Wet SEM: a novel method for rapid diagnosis of brain tumors

The authors present the application of wet SEM for histopathological assessment, a technology for imaging fully hydrated samples at atmospheric pressure in a scanning electron microscope (SEM). Both transmission and scanning electron microscopy techniques usually require long and complex sample preparation of the tissues. In marked contrast, a rapid preparation of tissues is described for evaluation by SEM imaging. The wet SEM technology successfully demonstrated both histological and ultrastructural features of several CNS tumors: Rosette formation and intracytoplasmic lumens were observed in ependymoma; numerous fibrillary processes in fibrillary astrocytoma; and focal rosette formation with no intracytoplasmic lumens in medulloblastoma. Application of this method simultaneously with frozen section may improve rapid intraoperative diagnosis of these intracranial tumors.     

A Novel Method for “Wet” SEM

Progress in the processing of wet tissues, without the need of fixation and complex preparation procedures, may facilitate the microscopic examination of tissues and cells. Microscopic examination of tissues is a central tool in clinical diagnosis as well as in diverse areas of research. The authors present the application of Wet SEM, a technology for imaging fully hydrated samples at atmospheric pressure in a scanning electron microscope (SEM). The technique is based on 2 principles. First, samples are imaged in sealed specimen capsules and are separated from the evacuated interior of the electron microscope by a thin, electron-transparent partition membrane that is strong enough to sustain a 1-atm pressure difference. Second, imaging is done in a SEM, based on detection of backscattered electrons, which penetrate a few microns into the specimen and thus give information on the cellular level.     

A novel method for "Wet" SEM

Progress in the processing of wet tissues, without the need of fixation and complex preparation procedures, may facilitate the microscopic examination of tissues and cells. Microscopic examination of tissues is a central tool in clinical diagnosis as well as in diverse areas of research. The authors present the application of Wet SEM, a technology for imaging fully hydrated samples at atmospheric pressure in a scanning electron microscope (SEM). The technique is based on 2 principles. First, samples are imaged in sealed specimen capsules and are separated from the evacuated interior of the electron microscope by a thin, electron-transparent partition membrane that is strong enough to sustain a 1-atm pressure difference. Second, imaging is done in a SEM, based on detection of backscattered electrons, which penetrate a few microns into the specimen and thus give information on the cellular level.     

Correlative evaluation of variable pressure-scanning electron microscopy to histology methods for vascular tissues

Abstract

For pathological investigation, vascular tissues implanted with biomaterials or medical devices require microscopic histology and scanning electron microscopy (SEM) assessment to evaluate biocompatibility with the vascular luminal surface. However, these valuable specimens are often susceptible to SEM and histology tissue preparation artifacts. Correlative variable pressure-scanning electron microscopy (VP-SEM) and histology methods were developed and evaluated, using current SEM technology, allowing direct imaging of hydrated samples without lengthy tissue processing, labor, equipment, and their sequela artifacts. Unstained and silver-stained, fixed swine carotid arteries were assessed using a cooling stage and an environmental secondary electron detector. After VP-SEM imaging, arteries were processed for histology evaluation. Assessment of luminal endothelial surface SEM images and tissue histology of the underlying mural wall was made to determine if low vacuum, cryo-freezing, electron beam exposure, and/or staining affected tissue morphology. Conventional SEM requires lengthy tissue processing with dedicated equipment for solvent dehydration, critical point drying, and metallic sputter coating in order to image under high vacuum for secondary electron imaging. This process causes notable artifacts when samples are later processed for microscopic histology. In contrast, silver staining of swine carotid arteries, followed by aldehyde fixation, facilitated VP-SEM imaging and histology preparation with results that were comparable to conventional SEM and had acceptable tissue morphology. VP-SEM with a cooling stage and an environmental secondary electron detector allows efficient SEM imaging and correlative histological assessment of vascular tissue samples. This correlative method may be further developed and implemented for research applications with biomaterial and medical device implanted tissues.     

