Serial Block-face Electron Microscopy Research Articles

Atypical flagella assembly and haploid genome coiling during male gamete formation in Plasmodium

Gametogenesis in Plasmodium spp. occurs within the Anopheles mosquito and is essential for sexual reproduction / differentiation and onwards transmission to mammalian hosts. To better understand the 3D organisation of male gametogenesis, we used serial block face scanning electron microscopy (SBF-SEM) and serial-section cellular electron tomography (ssET) of P. berghei microgametocytes to examine key structures during male gamete formation. Our data reveals an elaborate organisation of axonemes coiling around the nucleus in opposite directions forming a central axonemal band in microgametocytes. Furthermore, we discover the nucleus of microgametes to be tightly coiled around the axoneme in a complex structure whose formation starts before microgamete emergence during exflagellation. Our discoveries of the detailed 3D organisation of the flagellated microgamete and the haploid genome highlight some of the atypical mechanisms of axoneme assembly and haploid genome organisation during male gamete formation in the malaria parasite.

A high-throughput 3D X-ray histology facility for biomedical research and preclinical applications

Background The University of Southampton, in collaboration with the University Hospital Southampton (UHS) NHS Foundation Trust and industrial partners, has been at the forefront of developing three-dimensional (3D) imaging workflows using X-ray microfocus computed tomography (μCT) -based technology. This article presents the outcomes of these endeavours and highlights the distinctive characteristics of a μCT facility tailored explicitly for 3D X-ray Histology, with a primary focus on applications in biomedical research and preclinical and clinical studies. Methods The UHS houses a unique 3D X-ray Histology (XRH) facility, offering a range of services to national and international clients. The facility employs specialised μCT equipment explicitly designed for histology applications, allowing whole-block XRH imaging of formalin-fixed and paraffin-embedded tissue specimens. It also enables correlative imaging by combining μCT imaging with other microscopy techniques, such as immunohistochemistry (IHC) and serial block-face scanning electron microscopy, as well as data visualisation, image quantification, and bespoke analysis. Results Over the past seven years, the XRH facility has successfully completed over 120 projects in collaboration with researchers from 60 affiliations, resulting in numerous published manuscripts and conference proceedings. The facility has streamlined the μCT imaging process, improving productivity and enabling efficient acquisition of 3D datasets. Discussion & Conclusions The 3D X-ray Histology (XRH) facility at UHS is a pioneering platform in the field of histology and biomedical imaging. To the best of our knowledge, it stands out as the world's first dedicated XRH facility, encompassing every aspect of the imaging process, from user support to data generation, analysis, training, archiving, and metadata generation. This article serves as a comprehensive guide for establishing similar XRH facilities, covering key aspects of facility setup and operation. Researchers and institutions interested in developing state-of-the-art histology and imaging facilities can utilise this resource to explore new frontiers in their research and discoveries.

Size and shape analysis of micro- to nano-particles of quartz powders using advanced electron microscopy and laser diffraction methods

The paper deals with particle analysis and the evaluation of the shape and size distribution of micro- to nano-sized quartz particles as a key parameter for the evaluation of quality in many industrial fields, particularly for high-purity applications. Samples are analyzed by laser diffraction, the most commonly used method for determining the size of the particles; for precise measurement with the possibility of high-resolution imaging of particles, scanning electron microscopy was applied. For morphological and size analysis of electrically non-conductive micro- to nano-particles under environmentally compatible conditions of elevated gas pressure and humidity, environmental scanning electron microscopy was selected as the most suitable. Moreover, the high-end methods of focused ion beam scanning electron microscopy and serial block-face scanning electron microscopy enabled the analysis of particles in the epoxy resin volume including spatial imaging. The application of image post-processing as an important part of the electron microscopy workflow is also discussed.

