Embedment-free Sections Research Articles

Electron-translucency and partial defects of synaptic basal lamina in the electrocyte synapse of an electric ray (Narke japonica) in 3D embedment-free section electron microscopy.

The synaptic basal lamina of the electrocytes was disclosed to be electron-translucent to some extent when viewed in an en-face direction in embedment-free section transmission electron microscopy (EFS-TEM), and synaptic vesicles located close to the presynaptic membrane were seen through the synaptic basal lamina together with the presynaptic and postsynaptic membranes. This feature of translucency has the potential to analyze possible spatial interrelations in situ between bioactive molecules in the synaptic basal lamina and the synaptic vesicles in further studies. The synaptic basal lamina, appearing as an electron-dense line sandwiched by two parallel lines representing the presynaptic and postsynaptic membranes in ultrathin sections cut right to the synaptic junctional plane in conventional TEM, was not fully continuous but randomly intermittent along its trajectory. Compatible with the intermittent line appearance, the en-face 3D view in embedment-free section TEM revealed for the first time partial irregular defects of the synaptic basal lamina. Considering the known functional significance of several molecules contained in the synaptic basal lamina in the maintenance and exertion of the synapse, its partial defects may not represent its rigid structural features, but its immature structure under remodeling or its dynamic changes in consistency such as the sol/gel transition, whose validity needs further examination. RESEARCH HIGHLIGHTS: In embedment-free section TEM, a 3D en-face view of synaptic basal lamina in situ is reliably possible. The basal lamina en-face is electron-translucent, which makes it possible to analyze spatial interrelation between pre- and post-synaptic components. Partial irregular defects in the basal lamina are revealed in Torpedo electrocytes, suggesting its remodeling or dynamic changes in consistency.

Occurrence of fenestral diaphragms and knots in renal glomerular endothelia of diabetic mutant MafA-/-MafK+ mice as revealed in embedment-free transmission electron microscopy.

Using the advantages (high contrast and transparency and efficient 3D viewing) of embedment-free section transmission electron microscopy (TEM), the occurrence of numerous fenestral diaphragms was clearly shown in 3D en-face viewing of the renal glomerular capillary endothelium of severe overt diabetes mellitus mice, which were generally MafA-deficient and simultaneously MafK-overexpressed specifically in pancreatic β-cells. This presents another example of nephritis-induced diaphragmed fenestrae in the renal glomerular endothelium. In addition, knot-/umbilicus-like structures discrete from and larger than the central knots of regular diaphragms of fenestrated endothelium were clearly demonstrated to occur randomly in the renal glomerular endothelial fenestrae of mutant mice and wild ones. The knot-structures were revealed to be protrusions of underlining basement lamina in conventional TEM by section-tilting observation.

Advantages of embedment-free section transmission electron microscopy

The usefulness of embedment-free section transmission electron microscopy (TEM) is stressed for present and future morphological analyses, and several examples are demonstrated which are revealed in sections for the first time by this method: en-face views of slit diaphragm of renal glomerulus and fenestrated diaphragm of capillary endothelium, transparency of neural myelin, attenuated endothelium and some basement laminae, labyrinth architecture of vacuoles within lipid droplets, and enhanced 3D effect of ultrastructures, the latter of which is the case in electron tomography. In addition, the biological significance of structured appearance (microtrabecular lattices) of the cytoplasmic matrix, which is disclosed by this method, are briefly reviewed in relation to the sol-gel transition of cytoplasmic heterogenous proteins. Since the ultrastructures of various cells and tissues in this method are confirmed to be well correspondent to those in conventional epoxy section TEM except for isotropic dimensional changes, and because there is no necessity for any special expensive equipments other than those for the conventional TEM, the embedment-free section TEM method with these advantages, deserves much more wide application to the morphological research including electron tomography.

