones. Swollen mitochondria exhibit sites on the mitochondrial inner membrane which have undergone complex invagination resulting in membranous, electron-dense aggregates. Membrane contact occurs between the mitochondrion and the photoreceptor plasma membrane in the vicinity of these aggregates, and a series of subsequent membrane fusions results in expulsion of the mitochondrial aggregate from the photoreceptor. These events are primarily associated with rods. The potential fate of this purged material and consequences of its clearance by retinal pigment epithelia are discussed.

Bovine trophoblast giant cells maintain contact to the fetal basement membrane until birth – first evidence by serial block face scanning electron microscopy

Bovine trophoblast giant cells maintain contact to the fetal basement membrane until birth – first evidence by serial block face scanning electron microscopy

Contrast-Enhanced Tissue Processing of Fibrillin-Rich Elastic Fibres for 3D Visualization by Volume Scanning Electron Microscopy.

Elastic fibres constitute an important component of the extracellular matrix and currently are the subject of intensive study in order to elucidate their assembly, function and involvement in cell–matrix interactions and disease. However, few studies to date have investigated the 3D architecture of the elastic fibre system in bulk tissue. We describe a protocol for preparation of tissue samples, including primary fixation and backscatter electron contrast-enhancement steps, through dehydration into stable resin-embedded blocks for volume electron microscopy. The use of low molecular weight tannic acid and alcoholic lead staining are critical stages in this procedure. Block preparation by ultramicrotomy and evaporative metal coating prior to microscopical examination are also described. We present images acquired from serial block face scanning electron microscopy of cornea and aorta showing target structures clearly differentiated from cells and other matrix components. The processing method imparts high contrast to fibrillin-containing elastic fibres, thus facilitating their segmentation and rendering into 3D reconstructions by image analysis software from large serial image datasets.

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve.

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the surgical and instrumental expertise required to accurately quantify the structure and organization across multiple length scales have stunted its advancement. This work provides a detailed description of the murine dissection, en bloc staining, sample processing, and correlative imaging procedures for depicting the heart valve at different length scales. Hydrostatic transvalvular pressure was used to control the temporal heterogeneity by chemically fixing the heart valve conformation. Micro-computed tomography (µCT) was used to confirm the geometry of the heart valve and provide a reference for the downstream sample processing needed for the serial block face scanning electron microscopy (SBF-SEM). High-resolution serial SEM images of the extracellular matrix (ECM) were taken and reconstructed to provide a local 3D representation of its organization. µCT and SBF-SEM imaging methods were then correlated to overcome the spatial variation across the pulmonary valve. Though the work presented is exclusively on the pulmonary valve, this methodology could be adopted for describing the hierarchical organization in biological systems and is pivotal for the structural characterization across multiple length scales.

Serial block face scanning electron microscopy reveals novel organizational details of the retinal pigment epithelium.

Advances in imaging have led to the development of several new types of microscopes such as serial block face scanning electron microscopy (SBF-SEM), lightsheet microscopy, as well as X-ray micro-computed tomography (micro-CT), which enables the study of samples in fundamentally different ways. Significantly, these are now commercially available, which facilitates their widespread use in research. With SBF-SEM, fixed and resin-embedded specimens can be serially sectioned and imaged to construct a 3D dataset of the ultrastructure of cells and tissues at high resolution. We used this technique on perfusion-fixed C57BL/6 mouse eyes to image the outer retina. Our findings revealed novel organizational details of the retinal pigment epithelium (RPE) (Keeling et al., 2020b); a specialized cell monolayer that maintains the overlying photoreceptors and also forms the outer blood-retinal-barrier. RPE cells were found to look after far more photoreceptors than was widely assumed. 3D-data enabled measurements of the RPE cytoplasmic and nuclear volumes, the length and angle of microvilli on the apical RPE surface, as well as sub-RPE spaces under the basolateral membrane. The study also compared between mono-nucleate vs. bi-nucleate RPE cells, whilst the use of computing microinstructions (macros) provided information on interactions between adjacent cells in the RPE monolayer. Analysis of SBF-SEM stacks showed several hundred mitochondria which were rendered in 3D, providing information on their volume and spatial distribution in healthy RPE. Mitochondria were found in varying shapes and sizes, and predominantly localized to the mid and basal-zones of cells. The capabilities of SBF-SEM alongside other imaging techniques are being increasingly harnessed by investigators to gain novel insights into the organization of cells and tissues in the eye. These findings also help improve the current understanding of pathology linked with common blinding conditions such as age-related macular degeneration (AMD), as well as rare forms of retinopathy which leads to irreversible sight-loss