Partial inhibition of complex I restores mitochondrial morphology and mitochondria‐ER communication in hippocampus of APP/PS1 mice

AbstractBackgroundAlzheimer’s Disease (AD) has no cure. We showed that partial inhibition of mitochondrial complex I (MCI) with small molecule CP2 induced adaptive stress response activating multiple neuroprotective mechanisms. Chronic treatment reduced inflammation, improved synaptic and mitochondrial functions, and blocked neurodegeneration in symptomatic APP/PS1 mice, a translational model of AD.MethodsAnalysis of mitochondrial morphology and mitochondria‐endoplasmic reticulum (ER) contacts (MERCS) was done in the hippocampal CA1 tissue from APP/PS1 mice treated with CP2 and compared to APP/PS1 and non‐transgenic mice treated with vehicle using serial block‐face scanning electron microscopy (SBFSEM) and three‐dimensional (3D) EM reconstructions combined with western blot analysis and next generation RNA sequencing.ResultsWe demonstrate that CP2 treatment restores mitochondrial morphology and mitochondria‐ ER communication, reducing ER stress in the APP/PS1 mouse brain. Using 3D EM reconstructions, we found that mitochondria in AD dendrites existed primarily as mitochondria‐on‐a‐string (MOAS), a morphological phenotype that is difficult to detect using conventional 2D EM or fluorescence spectroscopy. Compared to other morphological phenotypes, MOAS are extensively enveloped in the ER membranes forming multiple MERCS known to contribute to abnormal lipid and calcium homeostasis, and ER stress in AD. CP2 treatment specifically reduced MOAS formation, consistent with improved energy homeostasis in the brain, with concomitant reduction in MERCS, ER stress, and improved lipid homeostasis.ConclusionsThese data provide novel information on the role MOAS play in AD. Our data demonstrate that in response to energetic stress, mitochondria in dendrites acquire MOAS phenotype with increased interaction with the ER, which may synergistically contribute to the AD development. Since CP2 treatment alleviated the ER stress and improved mitochondrial dynamics and function, these data further support the development of partial MCI inhibitors as disease modifying strategy for AD.

Modeling the 3-dimensional structure of the silkworm's spinning apparatus in silk production

The silk-spinning process of the silkworms transforms the liquid silk solution to a solid state under mild conditions, making it an attractive model for bioinspiration However, the precise mechanism behind silk expulsion remains largely unknown. Here we selected the silkworms as representative models to investigate the silk-spinning mechanism. We used serial block-face scanning electron microscopy (SBF-SEM) to reconstruct the three-dimensional structures of the spinnerets in silkworms at various stages and with different gene backgrounds. By comparing the musculature and duct deformation of these spinneret models during the spinning process, we were able to simulate the morphological changes of the spinneret. Based on the results, we proposed three essential factors for silkworm spinning: the pressure generated by the silk gland, the opening duct, and the pulling force generated by head movement. Understanding the silkworm spinning process provides insights into clarify the fluid-ejecting mechanism of a group of animals. Moreover, these findings are helpful to the development of biomimetic spinning device that mimics the push-and-pull dual-force system in silkworms. Statement of significanceThe silkworms’ spinning system produces fibers under mild conditions, making it an ideal candidate for bioinspiration. However, the mechanism of silk expulsion is unknown, and the three-dimensional structure of the spinneret is still uncertain. In this study, we reconstructed a detailed 3-dimensional model of the spinneret at near-nanometer resolution, and for the first time, we observed the changes that occur before and during the silk-spinning process. Our reconstructed models suggested that silkworms have the ability to control the spinning process by opening or closing the spinning duct. During the continuously spinning period, both the pressure generated by the silk gland and the pulling force resulting from head movement work in tandem to expel the silk solution. We believe that gaining a full understanding of the spinning process steps can advance our ability to spin synthetic fibers with properties comparable to those of native fibers by mimicking the natural spinning process.