Disclosure in 3D of slit‐membrane as well as ‐strands, and en‐face basal lamina in situ of renal glomerulus of normal rats in embedment‐free section transmission electron microscopy

With higher contrast and transparency due to the absence of epon and stereo-viewing effect due to thicker sections than conventional electron microscopy as methodological advantages, the renal glomerular slits were re-examined in embedment-free section electron microscopy. In addition to clear demonstration of strands bridging the slits in forms of ladders with highly irregular intervals and various extension-directions and length, this study disclosed clearly for the first time in the "section" TEM thin sheets which partially spanned the slit together with the strand-ladders. No strands were found to align in forms of typical zippers in normal kidney. Furthermore, en-face ultrastructure of the basal lamina in situ was clearly demonstrated in superimposed sites of the endothelial fenestrae with the slits.

A cytoplasmic gel network capable of mediating the conversion of chemical energy to mechanical work in diverse cell processes: A speculation

Enigmatic morphological features of the formation and fate of "dark" (hyper-basophilic, hyper-argyrophilic and hyper-electrondense) neurons suggest that the mechanical work causing their dramatic shrinkage (whole-cell ultrastructural compaction) is done by a previously "unknown" ultrastructural component residing in the spaces between their "known" (i.e. visible in the conventional transmission electron microscopy) ultrastructural constituents. Embedment-free section electron microscopy revealed in these spaces the existence of a continuous network of gel microdomains, which is embedded in a continuous network of fluid-filled lacunae. We gathered experimental facts suggesting that this gel network is capable of a volume-reducing phase-transition (an established physico-chemical phenomenon), which could be the motor of the whole-cell ultrastructural compaction. The present paper revisits our relevant observations and speculates how such a continuous whole-cell gel network can do both whole-cell and compartmentalized mechanical work.

What we have learned and will learn from cell ultrastructure in embedment‐free section electron microscopy

The limitations inherent in conventional electron microscopy (EM) using epoxy ultrathin sections for a clear recognition of biological entities having electron densities similar to or lower than that of epoxy resin have led to the development of embedment-free sectioning for EM. Embedment-free section EM is reliably performed using water-soluble polyethylene glycol (PEG) as a transient embedding medium, with subsequent de-embedment of PEG by immersion into water, followed by critical point-drying (CPD) of the embedment-free section. The present author has stressed that this approach clearly discloses structures whose contours and/or appearance are accordingly vague and/or fuzzy in conventional EM, but does not reveal any new structures. Based on embedment-free electron microscopy (PEG-EM), this article presents five major findings regarding strand- or microtrabecular lattices which have been clearly revealed to occur in the cytoplasmic matrix-an impossibility with conventional EM. These are (1) the appearance of lattices of different compactness in various cells and in intracellular domains of a given cell; (2) the faithful reproduction from an albumin solution in vitro of strand-lattices with correspondingly increasing compactness following increasing concentrations; (3) the appearance of more compact lattices from gelated gelatin than from solated gelatin at a given concentration in vitro; (4) the appearance of either greater or less lattice-compactness by hyper- or hypotonic pretreatments of cells; and (5) the appearance of certain intracellular proteins confined to the centripetal demilune-domain of centrifuged ganglion cells which is occupied with strand-lattices of a substantial compactness. From these findings, questions now arise as to the biological significance of the individual strand itself in the microtrabecular lattices in PEG-EM. In addition, it may be that the appearance of strand-lattices in a given biological domain represents the presence of soluble proteins; the lattice-compactness indicates the concentration of soluble proteins in the domain, and the aqueous cytoplasm is equivalent to the aqueous solution. Further, the appearance of two contiguous lattice domains exhibiting differing degrees of compactness in a given cell indicates that cytoplasmic proteins are solated in a domain with less compact lattices, whereas they are gelated in the other domain. These proposed interpretations need to be confirmed by further studies. If confirmed, the control mechanisms of the localization and movement of intracellular organelles could then be understood on the basis not only of information about the cytoskeletons but also of cell ultrastructure-related information on the concentration and sol-gel states of intracellular proteins. In addition, possible interpretations of the significance of strand-lattices in PEG-EM are also applicable to the nucleoplasm, especially extra-heterochromatin (euchromatin) areas. Finally, several potential uses/advantages of PEG-EM in the cell-ultrastructure have also been demonstrated, especially in three-dimensional reconstructions of nonmembranous structures including stereo-viewing using a pair of EM images with appropriate tilting as well as electron microscopic tomography.