Fabrication of Nanoporous Al by Vapor-Phase Dealloying: Morphology Features, Mechanical Properties and Model Predictions

The physical and chemical properties shown by nanoporous metals, related to their unique structure, make them very promising for application in several fields. Recently, vapor-phase dealloying has been reported as a method for the preparation of several non-noble nanoporous metals, alternatively to dealloying in aqueous solutions. Using this approach, we have successfully fabricated nanoporous Al starting from an Al20Zn80 nanocomposite obtained by ball milling. The nanocomposite was annealed at 550 °C under high-vacuum conditions, and the difference in the vapor pressures allowed the selective removal of Zn by vapor-phase dealloying. The morphology of the resulting nanoporous material was analyzed by Scanning Electron Microscopy showing pores from few to thousands of nm; moreover, the nanoporous 3D structure was observed through Serial Block Face-Scanning Electron Microscopy. A specific surface area as high as 73 m2 g−1 was estimated by N2 physisorption measurements. In addition, a fractal model able to well reproduce the morphology of nanoporous Al was built. This model has been used for predicting mechanical properties which are in good agreement with experimental data obtained by nanoindentation.

Large-scale electron microscopic volume imaging of interfascicular oligodendrocytes in the mouse corpus callosum.

Single oligodendrocytes produce myelin sheaths around multiple axons in the central nervous system. Interfascicular oligodendrocytes (IOs) facilitate nerve conduction, but their detailed morphologies remain largely unknown. In the present study, we three-dimensionally reconstructed IOs in the corpus callosum of adult mouse using serial block face scanning electron microscopy. The cell bodies of IOs were morphologically polarized and extended thick processes from the cytoplasm-rich part of the cell. Processes originating from the cell body of each IO can be classified into two types: one myelinates an axon without branching, while the other type branches and each branch myelinates a distinct axon. Myelin sheaths originating from a particular IO have biased thicknesses, wrapping axons of a limited range of diameters. Consistent with this finding, IOs transduced and visualized with a rabies viral vector expressing GFP showed statistically significant variation in their myelination patterns. We further reconstructed the sheath immediately adjacent to that derived from each of the analyzed IOs; the thicknesses of the pair of sheaths were significantly correlated despite emanating from different IOs. These results suggest that a single axon could regulate myelin sheath thicknesses, even if the sheaths are derived from distinct IOs. Collectively, our results indicate that the IOs have their own myelin profiles defined by myelin thickness and axonal diameter although axons may regulate thickness of myelin sheath.

Maturation of Heterogeneity in Afferent Synapse Ultrastructure in the Mouse Cochlea.

Auditory nerve fibers (ANFs) innervating the same inner hair cell (IHC) may have identical frequency tuning but different sound response properties. In cat and guinea pig, ANF response properties correlate with afferent synapse morphology and position on the IHC, suggesting a causal structure-function relationship. In mice, this relationship has not been fully characterized. Here we measured the emergence of synaptic morphological heterogeneities during maturation of the C57BL/6J mouse cochlea by comparing postnatal day 17 (p17, ∼3 days after hearing onset) with p34, when the mouse cochlea is mature. Using serial block face scanning electron microscopy and three-dimensional reconstruction we measured the size, shape, vesicle content, and position of 70 ribbon synapses from the mid-cochlea. Several features matured over late postnatal development. From p17 to p34, presynaptic densities (PDs) and post-synaptic densities (PSDs) became smaller on average (PDs: 0.75 to 0.33; PSDs: 0.58 to 0.31 μm2) and less round as their short axes shortened predominantly on the modiolar side, from 770 to 360 nm. Membrane-associated synaptic vesicles decreased in number from 53 to 30 per synapse from p17 to p34. Anatomical coupling, measured as PSD to ribbon distance, tightened predominantly on the pillar side. Ribbons became less spherical as long-axes lengthened only on the modiolar side of the IHC, from 372 to 541 nm. A decreasing gradient of synaptic ribbon size along the modiolar-pillar axis was detected only at p34 after aligning synapses of adjacent IHCs to a common reference frame (median volumes in nm3 × 106: modiolar 4.87; pillar 2.38). The number of ribbon-associated synaptic vesicles scaled with ribbon size (range 67 to 346 per synapse at p34), thus acquiring a modiolar-pillar gradient at p34, but overall medians were similar at p17 (120) and p34 (127), like ribbon surface area (0.36 vs. 0.34 μm2). PD and PSD morphologies were tightly correlated to each other at individual synapses, more so at p34 than p17, but not to ribbon morphology. These observations suggest that PDs and PSDs mature according to different cues than ribbons, and that ribbon size may be more influenced by cues from the IHC than the surrounding tissue.