3D reconstructions of parasite development and the intracellular niche of the microsporidian pathogen Encephalitozoon intestinalis

Microsporidia are an early-diverging group of fungal pathogens with a wide host range. Several microsporidian species cause opportunistic infections in humans that can be fatal. As obligate intracellular parasites with highly reduced genomes, microsporidia are dependent on host metabolites for successful replication and development. Our knowledge of microsporidian intracellular development remains rudimentary, and our understanding of the intracellular niche occupied by microsporidia has relied on 2D TEM images and light microscopy. Here, we use serial block-face scanning electron microscopy (SBF-SEM) to capture 3D snapshots of the human-infecting species, Encephalitozoon intestinalis, within host cells. We track E. intestinalis development through its life cycle, which allows us to propose a model for how its infection organelle, the polar tube, is assembled de novo in developing spores. 3D reconstructions of parasite-infected cells provide insights into the physical interactions between host cell organelles and parasitophorous vacuoles, which contain the developing parasites. The host cell mitochondrial network is substantially remodeled during E. intestinalis infection, leading to mitochondrial fragmentation. SBF-SEM analysis shows changes in mitochondrial morphology in infected cells, and live-cell imaging provides insights into mitochondrial dynamics during infection. Our data provide insights into parasite development, polar tube assembly, and microsporidia-induced host mitochondria remodeling.

Probing the in situ volumes of Arabidopsis leaf plastids using three-dimensional confocal and scanning electron microscopy.

Leaf plastids harbor a plethora of biochemical reactions including photosynthesis, one of the most important metabolic pathways on Earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols delivering in situ volumes based on: (i) chlorophyll fluorescence emerging from the thylakoid membrane, (ii) a CFP marker embedded in the envelope, and (iii) calculations from serial block-face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild-type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches, the average volume of a mature Col-0 wild-type mesophyll chloroplasts is 93 μm3 . Wild-type guard cell plastids are approximately 18 μm3 . Lastly, our comparative analysis shows that the chlorophyll fluorescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or costly SBFSEM equipment.

High Fat Diet-Induced Obesity Dysregulates Splenic B Cell Mitochondrial Activity.

Diet-induced obesity impairs mitochondrial respiratory responses in tissues that are highly metabolically active, such as the heart. However, less is known about the impact of obesity on the respiratory activity of specific cell types, such as splenic B cells. B cells are of relevance, as they play functional roles in obesity-induced insulin resistance, inflammation, and responses to infection. Here, we tested the hypothesis that high-fat-diet (HFD)-induced obesity could impair the mitochondrial respiration of intact and permeabilized splenic CD19+ B cells isolated from C57BL/6J mice and activated ex vivo with lipopolysaccharide (LPS). High-resolution respirometry was used with intact and permeabilized cells. To reveal potential mechanistic targets by which HFD-induced obesity dysregulates B cell mitochondria, we conducted proteomic analyses and 3D serial block face scanning electron microscopy (SBFEM). High-resolution respirometry revealed that intact LPS-stimulated B cells of obese mice, relative to controls, displayed lower ATP-linked, as well as maximal uncoupled, respiration. To directly investigate mitochondrial function, we used permeabilized LPS-stimulated B cells, which displayed increased H2O2 emission and production with obesity. We also examined oxidative phosphorylation efficiency simultaneously, which revealed that oxygen consumption and ATP production were decreased in LPS-stimulated B cells with obesity relative to controls. Despite minimal changes in total respiratory complex abundance, in LPS-stimulated B cells of obese mice, three of the top ten most downregulated proteins were all accessory subunits of respiratory complex I. SBFEM showed that B cells of obese mice, compared to controls, underwent no change in mitochondrial cristae integrity but displayed increased mitochondrial volume that was linked to bioenergetic function. Collectively, these results establish a proof of concept that HFD-induced obesity dysregulates the mitochondrial bioenergetic metabolism of activated splenic B cells.

3D reconstruction of murine mitochondria reveals changes in structure during aging linked to the MICOS complex.