First Disclosure of Lipid Droplet Substructure and Myelin Translucency in Embedment-Free Section Electron Microscopy

In conventional transmission electron microscopy (EM), thinly sectioned specimens embedded in epoxy resin are observed. However, because of a substantial level of electron density of epoxy resin, the possibility cannot be ruled out that bio-structures having electron density similar to that of epoxy resin are not clearly recognized and thus are neglected or misinterpreted in conventional EM. This was the reason to require for embedment-free EM. Embedment-free sections have already been made available reliably by transient embedding in polyethylene glycol (PEG) and subsequent de-embedding through immersion in water, and further by critical-point drying, and this embedment-free EM is thus termed PEG-EM. However, this PEG-EM has not been successful to attract reasonable attention from electron microscopists and instead been misunderstood as a non-reliable method. In this paper, the remarkably enhanced contrast and electron translucency of any observation targets in PEG-EM are clearly demonstrated by comparing with images in conventional EM of adipocytes and neural myelin as examples. These features of PEG-EM, together with faithful correspondence in EM images of any individual substructures between the two methods, confirm the reliability of PEG-EM. Furthermore, the much higher thickness of embedment-free sections together with these features makes the PEG-EM more advantageous than the conventional EM for three-dimensional appreciation of structural elements, which is made by stereo-viewing of sections or by EM tomography. Therefore, the PEG-EM is regarded as an important adjunct to the conventional EM for histological studies and wide application of this method may unravel a new level of histology.

Embedment-free section electron microscopy

Because of potential hindrance of clear viewing in epoxy sections of biological entities having an electron density similar to and lower than that of epoxy resin, the author has stressed that the embedment-free section electron microscopy is necessary for re-examination and/or clarification of biological specimen structures, and that the embedment-free electron microscopy is reliably done by using water-soluble polyethylene glycol (PEG) as a transient embedding media and by critical point-drying of embedment-free sections after de-embedment of PEG by immersion of semithin sections into water. With the embedment-free electron microscopy, the author has presented five major findings: the appearance of microtrabecular lattices with different compactnesses in various cells and in intracellular domains of a given cell, the faithful reproduction of microtrabecula-like strand lattices in vitro with increasing compactnesses from artificial protein solutions at correspondingly increasing concentrations, the appearance of more compact lattices from gelated gelatin than from solated gelatin at a given concentration in vitro, the changeability in compactnesses of the microtrabecular lattices by hyper- or hypotonic shock treatments of cells, and the confined appearance of an intracellular protein in the centripetal demilune of centrifuged ganglion cells which is occupied with the microtrabecular lattices of a substantial compactness. From these findings, several conclusions are drawn: individual strands themselves of the microtrabeculae are meaningless, the appearance of microtrabeculae represents the presence of proteins at a certain concentration, and it is therefore likely that the aqueous cytoplasm is equivalent to the aqueous solution. In addition, it is possible that the appearance of two contiguous lattice domains exhibiting different compactnesses in a given cell may represent the occurrence of a contiguity of sol to gel states of cytoplasmic domains. It is thus proposed that the localization and movement of intracellular organelles are controlled not only by the cytoskeletons but also by the concentration and sol/gel states of intracellular proteins. In addition, several potential usefulness of the embedment-free electron microscopy has also been demonstrated.

Nuclear matrix of the most primitive eukaryoteArchezoa

Accumulating evidence suggests that unicellular Archezoa are the most primitive eukaryotes and their nuclei are of significance to the study of evolution of the eukaryotic nucleus. Nuclear matrix is an ubiquitous important structure of eukaryotic nucleus; its evolution is certainly one of the most important parts of the evolution of nucleus. To study the evolution of nuclear matrix, nuclear matrices of Archezoa are investigated.Giardia lamblia cells are extracted sequentially. Both embedment-free section EM and whole mount cell EM of the extracted cells show that, like higher eukaryotes, this species has a residual nuclear matrix in its nucleus and rich intermediate filaments in its cytoplasm, and the two networks connect with each other to form a united network. But its nuclear matrix does not have nucleolar matrix and its lamina is not as typical as that of higher eukaryotes; Western blotting shows that lamina of Giardia and two other Archezoa Entanzoeba invadens and Trichomonas vaginali all contain only one polypeptide each which reacts with a mammalia anti-lamin polyclonal serum and is similar to lamin B (67 ku) of mammalia in molecular weight. According to the results and references, it is suggested that nuclear matrix is an early acquisition of the eukaryotic nucleus, and it and the "eukaryotic chromatin" as a whole must have originated very early in the process of evolution of eukaryotic cell, and their origin should be an important prerequisite of the origin of eukaryotic nucleus: in the lamin (gene) family, B-type lamins (gene) should be the ancestral type and that A-type lamins (gene) might derive therefrom.