Techniques used to assess intussusceptive angiogenesis: A systematic review.

Intussusceptive angiogenesis (IA) is an important physiological form of angiogenesis in which an existing vessel splits in two by the formation of an intraluminal tissue pillar. The presence of these intraluminal pillars form the hallmark of ongoing IA in growing vascular beds. However, their visualization is technically challenging. The goal of this systematic review was to investigate which techniques are being used to identify intraluminal pillars and to formulate important points to keep in mind when studying IA. A systematic literature search resulted in 154 evaluated articles of which the majority (65%) provided sufficient data to unambiguously demonstrate the presence of intraluminal pillars. Scanning electron microscopy imaging of vascular corrosion casts and serial sectioning of ultrathin sections are the most used techniques. New methods such as serial block face scanning electron microscopy and micro computed tomography (μCT) are gaining importance. Moreover, our results indicate that IA was studied in a variety of animals and tissues. IA is a biologically very relevant form of angiogenesis. Techniques to visualize intraluminal pillars need to have a minimal resolution of 1μm and should provide information on the 3D-nature of the pillars. Optimally, several techniques are combined to demonstrate ongoing IA.

Volumetric Semantic Instance Segmentation of the Plasma Membrane of HeLa Cells.

In this work, an unsupervised volumetric semantic instance segmentation of the plasma membrane of HeLa cells as observed with serial block face scanning electron microscopy is described. The resin background of the images was segmented at different slices of a 3D stack of 518 slices with 8192 × 8192 pixels each. The background was used to create a distance map, which helped identify and rank the cells by their size at each slice. The centroids of the cells detected at different slices were linked to identify them as a single cell that spanned a number of slices. A subset of these cells, i.e., the largest ones and those not close to the edges were selected for further processing. The selected cells were then automatically cropped to smaller regions of interest of 2000 × 2000 × 300 voxels that were treated as cell instances. Then, for each of these volumes, the nucleus was segmented, and the cell was separated from any neighbouring cells through a series of traditional image processing steps that followed the plasma membrane. The segmentation process was repeated for all the regions of interest previously selected. For one cell for which the ground truth was available, the algorithm provided excellent results in Accuracy (AC) and the Jaccard similarity Index (JI): nucleus: JI , AC , cell including nucleus JI , AC , cell excluding nucleus JI , AC . A limitation of the algorithm for the plasma membrane segmentation was the presence of background. In samples with tightly packed cells, this may not be available. When tested for these conditions, the segmentation of the nuclear envelope was still possible. All the code and data were released openly through GitHub, Zenodo and EMPIAR.

A belt for the cell: cellulosic wall thickenings and their role in morphogenesis of the 3D puzzle cells in walnut shells.

Walnut (Juglans regia) kernels are protected by a tough shell consisting of polylobate sclereids that interlock into a 3D puzzle. The shape transformations from isodiametric to lobed cells is well documented for 2D pavement cells, but not for 3D puzzle sclereids. Here, we study the morphogenesis of these cells by using a combination of different imaging techniques. Serial face-microtomy enabled us to reconstruct tissue growth of whole walnut fruits in 3D, and serial block face-scanning electron microscopy exposed cell shapes and their transformation in 3D during shell tissue development. In combination with Raman and fluorescence microscopy, we revealed multiple loops of cellulosic thickenings in cell walls, acting as stiff restrictions during cell growth and leading to the lobed cell shape. Our findings contribute to a better understanding of the 3D shape transformation of polylobate sclereids and the role of pectin and cellulose within this process.