During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized. We measured changes in mitochondrial morphology in aged murine gastrocnemius, soleus, and cardiac tissues using serial block-face scanning electron microscopy and 3D reconstructions. We also used reverse transcriptase-quantitative PCR, transmission electron microscopy quantification, Seahorse analysis, and metabolomics and lipidomics to measure changes in mitochondrial morphology and function after loss of mitochondria contact site and cristae organizing system (MICOS) complex genes, Chchd3, Chchd6, and Mitofilin. We identified significant changes in mitochondrial size in aged murine gastrocnemius, soleus, and cardiac tissues. We found that both age-related loss of the MICOS complex and knockouts of MICOS genes in mice altered mitochondrial morphology. Given the critical role of mitochondria in maintaining cellular metabolism, we characterized the metabolomes and lipidomes of young and aged mouse tissues, which showed profound alterations consistent with changes in membrane integrity, supporting our observations of age-related changes in muscle tissues. We found a relationship between changes in the MICOS complex and aging. Thus, it is important to understand the mechanisms that underlie the tissue-dependent 3D mitochondrial phenotypic changes that occur in aging and the evolutionary conservation of these mechanisms between Drosophila and mammals.

Implementing new computational methods for the study of JCT and SC inner wall basement membrane biomechanics and hydrodynamics

PurposeThe conventional aqueous outflow pathway, which includes the trabecular meshwork (TM), juxtacanalicular tissue (JCT), and the inner wall endothelium of Schlemm's canal (SC), regulates intraocular pressure (IOP) by controlling the aqueous humor outflow resistance. Despite its importance, our understanding of the biomechanics and hydrodynamics within this region remains limited. Fluid-structure interaction (FSI) offers a way to estimate the biomechanical properties of the JCT and SC under various loading and boundary conditions, providing valuable insights that are beyond the reach of current imaging techniques. MethodsIn this study, a normal human eye was fixed at a pressure of 7 mm Hg, and two radial wedges of the TM tissues, which included the SC inner wall basement membrane and JCT, were dissected, processed, and imaged using 3D serial block-face scanning electron microscopy (SBF-SEM). Four different sets of images were used to create 3D finite element (FE) models of the JCT and inner wall endothelial cells of SC with their basement membrane. The outer JCT portion was carefully removed as the outflow resistance is not in that region, leaving only the SCE inner wall and a few µm of the tissue, which does contain the resistance. An inverse iterative FE algorithm was then utilized to calculate the unloaded geometry of the JCT/SC complex at an aqueous humor pressure of 0 mm Hg. Then in the model, the intertrabecular spaces, pores, and giant vacuole contents were replaced by aqueous humor, and FSI was employed to pressurize the JCT/SC complex from 0 to 15 mm Hg. ResultsIn the JCT/SC complex, the shear stress of the aqueous humor is not evenly distributed. Areas proximal to the inner wall of SC experience larger stresses, reaching up to 10 Pa, while those closer to the JCT undergo lower stresses, approximately 4 Pa. Within this complex, giant vacuoles with or without I-pore behave differently. Those without I-pores experience a more significant strain, around 14%, compared to those with I-pores, where the strain is roughly 9%. ConclusionsThe distribution of aqueous humor wall shear stress is not uniform within the JCT/SC complex, which may contribute to our understanding of the underlying selective mechanisms in the pathway.

A Novel Sandwich Method for Serial Block Face SEM Imaging of Airway Multiciliated Epithelium.

Volume electron microscopy technologies such as serial block face scanning electron microscopy (SBF-SEM) allow the characterization of tissue organization and cellular content in three dimensions at nanoscale resolution. Here, we describe the procedure to process and image an air-liquid interface culture of human or mouse airway epithelial cells for visualization of the multiciliated epithelium by SBF-SEM in vertical or horizontal cross section.