INTRANUCLEAR NETWORK OF 3–5 nm AND 8–10 nm FIBERS IN EL-4 LYMPHOMA CELLS

While much evidence indicates a high degree of spatial organization in the nucleus, the underlying molecular structures that support it remain poorly characterized. By extracting with high concentrations of RNase A in a modification of the sequential extraction protocol of Penman, we have identified a novel intranuclear network in the mouse lymphoma cell line, EL-4. Micrographs of embedment-free sections of extracted cells reveal anastomosing filaments of two different diameters: 3–5nm and 8–10nm. The 3–5-nm filaments are interconnected in many junctions and appear to blend smoothly into each other. The 8–10-nm fibers frequently split into two 3–5-nm filaments. Some 3–5-nm fibers appear to be connected at 90° angles with the 8–10-nm fibers. All junctions are smooth with no apparent junction protein. Flow cytometric analysis of RNase A- (and DNase I-) extracted nuclear matrices indicates that they do not contain significant amounts of protein that react with anti-actin and anti-vimentin monoclonal antibodies. Extraction of EL-4 nuclear matrices with high salt does not reveal 8–10-nm core filaments described after similar treatment of tumor cell lines of cervical and mammary origin. The novel characteristics of the core filaments in EL-4 lymphoma cells may reflect cell-type specificity of the nuclear matrix.

Detection of dense intra- and perinuclear 10 nm filament systems by whole mount and embedment-free electron microscopy in several species of the green algal order Dasycladales

In contrast to the immense body of evidence supporting the occurrence of intermediate filament (IF) proteins in the animal kingdom, there is only limited information on their distribution in plants. Nevertheless, a number of immunocytochemical and electron microscopical observations indicate that particularly in higher plant cells IFs contribute to the construction of the cyto- and karyoskeleton. Here we show by whole mount electron microscopy of the giant nuclei extruded together with adhering cytoplasm from the rhizoids of some species of the algal order Dasycladales that cytoplasmic 10 nm filament networks also occur in unicellular, mononucleated green organisms of early evolutionary origin. The filament systems were associated with the residual nuclear envelope which consisted of a dense arrangement of pore complexes suspended by a meshwork of short 5 to 6 nm filaments; structurally it was very similar to the nuclear envelopes obtained from mammalian cells. When the Dasycladales nuclei were processed side by side with mouse skin fibroblasts, the algal filament systems were physically almost indistinguishable from the mammalian vimentin filament network. Embedment-free thin sections of rhizoids have not only confirmed the existence of the perinculear 10 nm filaments and their seamless association with the nuclear envelope, but have demonstrated the existence of an extensive intranuclear meshwork of 10 nm filaments. The latter were morphologically indistinguishable from the perinuclear 10 nm filaments and seem to be connected to these via the nuclear envelope to form a continuum. Among a variety of antibodies directed against mammalian IF proteins, only polyclonal anti-mouse lamin B antibodies decorated the cytoplasmic filaments of the Dasycladales cells. Surprisingly, none of the antibodies decorated the thinner filaments of the nuclear envelope, which possibly represent the nuclear lamina. In accord with this observation, one anti-lamin B antibody recognized in Western blot analysis of a urea extract ofAcetabularia acetabulum rhizoids three polypeptides with Mrs of approximately 47,000, 64,000, and 76,000. The proteins did not react with the α-IFA antibody. Since the Dasycladales have a fossil record of nearly 600 million years — an extant genus, Acicularia, also investigated here, evolved about 170 million years ago -, the molecular characterization of the subunit proteins of their cytoplasmic filament systems might throw further light on the evolution and biological role of IFs.