Electron microscopy of wet tissues: a case study in renal pathology

In this report we introduce wet-tissue scanning electron microscopy, a novel technique for direct imaging of wet tissue samples using backscattered electrons. Samples placed in sealed capsules are imaged through a resilient, electron-transparent membrane. The contrast of the imaged samples may be enhanced by chemical staining. The samples several millimeters thick and imaged without sectioning, makes this technique suitable for rapid analysis of tissue specimens. We applied this technique to D-limonene-induced nephropathy where accumulation of hyaline protein droplets is induced in proximal and distal convoluted tubules of the kidney. Images obtained by scanning electron microscopy of hydrated kidney specimens exhibited superior resolution, contrast, and magnification compared with those obtained by conventional light microscopy of paraffin sections. The electron micrographs can be obtained within an hour of tissue removal, whereas preparation for light microscopy requires at least 1 day. These advantages of the wet scanning electron microscopy technique indicate its potential utility in a wide range of applications in histopathology and toxicology.     

Adenocarcinoma of the lung: a comparative diagnostic study using light and electron microscopy

Several techniques for diagnosing adenocarcinoma of the lung are commonly available, but the frequency of their use and diagnostic sensitivity may vary. Twenty cases of primary lung adenocarcinoma obtained at surgery were studied by the following four routine techniques: light microscopy (LM) using hematoxylin-eosin (H&E) stain, mucicarmine stain, and PAS-diastase stain, and electron microscopy (EM). Three observers independently determined the positivity (0 [none], +/- [equivocal], 1 + [slight], 2 + [moderate], 3 + [marked]) of each of these cases for lumen formation in H&E-stained sections (LM lumens), intracytoplasmic (cytoplasmic mucicarmine) or intraluminal (luminal mucicarmine) mucicarmine, intracytoplasmic (cytoplasmic PAS) or intraluminal (luminal PAS) PAS-diastase, and lumen formation (EM lumens) or microvilli (EM microvilli) on electron microscopy. Comparative matching of these seven microscopic determinants (using Wilcoxon signed-rank test) demonstrated significant (P less than .01) sensitivity of EM microvilli over EM lumens, EM microvilli over luminal mucicarmine, cytoplasmic PAS over luminal mucicarmine, EM microvilli over cytoplasmic mucicarmine, cytoplasmic PAS over cytoplasmic mucicarmine, and EM microvilli over LM lumens, and a significant (P less than .05) sensitivity of cytoplasmic PAS over LM lumens, EM microvilli over luminal PAS, luminal PAS over luminal mucicarmine, and cytoplasmic PAS over EM lumens. Friedman's nonparametric test (P less than .05) indicated a significant difference among the microscopic determinants. The most sensitive was EM microvilli (mean rank score, 5.17) followed by cytoplasmic PAS (4.77), luminal PAS (4.02), cytoplasmic mucicarmine (3.62), LM lumens (3.52), EM lumens (3.47), and luminal mucicarmine (3.40). However, each of the diagnostic techniques had case examples positive for one, but not for the others, indicating that maximum yield of adenocarcinoma diagnoses will be obtained by performing all four techniques (H&E, mucicarmine, PAS-diastase, and electron microscopy.     

A new method of wet scanning electron microscopy for the analysis of myelination in EAE mouse model of multiple sclerosis

Development of effective therapies for multiple sclerosis (MS) is dependent on the advancement of improved tools for evaluation of progression of this disease in animal models. We present a novel technique utilizing scanning electron microscopy (SEM) for imaging wet biological specimens thus enabling rapid and high-resolution imaging of myelin in mouse spinal cord (SC). We demonstrate the advantages of using the wet SEM technique to image myelin in a murine model of MS, experimental autoimmune encephalomyelitis (EAE) induced in the Biozzi (antibody-high) mouse, by sensitization with spinal cord homogenate (SCH) in adjuvant. Our studies show that the methodology allows easy identification of normal and pathological components with great clarity, which is then correlated with light microscopy (LM) and validated thereby. Furthermore, we demonstrate gold immunolabeling of specific epitopes. We conclude that the new technique provides a quick, accurate, and detailed structural evaluation of the SC that can be applied to advance the research of MS.     