3D reconstruction of structures of hatched larva and young juvenile of the larvacean Oikopleura dioica using SBF-SEM

The larvacean Oikopleura dioica is a planktonic chordate and an emerging model organism with a short life cycle of 5 days that belongs toTunicata (Urochordata), the sister clade of vertebrates. It is characterized by the rapid development of a tadpole-shaped body. Organ formation in the trunk proceeds within 7 h after the hatching of the tailbud larvae at 3 h after fertilization (hpf) and is completed at 10 hpf, giving rise to fully functional juveniles as miniature adult form. Serial block face scanning electron microscopy was used to acquire ~ 2000 serial transverse section images of a 3 hpf larva and a 10 hpf juvenile to characterize the structures and cellular composition of the trunk and organs using 3D images and movies. Germ cells were found to fuse and establish a central syncytial cell in the gonad as early as 10 hpf. Larval development gave rise to functional organs after several rounds of cell division through trunk morphogenesis. The feature would make O. dioica ideal for analyzing cellular behaviors during morphogenetic processes using live imaging. The detailed descriptions of the larvae and juveniles provided in this study can be utilized as the start and end points of organ morphogenesis in this rapidly developing organism.

A multifaceted analysis reveals two distinct phases of chloroplast biogenesis during de-etiolation in Arabidopsis.

Light triggers chloroplast differentiation whereby the etioplast transforms into a photosynthesizing chloroplast and the thylakoid rapidly emerges. However, the sequence of events during chloroplast differentiation remains poorly understood. Using Serial Block Face Scanning Electron Microscopy (SBF-SEM), we generated a series of chloroplast 3D reconstructions during differentiation, revealing chloroplast number and volume and the extent of envelope and thylakoid membrane surfaces. Furthermore, we used quantitative lipid and whole proteome data to complement the (ultra)structural data, providing a time-resolved, multi-dimensional description of chloroplast differentiation. This showed two distinct phases of chloroplast biogenesis: an initial photosynthesis-enabling 'Structure Establishment Phase' followed by a 'Chloroplast Proliferation Phase' during cell expansion. Moreover, these data detail thylakoid membrane expansion during de-etiolation at the seedling level and the relative contribution and differential regulation of proteins and lipids at each developmental stage. Altogether, we establish a roadmap for chloroplast differentiation, a critical process for plant photoautotrophic growth and survival.

3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina.

The retinal pigment epithelium (RPE) is located between the neuroretina and the choroid, and plays a critical role in vision. RPE cells internalise outer segments (OS) from overlying photoreceptors in the daily photoreceptor renewal. Changes to RPE structure are linked with age and retinopathy, which has been described in the past by conventional 2D electron microscopy. We used serial block face scanning electron microscopy (SBF-SEM) to reconstruct RPE cells from the central mouse retina. Three-dimensional-reconstructed OS revealed the RPE to support large numbers of photoreceptors (90–216 per RPE cell). Larger bi-nucleate RPE maintained more photoreceptors, although their cytoplasmic volume was comparable to smaller mono-nucleate RPE supporting fewer photoreceptors. Scrutiny of RPE microvilli and interdigitating OS revealed the angle and surface area of contact between RPE and photoreceptors. Bi-nucleate RPE contained more mitochondria compared to mono-nucleate RPE. Furthermore, bi-nucleate cells contained larger sub-RPE spaces, supporting a likely association with disease. Use of perfusion-fixed tissues ensured the highest possible standard of preservation, providing novel insights into the 3D RPE architecture and changes linked with retinopathy. This study serves as a benchmark for comparing retinal tissues from donor eyes with age-related macular degeneration (AMD) and other retinopathies.

Bleeding Cessation in a Mouse Jugular Vein Puncture Wound Model Is Caused By Extravascular Capping, Not Hole Infill