Call to action to properly utilize electron microscopy to measure organelles to monitor disease

This review provides an overview of the current methods for quantifying mitochondrial ultrastructure, including cristae morphology, mitochondrial contact sites, and recycling machinery and a guide to utilizing electron microscopy to effectively measure these organelles. Quantitative analysis of mitochondrial ultrastructure is essential for understanding mitochondrial biology and developing therapeutic strategies for mitochondrial-related diseases. Techniques such as transmission electron microscopy (TEM) and serial block face-scanning electron microscopy, as well as how they can be combined with other techniques including confocal microscopy, super-resolution microscopy, and correlative light and electron microscopy are discussed. Beyond their limitations and challenges, we also offer specific magnifications that may be best suited for TEM analysis of mitochondrial, endoplasmic reticulum, and recycling machinery. Finally, perspectives on future quantification methods are offered.

Beyond the surface: Investigation of tumorsphere morphology using volume electron microscopy

The advent of volume electron microscopy (vEM) has provided unprecedented insights into cellular and subcellular organization, revolutionizing our understanding of cancer biology. This study presents a previously unexplored comparative analysis of the ultrastructural disparities between cancer cells cultured as monolayers and tumorspheres. By integrating a robust workflow that incorporates high-pressure freezing followed by freeze substitution (HPF/FS), serial block face scanning electron microscopy (SBF-SEM), manual and deep learning-based segmentation, and statistical analysis, we have successfully generated three-dimensional (3D) reconstructions of monolayer and tumorsphere cells, including their subcellular organelles. Our findings reveal a significant degree of variation in cellular morphology in tumorspheres. We observed the increased prevalence of nuclear envelope invaginations in tumorsphere cells compared to monolayers. Furthermore, we detected a diverse range of mitochondrial morphologies exclusively in tumorsphere cells, as well as intricate cellular interconnectivity within the tumorsphere architecture. These remarkable ultrastructural differences emphasize the use of tumorspheres as a superior model for cancer research due to their relevance to in vivo conditions. Our results strongly advocate for the utilization of tumorsphere cells in cancer research studies, enhancing the precision and relevance of experimental outcomes, and ultimately accelerating therapeutic advancements.

Ultrastructural Characteristics and Synaptic Connectivity of Photoreceptors in the Simplex Retina of Little Skate (Leucoraja erinacea).

The retinas of the vast majority of vertebrate species are termed "duplex," that is, they contain both rod and cone photoreceptor neurons in different ratios. The retina of little skate (Leucoraja erinacea) is a rarity among vertebrates because it contains only a single photoreceptor cell type and is thus "simplex." This unique retina provides us with an important comparative model and an exciting opportunity to study retinal circuitry within the context of a visual system with a single photoreceptor cell type. What is perhaps even more intriguing is the fact that the Leucoraja retina is able use that single photoreceptor cell type to function under both scotopic and photopic ranges of illumination. Although some ultrastructural characteristics of skate photoreceptors have been examined previously, leading to a general description of them as "rods" largely based on outer segment (OS) morphology and rhodopsin expression, a detailed study of the fine anatomy of the entire cell and its synaptic connectivity is still lacking. To address this gap in knowledge, we performed serial block-face electron microscopy imaging and examined the structure of skate photoreceptors and their postsynaptic partners. We find that skate photoreceptors exhibit unusual ultrastructural characteristics that are either common to rods or cones in other vertebrates (e.g., outer segment architecture, synaptic ribbon number, terminal extensions), or are somewhere in between those of a typical vertebrate rod or cone (e.g., number of invaginating contacts, clustering of multiple ribbons over a single synaptic invagination). We suggest that some of the ultrastructural characteristics we observe may play a role in the ability of the skate retina to function across scotopic and photopic ranges of illumination. Our findings have the potential to reveal as yet undescribed principles of vertebrate retinal design.

Ultrastructure and 3D reconstruction of a diplonemid protist (Diplonemea) and its novel membranous organelle.