VASCULARIZATION IN INFANTILE BRAIN TUMORS INDUCED BY HUMAN ADENOVIRUS TYPE 12 IN RATS

Early vascular changes in undifferentiated infantile brain tumors induced by human adenovirus type 12 (Ad 12) in rats were studied using the India ink injection method, scanning electron microscopy (SEM) of vascular corrosion casts, transmission electron microscopy, and the peroxidase anti peroxidase method for glial fibrillary acidic protein detection. The process of vascularization of solid type tumors was divided into five phases : Stage O, no remarkable changes; Stage I, dilatation of vessels; Stage II, formation of vascular network; Stage III, fine branching in the vascular network; and Stage IV, necrosis. SEM of vascular replica in Stage III suggested compression of the vessels by proliferating tumor cells. In the tapetum region, Stage I was recognized in solid type tumors 100 to 230 (m = 180) μm in diameter, while in the olfactory lobes and in the anterior horn of lateral ventricle closely adjacent to the vascular gray matter, Stage I occurred in much larger tumors. In the diffuse type tumors, the dilatation of vessels was first noticed when the tumors were about 1600 μm in diameter. The solid type tumors mainly developed in the poorly vascular subventricular zone, while the diffuse type ones were usually found deep in the vascular gray matter. The results indicate that vascularization is closely related to the local vascularity and the growth pattern of the microtumors, and that of the solid type tumors induced by Ad 12 may be a model of vascular changes in infantile brain tumors which mostly develop and grow rapidly in the subventricular zone which have poor vascular development. ACTA PATHOL. JPN. 34: 1343–1354, 1984.     

Vascularization in infantile brain tumors induced by human adenovirus type 12 in rats

Early vascular changes in the undifferentiated infantile brain tumors induced by human adenovirus type 12 (Ad 12) in rats were studied using the India ink injection method, scanning electron microscopy (SEM) of vascular corrosion casts, transmission electron microscopy, and the peroxidase anti peroxidase method for glial fibrillary acidic protein detection. The process of vascularization of solid type tumors was divided into five phases: Stage O, no remarkable changes; Stage I, dilatation of vessels; Stage II, formation of vascular network; Stage III, fine branching in the vascular network; and Stage IV, necrosis. SEM of vascular replica in Stage III suggested compression of the vessels by proliferating tumor cells. In the tapetum region, Stage I was recognized in solid type tumors 100 to 230 (m = 180) micrometer in diameter, while in the olfactory lobes and in the anterior horn of lateral ventricle closely adjacent to the vascular gray matter, Stage I occurred in much larger tumors. In the diffuse type tumors, the dilatation of vessels was first noticed when the tumors were about 1600 micrometers in diameter. The solid type tumors mainly developed in the poorly vascular subventricular zone, while the diffuse type ones were usually found deep in the vascular gray matter. The results indicate that vascularization is closely related to the local vascularity and the growth pattern of the microtumors, and that of the solid type tumors induced by Ad 12 may be a model of vascular changes in infantile brain tumors which mostly develop and grow rapidly in the subventricular zone which have poor vascular development.     

Tanycytic Ependymoma

Tanycytic ependymoma is an uncommon fibrillar variant of ependymoma characterized by streams of piloid, or hair-like, cells having “ependymal” nuclei. True ependymal rosettes are absent, and perivascular rosettes are inconspicuous. Misinterpretation as schwannoma or astrocytoma is a diagnostic problem and well-documented cases are scarce. The purpose of this report is to document the ependymal features of the neoplasm and to increase awareness of the entity's existence. Biopsy tissues from three patients with tanycytic ependymoma were examined. All tumors consisted of sheets of spindle cells that were positive for glial fibrillary acidic and S-100 proteins. Ultrastructural examination showed characteristic ependymal features, including intracytoplasmic intermediate filaments, prominent intercellular junctions, numerous slender surface microvilli, and microvilli-lined lumina. Accurate recognition of the ependymal nature of this spindle neoplasm requires a high index of suspicion. Because the spindle cells are immu-noreactive with antibodies to both glial fibrillary acidic and S-100 proteins, ultrastructural confirmation of ependymal features is necessary.     

Simulation of bone resorption-repair coupling in vitro

In the normal adult human skeleton, new bone formation by osteoblasts restores the contours of bone surfaces following osteoclastic bone resorption, but the evidence for resorption-repair coupling remains circumstantial. To investigate whether sites of prior resorption, more than the surrounding unresorbed surface, attract osteoblasts or stimulate them to proliferate or make new matrix, we developed a simple in vitro system in which resorption-repair coupling occurs. Resorption pits were produced in mammalian dentine or bone slabs by culturing chick bone-derived cells on them for 2–3 days. The chick cells were swept off and the substrata reseeded with rat calvarial osteoblastic cells, which make bone nodules in vitro, for periods of up to 8 weeks. Cell positions and new bone formation were investigated by ordinary light microscopy, fluorescence and reflection confocal laser microscopy, and SEM, in stained and unstained samples. There was no evidence that the osteoblasts were especially attracted to, or influenced by, the sites of resorption in dentine or bone before cell confluence was reached. Bone formation was identified by light microscopy by the accumulation of matrix, staining with alizarin and calcein and by von Kossa's method, and confirmed by scanning electron microscopy (SEM) by using backscattered electron (BSE) and transmitted electron imaging of unembedded samples and BSE imaging of micro-milled embedded material. These new bone patches were located initially in the resorption pits. The model in vitro system may throw new light on the factors that control resorption-repair coupling in the mineralised tissues in vivo.This work was presented at the 5th Workshop on Cells and Cytokines in Bone and Cartilage, Davos, April 1994     