Atomic force microscopy of embedment-free sections of cells and tissues.

The atomic force microscope (AFM) was applied for the first time to embedment-free biological sections. Aldehyde-fixed tissues (kidney, liver etc.) of mice were postfixed with osmium tetroxide and cut into 500-700 nm thick sections after embedding of the tissue block in polyethylene glycol (PEG); the sections mounted on glass slides were deembedded and critical point-dried. The AFM images were collected in air from those tissue sections in a dynamic force mode. Solid-height-mode images, which were comparable to transmission electron microscope (TEM) images, seemed to provide us more useful information than the solid-mode images which resembled scanning electron microscope (SEM) images. Internal structures including chromatin fibers in the nuclei and cytoplasmic organelles were clearly recognizable by AFM. Minute surface structures including the end-feet of renal podocytes were demonstrated. We confirm that a vertical resolution of 0.1 nm and a lateral resolution of 5-10 nm are attainable with the dynamic force mode of AFM using thin sections of tissues. The present paper proposes the usefulness of AFM for observation of biological materials without metal coating in a non-vacuous environment.

Ultrastructural filament organization of hepatocyte cytoplasm revealed by embedment-free sections of rat liver.

The cytoplasmic matrix of rat liver cells was examined by embedment-free electron microscopy of polyethylene glycol (PEG) embedded tissue from which the PEG had been removed subsequently. Non-ionic detergent extraction of fresh blocks of tissue permitted the major cytoskeletal components to be visualized in relatively thick embedment-free sections. Immunolabelling confirmed the presence of the major cytoskeletal proteins (actin, cytokeratin and tubulin) in these sections. Hence, this method permits the examination of the ultrastructural organization of the cytoskeleton of the hepatocyte in blocks of fresh hepatic tissue.

3-D examination of the cytoskeletal sheets of mammalian eggs

Mammalian eggs and embryos contain an elaborate cytoskeletal network of “sheets” which are distributed throughout the entire cell cytoplasm. Cytoskeletal sheets are long, planar structures unlike the cytoskeletal networks typical of somatic cells (actin filaments, microtubules, and intermediate filaments), which are filamentous. These sheets are not found in mammalian somatic cells nor are they found in nonmammalian eggs or embryos. Evidence that they are, indeed, cytoskeletal in nature is derived from studies demonstrating that 1) the sheets are retained in the detergent-resistant cytoskeleton fraction; 2) there are no associated membranes (determined by freeze-fracture); and 3) the sheets dissociate into filaments at the blastocyst stage of embryogenesis. Embedment-free sections of hamster eggs viewed at 60 kV show sheets running across the egg cytoplasm (Fig. 1). Although this approach provides excellent global views of the sheets and their reorganization during development, the mechanism of image formation for embedment-free sections does not permit evaluation of the sheets at high resolution.

Intermediate filaments in higher plant cells and their assembly in a cell-free system

Networks of 10 nm filaments were detected in suspension cells ofDaucus carota var.sativa DC, root tip cells ofPisum sativum, mesophyll cells ofBrassica pekinensis and pollen ofNicotiana tabacum by selective extraction combined with whole mount and embedment-free section using diethylene glycol distearate plastic for electron microscopy. Western blot analysis showed that the major filament components are 3 polypeptides of 64kDa, 58kDa, 52kDa, which react strongly with AE3, a monoclonal antibody that recognizes relatively basic keratin — the type II intermediate filaments (IFs) of animal cells -, and 1 polypeptide of 50kDa, which reacts strongly with AE 1, a monoclonal antibody that recognizes relatively acidic keratin — the type I IFs of animal cells. The 10nm plant filaments could be reassembled in a cell-free system. The assembled filaments are indistinguishable from native plant intermediate filament in morphology and protein composition. Thus we demonstrated that keratin intermediate filaments exist in the cytoplasm of higher plants. We also found no obvious tissue or species specificity in the plant IF proteins.