The knowledge of cell biology of a eukaryotic group is essential for correct interpretation of ecological and molecular data. Although diplonemid protists are one of the most species-rich lineages of marine eukaryotes, only very fragmentary information is available about the cellular architecture of this taxonomically diverse group. Here, a large serial block-face scanning electron microscopy data set complemented with light and fluorescence microscopy allowed the first detailed three-dimensional reconstruction of a diplonemid species. We describe numerous previously unknown peculiarities of the cellular architecture and cell division characteristic for diplonemid flagellates, and illustrate the obtained results with multiple three-dimensional models, comprehensible for non-specialists in protist ultrastructure.

Bacterial contact induces polar plug disintegration to mediate whipworm egg hatching.

The bacterial microbiota promotes the life cycle of the intestine-dwelling whipworm Trichuris by mediating hatching of parasite eggs ingested by the mammalian host. Despite the enormous disease burden associated with Trichuris colonization, the mechanisms underlying this transkingdom interaction have been obscure. Here, we used a multiscale microscopy approach to define the structural events associated with bacteria-mediated hatching of eggs for the murine model parasite Trichuris muris. Through the combination of scanning electron microscopy (SEM) and serial block face SEM (SBFSEM), we visualized the outer surface morphology of the shell and generated 3D structures of the egg and larva during the hatching process. These images revealed that exposure to hatching-inducing bacteria catalyzed asymmetric degradation of the polar plugs prior to exit by the larva. Unrelated bacteria induced similar loss of electron density and dissolution of the structural integrity of the plugs. Egg hatching was most efficient when high densities of bacteria were bound to the poles. Consistent with the ability of taxonomically distant bacteria to induce hatching, additional results suggest chitinase released from larva within the eggs degrade the plugs from the inside instead of enzymes produced by bacteria in the external environment. These findings define at ultrastructure resolution the evolutionary adaptation of a parasite for the microbe-rich environment of the mammalian gut.

Cell-Cell and Cell-Matrix Interactions at the Presumptive Stem Cell Niche of the Chick Corneal Limbus.

(1) Background: Owing to its ready availability and ease of acquisition, developing chick corneal tissue has long been used for research purposes. Here, we seek to ascertain the three-dimensional microanatomy and spatiotemporal interrelationships of the cells (epithelial and stromal), extracellular matrix, and vasculature at the corneo-scleral limbus as the site of the corneal stem cell niche of the chicken eye. (2) Methods: The limbus of developing (i.e., embryonic days (E) 16 and 18, just prior to hatch) and mature chicken eyes was imaged using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and the volume electron microscopy technique, serial-block face SEM (SBF-SEM), the latter technique allowing us to generate three-dimensional reconstructions from data sets of up to 1000 serial images; (3) Results: Data revealed that miniature limbal undulations of the embryonic basement membrane, akin to Palisades of Vogt (PoV), matured into distinct invaginations of epithelial cells that extended proximally into a vascularized limbal stroma. Basal limbal epithelial cells, moreover, occasionally exhibited a high nuclear:cytoplasmic ratio, which is a characteristic feature of stem cells. SBF-SEM identified direct cell-cell associations between corneal epithelial and stromal cells at the base of structures akin to limbal crypts (LCs), with cord-like projections of extracellular matrix extending from the basal epithelial lamina into the subjacent stroma, where they made direct contact with stomal cells in the immature limbus. (4) Conclusion: Similarities with human tissue suggest that the corneal limbus of the mature chicken eye is likely the site of a corneal stem cell niche. The ability to study embryonic corneas pre-hatch, where we see characteristic niche-like features emerge, thus provides an opportunity to chart the development of the limbal stem cell niche of the cornea.

Biomechanics of the JCT and SC Inner Wall Endothelial Cells with Their Basement Membrane Using 3D Serial Block-Face Scanning Electron Microscopy.