Macrophagic Myofasciitis in Childhood: The Role of Scanning Electron Microscopy/Energy-Dispersive Spectroscopy for Diagnosis

Macrophagic myofasciitis (MMF) is an inflammatory myopathy related to aluminum-containing vaccines. Described in 1998, most cases were reported in adults, with only 22 cases being reported in children. Three children aged between 13 months and 3½ years were investigated in our institution for neuromuscular symptoms. They underwent thorough clinical, familial, and laboratory investigations, electroneuromyography, muscle biopsy with transmission electron microscopy, scanning electron microscopy/energy dispersive spectroscopy (SEM/EDS), and, in one case, brain magnetic resonance imaging. They had received regular immunizations. Two patients were hypotonic and one presented with myotonia. Muscle biopsy of all patients presented macrophagic infiltrates with intracytoplasmic aluminum content as revealed by SEM/EDS analysis. Their diverse clinical picture does not support a direct relationship between local morphologic findings and systemic symptoms. The atypical clinical presentation of these children may not result from the superposition of MMF upon a background systemic neuromyopathy, suggesting instead that they are two coincident and independent conditions. Although the finding of macrophage infiltrates in muscle tissue is not new, the identification of aluminum content is recent. The use of tissue sections for aluminum detection and mapping by SEM/EDS is conclusive for, diagnosis; it has not been reported previously in a pathology journal, to the authors' knowledge.     

Light microscopic, ultrastructural and immunocytochemical spectrum of malignant lacrimal and salivary gland tumors, including malignant mixed tumors.

Ten malignant myoepithelial tumors of the salivary glands and one of lacrimal gland origin were studied by light, electron microscopy and immunocytochemistry. The light microscopic appearance of the tumors varied from primarily spindle cell neoplasms (two cases), to others with predominantly epithelial components (four cases) and mixed varieties (five cases). Therefore, they can be confused with other epithelial and mesenchymal neoplasms. The electron microscopic spectrum varied from tumors with widespread and typical myoepithelial differentiation (i.e. myofilament bundles at the cell periphery, attachment plaques and intercellular junctions) to some with diffusely distributed filaments, without associated spindle densities but with attachment plaques, and others with evidence of duct formation and containing scattered cells showing intracytoplasmic tonofilaments. Often the tumors revealed mixed ultrastructural features; the relative numbers of the different cellular components was variable. The eleven neoplasms were S-100 protein, actin and keratin positive, either focally or diffusely, with varying degrees of intensity. Ten of the eleven tumors were positive for vimentin and nine of ten tested expressed carcinoembryonic antigen. Only two of nine were focally positive for glial fibrillary acidic protein. The study emphasizes the variable light microscopic appearances of these neoplasms and their immunocytochemical and ultrastructural spectrum. Accurate determination of myoepithelial differentiation sometimes requires careful evaluation of the light, ultrastructural and immunocytochemical findings. If all three diagnostic modalities are not utilized, it is likely that some of these neoplasms will be improperly classified.     

Macrophagic myofasciitis in childhood: the role of scanning electron microscopy/energy-dispersive spectroscopy for diagnosis