Rat kidney glomerular basement membrane visualized in situ by embedment‐free sectioning and subsequent platinum‐carbon replication

Following the removal of polyethylene glycol (PEG) from thin sections, and viewing through the endothelial fenestrae and/or the interpedicel spaces, the rat renal glomerular basement membrane in situ was revealed to consist of meshworks and to be electron-transparent when examined at right angles to the plane of the membrane. By subsequent platinum replication of the embedment-free sections, the lamina densa of the basement membrane appeared as a veil composed of rather closely packed particles. The architecture of the split diaphragm and the surface morphology of the endothelial cell membrane were also clearly revealed. The present results indicate that the PEG method, with or without replication, can provide valuable information on basement membrane morphology.

Specialized cytoskeletal elements in mammalian eggs: Structural and biochemical evidence for their composition

Mammalian eggs and embryos contain an extensive detergent-resistant cytoskeletal network, including many elements which have been referred to as sheets in hamster eggs. In this study we examined the structure of the sheet-like components by using embedment-free sections and freeze-fracture electron microscopy and found that the sheets are composed of both filamentous and particulate components. In addition, exposure to a high salt extraction medium resulted in the disappearance of the sheets at the ultrastructural level. SDS-polyacrylamide gel electrophoresis of the cell fractions revealed four stainable proteins solubilized by the high salt extraction with one of the proteins being greatly enriched. Because these cytoskeletal sheets undergo an extensive reorganization coincident with key events during early development they serve as internal markers for the establishment of polarity and subsequent differentiation of the first embryonic epithelium, the trophectoderm.

Visualization of the inner and outer surfaces of the cell membrane and cytoskeleton by polyethylene glycol embedding, subsequent deembedding, and rotary replication with platinum.

Using rotary replication with platinum and carbon to embedment-free sections of polyethylene glycol (PEG)-embedded tissue from which PEG had been removed, the membrane specializations on the outer (exoplasmic) and inner (protoplasmic) cell surface as well as the organization of the cytoskeleton was demonstrated. The high quality and excellent preservation were comparable to that obtained by the rapid-freezing, deep-etched replica method. The present results indicate that the PEG method is essentially acceptable as a reliable morphological technique. Because either sectioned or replica images from the same tissue cells can be observed simultaneously, the PEG method with rotary replication should provide valuable information on cell ultrastructure.

Alterations in nuclear matrix structure after adenovirus infection.

Infection of HeLa cells with adenovirus serotype 2 causes rearrangements in nuclear matrix morphology which can best be seen by gentle cell extraction and embedment-free section electron microscopy. We used these techniques to examine the nuclear matrices and cytoskeletons of cells at 6, 13, 28, and 44 h after infection. As infection progressed, chromatin condensed onto the nucleoli and the nuclear lamina. Virus-related inclusions appeared in the nucleus, where they partitioned with the nuclear matrix. These virus centers consisted of at least three distinguishable areas: amorphously dense regions, granular regions whose granulations appeared to be viral capsids, and filaments connecting these regions to each other and to the nuclear lamina. The filaments became decorated with viral capsids of two different densities, which may be empty capsid shells and capsids with DNA-protein cores. The interaction of some capsids with the filaments persisted even after lysis of the cell. We propose that granulated virus-related structures are sites of capsid assembly and storage and that the filaments may be involved in the transport of capsids and capsid intermediates. The nuclear lamina became increasingly crenated after infection, with some extensions appearing to bud off and form blebs of nuclear material in the cytoplasm. The perinuclear cytoskeleton became rearranged after infection, forming a corona of decreased filament number around the nucleus. In summary, we propose that adenovirus rearranges the nuclear matrix and cytoskeleton to support its own replication.

Ultrastructural localization of actin in the intermediate lobe of rat hypophysis.

The localization of actin in the cells of the pars intermedia of rat hypophysis was studied by means of the polyethylene glycol (PEG) embedding and subsequent de-embedding method together with FITC and IgG-colloidal gold immunolabelling techniques. Actin immunofluorescence was detected to be punctate throughout the entire cytoplasm, except for the nuclear region. In electron microscopy actin gold-labelling was localized on portions of microtrabeculae in close association with the secretory granules, but not within the secretory granules themselves. This close association of actin with the secretory granules strongly suggests the involvement of the contractile protein in the cellular secretory process. Several restrictions due to the PEG-method itself are also discussed to interpret clearly immunoelectron microscope images from the embedment-free sections.