More than ~70% of the aqueous humor exits the eye through the conventional aqueous outflow pathway that is comprised of the trabecular meshwork (TM), juxtacanalicular tissue (JCT), the inner wall endothelium of Schlemm's canal (SC). The flow resistance in the JCT and SC inner wall basement membrane is thought to play an important role in the regulation of the intraocular pressure (IOP) in the eye, but current imaging techniques do not provide enough information about the mechanics of these tissues or the aqueous humor in this area. A normal human eye was perfusion-fixed and a radial wedge of the TM tissue from a high-flow region was dissected. The tissues were then sliced and imaged using serial block-face scanning electron microscopy. Slices from these images were selected and segmented to create a 3D finite element model of the JCT and SC cells with an inner wall basement membrane. The aqueous humor was used to replace the intertrabecular spaces, pores, and giant vacuoles, and fluid-structure interaction was employed to couple the motion of the tissues with the aqueous humor. Higher tensile stresses (0.8-kPa) and strains (25%) were observed in the basement membrane beneath giant vacuoles with open pores. The volumetric average wall shear stress was higher in SC than in JCT/SC. As the aqueous humor approached the inner wall basement membrane of SC, the velocity of the flow decreased, resulting in the formation of small eddies immediately after the flow left the inner wall. Improved modeling of SC and JCT can enhance our understanding of outflow resistance and funneling. Serial block-face scanning electron microscopy with fluid-structure interaction can achieve this, and the observed micro-segmental flow patterns in ex vivo perfused human eyes suggest a hypothetical mechanism.

Minimal resin embedding of SBF-SEM samples reduces charging and facilitates finding a surface-linked region of interest

BackgroundFor decoding the mechanism of how cells and organs function information on their ultrastructure is essential. High-resolution 3D imaging has revolutionized morphology. Serial block face scanning electron microscopy (SBF-SEM) offers non-laborious, automated imaging in 3D of up to ~ 1 mm3 large biological objects at nanometer-scale resolution. For many samples there are obstacles. Quality imaging is often hampered by charging effects, which originate in the nonconductive resin used for embedding. Especially, if the imaged region of interest (ROI) includes the surface of the sample and neighbours the empty resin, which insulates the object. This extra resin also obscures the sample’s morphology, thus making navigation to the ROI difficult.ResultsUsing the example of small arthropods and a fish roe we describe a workflow to prepare samples for SBF-SEM using the minimal resin (MR) embedding method. We show that for imaging of surface structures this simple approach conveniently tackles and solves both of the two major problems—charging and ROI localization—that complicate imaging of SBF-SEM samples embedded in an excess of overlying resin. As the surface ROI is not masked by the resin, samples can be precisely trimmed before they are placed into the imaging chamber. The initial approaching step is fast and easy. No extra trimming inside the microscope is necessary. Importantly, charging is absent or greatly reduced meaning that imaging can be accomplished under good vacuum conditions, typically at the optimal high vacuum. This leads to better resolution, better signal to noise ratio, and faster image acquisition.ConclusionsIn MR embedded samples charging is minimized and ROI easily targeted. MR embedding does not require any special equipment or skills. It saves effort, microscope time and eventually leads to high quality data. Studies on surface-linked ROIs, or any samples normally surrounded by the excess of resin, would benefit from adopting the technique.

The three-dimensional conformation and activity of mitochondria in syncytial male germ line-cysts of medicinal leeches

We studied the spatial conformation and activity of mitochondria in the developing syncytial male germline cysts during spermatogenesis of the medicinal leeches using light, fluorescent, transmission electron microscopy, and serial block-face scanning electron microscopy. In cysts with spermatogonia and spermatocytes, mitochondria form networks and are in a dynamic hyperfusion state, while in cysts with spermatids, a single huge mitochondrion is observed. As spermiogenesis progresses, this huge mitochondrion is finally located in the future midpiece. The highest activity, in terms of membrane potential, of the mitochondria in H. medicinalis germline cysts was observed in cysts with spermatocytes; the lowest was in cysts with late elongated spermatids.