Introduction: Vascular damage comes in many forms with the puncture wound likely the longest known to humans. Experimentally, vascular damage has typically been visualized in mouse models in which there is little bleeding. Under these conditions, damage is limited mostly to the endothelial layer lining the vessel. Visualization has varied from light to scanning electron microscopy. Interpretation has been dominated by intravital microscopy outcomes in which an initial layer of p-selection-exposed, i.e., α-granule secretion-positive, platelets is deposited in association with the damaged vessel wall and extended by the accumulation of a less activated outer layer of platelets. These results have given rise to a Core and Shell model of platelet-rich thrombus formation [Tomaiuolo et al., 2017]. Recently, new mouse models have been presented in which the vessel is punctured to create a 300 to 600 micron wound hole [Tomoiuolo et al., 2019]. Bleeding is now profuse. The puncture wound results have been interpreted within a Core and Shell model. However, two important aspects of the experimental data [Tomoiuolo et al., 2019] suggest that the existing model may not explain the actual results. First, p-selectin expression as a marker for α-granule secretion and platelet activation was present in limited areas towards the periphery of the resulting thrombus, not as well-defined Core. Second, the hemostatic thrombus when viewed at early stages, ex vivo, showed a pebbly distribution of platelet aggregates suggestive of nucleated platelet accumulation rather than the smooth layers that would follow from a Core and Shell model. We hypothesize that nucleated accumulation of platelet aggregates within the puncture hole could provide pedestals upon which localized accumulation of platelets form the infrastructure of a vaulted thrombus whose extravascular capping leads to bleeding cessation. Methods: To test the proposed hypothesis, we visualized the interior and overall structure of the forming puncture wound thrombus in full 3D at sub-platelet level resolution. To achieve this end, we took our proven serial block face scanning electron microscopy (SBF-SEM) protocols for visualizing platelet organelles in 3D [Pokrovskaya et al., 2018] and adapted them to the visualization of forming thrombi over 1000s of image. To localize samples for electron microscopy, we used in vivo antibody labeling [Tomaiuolo et al., 2019]. This approach had the added advantage of enabling correlative light microscopy mapping overall p-selectin, a marker of platelet secretion, and fibrin distributions against 3D, platelet resolution, thrombus morphology. Results: We found that a 1 min thrombus, pre-bleeding cessation, was structured about the localized accumulation of pedestal-like platelet aggregates along the sides of the puncture hole, extended and spaced along the extravascular adventitia. Subsequent pedestal extension formed a "pontoon" bridge that "capped" extravascularly the puncture hole. At 5 min, full bleeding cessation, we found that forming platelet thrombus had a Swiss cheese-like interior of vaults that were continuous with the intravascular vessel lumen and framed by columns of platelets, presumed pedestal extensions. The thrombus was sealed on the extravascular side by a platelet "cap" (Figure). As expected after bleeding cessation, red blood cells accumulated on the intravascular side of the cap. Formation of a tightly sealed cap was dependent on α-granule secretion as indicated by the effect of knockout of VAMP-8, the primary SNARE protein involved in a-granule release. Based upon morphology, vaults within the forming thrombus were lined with apparent procoagulant platelets providing a potential protected surface for coagulation factor activation. Conclusions: We conclude that bleeding cessation in a true puncture wound occurs from the extravascular side of the thrombus rather than through the formation of a platelet plug that fills the hole. We propose an alternative model of bleeding cessation in which localized platelet aggregates are the starting pedestal upon which all subsequent steps in puncture thrombus formation build, i.e., "Cap and Build". The extent to which properties differ among systems remains an open question. Tomaiuolo et al. 2017. Intervent. Cardiol. Clin. 6: 1-12. Pokrovskaya et al. 2018. Blood Adv. 2: 2947-2958 Tomaiuolo et al. 2019. Proc. Natl. Acad. Sci. USA 116:2 243-2252 Figure Disclosures No relevant conflicts of interest to declare.

Quantitative assessment of dentine mineralization and tubule occlusion by NovaMin and stannous fluoride using serial block face scanning electron microscopy

Dentine hypersensitivity (DH) is one of the most common dental conditions affecting most adults during their lifetime. Tubule occlusion is a widely accepted method for treating DH. Current in-vitro techniques such as focused ion beam, scanning electron microscopy (SEM), or hydraulic conductance that are used to determine tubule occlusion do not provide the depth of occlusion, are time-consuming, expensive and the volume of dentine tested is limited. The presented study aimed to assess the ability of serial block-face SEM (SBF-SEM) to section dentine, to quantify the number of occluded tubules including the depth of penetration by NovaMin and stannous fluoride (SnF2 ) and to compare mineral density between the control and treated dentine. Results demonstrated that NovaMin provided a better occlusion with 100% of the tubules blocked at the surface compared to 83% for SnF2 . The grayscale value (230.42) was significantly higher (p ≤ 0.05) after treatment with NovaMin compared to SnF2 (222.06) and the control (196.37), indicating increased mineral density and dentine mineralization. SBF-SEM has the potential to be used for large volume analysis of bone-like materials at high resolution with minimal sample preparation over a short period. It can be significantly useful in the development and research of new biomaterials.

The Growth of Eimeria tenella: Characterization and Application of Quantitative Methods to Assess Sporozoite Invasion and Endogenous Development in Cell Culture.

In vitro development of the complete life cycle of Eimeria species has been achieved in primary cultures of avian epithelial cells with low efficiency. The use of immortalized cell lines simplifies procedures but only allows partial development through one round of parasite invasion and intracellular replication. We have assessed the suitability of Madin-Darby Bovine Kidney (MDBK) cells to support qualitative and quantitative studies on sporozoite invasion and intracellular development of Eimeria tenella. Analysis of parasite ultrastructure by transmission electron microscopy and serial block face—scanning electron microscopy proved the suitability of the system to generate good quality schizonts and first-generation merozoites. Parasite protein expression profiles elucidated by mass spectrometry corroborated previous findings occurring during the development of the parasite such as the presence of alternative types of surface antigen at different stages and increased abundance of proteins from secretory organelles during invasion and endogenous development. Quantitative PCR (qPCR) allowed the tracking of development by detecting DNA division, whereas reverse transcription qPCR of sporozoite- and merozoite-specific genes could detect early changes before cell division and after merozoite formation, respectively. These results correlated with the analysis of development using ImageJ semi-automated image analysis of fluorescent parasites, demonstrating the suitability and reproducibility of the MDBK culture system. This systems also allowed the evaluation of the effects on invasion and development when sporozoites were pre-incubated with anticoccidial drugs, showing similar effects to those reported before. We have described through this study a series of methods and assays for the further application of this in vitro culture model to more complex studies of Eimeria including basic research on parasite cell biology and host-parasite interactions and for screening anticoccidial drugs.

Semantic segmentation of HeLa cells: An objective comparison between one traditional algorithm and four deep-learning architectures.

The quantitative study of cell morphology is of great importance as the structure and condition of cells and their structures can be related to conditions of health or disease. The first step towards that, is the accurate segmentation of cell structures. In this work, we compare five approaches, one traditional and four deep-learning, for the semantic segmentation of the nuclear envelope of cervical cancer cells commonly known as HeLa cells. Images of a HeLa cancer cell were semantically segmented with one traditional image-processing algorithm and four three deep learning architectures: VGG16, ResNet18, Inception-ResNet-v2, and U-Net. Three hundred slices, each 2000 × 2000 pixels, of a HeLa Cell were acquired with Serial Block Face Scanning Electron Microscopy. The first three deep learning architectures were pre-trained with ImageNet and then fine-tuned with transfer learning. The U-Net architecture was trained from scratch with 36, 000 training images and labels of size 128 × 128. The image-processing algorithm followed a pipeline of several traditional steps like edge detection, dilation and morphological operators. The algorithms were compared by measuring pixel-based segmentation accuracy and Jaccard index against a labelled ground truth. The results indicated a superior performance of the traditional algorithm (Accuracy = 99%, Jaccard = 93%) over the deep learning architectures: VGG16 (93%, 90%), ResNet18 (94%, 88%), Inception-ResNet-v2 (94%, 89%), and U-Net (92%, 56%).

Observations on nascent matrix structures in embryonic cornea: Important in cell interactions, or merely vestiges of the lens surface?

Here we present some new observations on early stages in chick corneal development obtained by remining of datasets obtained via serial block face scanning electron microscopy. We focus on matrix cords, proteoglycan-rich structures of apparent ectodermal origin, emerging from the epithelial basal lamina, which extend proximally into the growing collagenous matrix destined to become the corneal stroma. Cords have no known function. In their earliest manifestation, we describe how they appear to run continuously from epithelium to the lens, in contact with both tissues and may therefore be simply vestigial structures, remaining from the earlier detachment of the lens from its parent ectoderm. However, neural crest cells migrating to form the corneal endothelial monolayer appear to form close associations with cords via elaborate pseudopodial extensions. Presumptive endothelium and keratocytes, in the subsequent wave of neural crest cell influx, may conceivably utilise cords, as well as utilising collagenous fibrils of the interstitial matrix, as substrate cues in cell guidance, attachment and migration. The possibility also exists that cords fulfil a functional role in corneal morphogenesis via mechanotransduction through cell matrix interactions.