Macrophagic myofasciitis (MMF) is an inflammatory myopathy related to aluminum-containing vaccines. Described in 1998, most cases were reported in adults, with only 22 cases being reported in children. Three children aged between 13 months and 3(1/2) years were investigated in our institution for neuromuscular symptoms. They underwent thorough clinical, familial, and laboratory investigations, electroneuromyography, muscle biopsy with transmission electron microscopy, scanning electron microscopy/energy dispersive spectroscopy (SEM/EDS), and, in one case, brain magnetic resonance imaging. They had received regular immunizations. Two patients were hypotonic and one presented with myotonia. Muscle biopsy of all patients presented macrophagic infiltrates with intracytoplasmic aluminum content as revealed by SEM/EDS analysis. Their diverse clinical picture does not support a direct relationship between local morphologic findings and systemic symptoms. The atypical clinical presentation of these children may not result from the superposition of MMF upon a background systemic neuromyopathy, suggesting instead that they are two coincident and independent conditions. Although the finding of macrophage infiltrates in muscle tissue is not new, the identification of aluminum content is recent. The use of tissue sections for aluminum detection and mapping by SEM/EDS is conclusive for, diagnosis; it has not been reported previously in a pathology journal, to the authors' knowledge.     

“Deep-media culture condition” promoted lumen formation of endothelial cells within engineered three-dimensional tissues in vitro

In the field of tissue engineering, the induction of microvessels into tissues is an important task because of the need to overcome diffusion limitations of oxygen and nutrients within tissues. Powerful methods to create vessels in engineered tissues are needed for creating real living tissues. In this study, we utilized three-dimensional (3D) highly cell dense tissues fabricated by cell sheet technology. The 3D tissue constructs are close to living-cell dense tissue in vivo. Additionally, creating an endothelial cell (EC) network within tissues promoted neovascularization promptly within the tissue after transplantation in vivo. Compared to the conditions in vivo, however, common in vitro cell culture conditions provide a poor environment for creating lumens within 3D tissue constructs. Therefore, for determining adequate conditions for vascularizing engineered tissue in vitro, our 3D tissue constructs were cultured under a “deep-media culture conditions.” Compared to the control conditions, the morphology of ECs showed a visibly strained cytoskeleton, and the density of lumen formation within tissues increased under hydrostatic pressure conditions. Moreover, the increasing expression of vascular endothelial cadherin in the lumens suggested that the vessels were stabilized in the stimulated tissues compared with the control. These findings suggested that deep-media culture conditions improved lumen formation in engineered tissues in vitro.     

Matrix crystals in cytologic urine specimens: Observations on their mineral composition by energy dispersive X-ray microanalysis and morphologic scanning electron microscopy

Crystals consisting by light microscopy of organic matrix (matrix crystals) encountered in cytologic urine specimens of 8 patients were examined for mineral phase components by scanning electron microscopy with energy dispersive X-ray microanalysis (SEM-EDX) and by morphologic scanning electron microscopy (SEM) performed separately in four of the eight cases. Whenever possible (three cases) mineralized crystals present in these specimens were examined separately by SEM-EDX for comparison of mineral phase composition with that of the corresponding matrix forms. Although by SEM-EDX components of matrix, glass and slide preparation media interfere with the precise estimation of the mineral phase components, the results of this method supported by the SEM morphology suggest that crystals consisting of organic matrix include a mineral phase, the lattice structure of which provides them from the early stages of formation with the characteristic morphology of the fully mineralized forms. This also suggests that organic matrix plays a role in the nucleation of minerals during the formation of certain urinary crystals. Diagn Cytopathol 1994; 11:38–46. © 1994 Wiley-Liss, Inc.     

Coexistence of Intracytoplasmic Lumens and Membrane-bound Vesicles in an Invasive Carcinoma Arising in a Cystosarcoma Phyllodes

An unusual invasive breast carcinoma, arising in a cystosarcoma phyllodes and characterized by a variable cytoplasmic appearance and mucin content, was evaluated to determine the nature of the secretory material within the cells as well as the type of secretory organelle at the ultrastructural level. Histochemical studies revealed both acidic (sialic acid) and neutral mucin within the tumor cells. Ultrastructural analysis revealed secretory material within membrane-bound vesicles in some cells and within intracytoplasmic lumens in others; some cells contained both membrane-bound vesicles and intracytoplasmic lumens simultaneously. The Golgi derivation of the intracytoplasmic lumens was supported by their presence within or near hyperplastic Golgi complexes. The histochemical characteristics of the secretory material is correlated with their ultrastructural site of accumulation.     

Coexistence of Intracytoplasmic Lumens and Membrane-bound Vesicles in an Invasive Carcinoma Arising in a Cystosarcoma Phyllodes

An unusual invasive breast carcinoma, arising in a cystosarcoma phyllodes and characterized by a variable cytoplasmic appearance and mucin content, was evaluated to determine the nature of the secretory material within the cells as well as the type of secretory organelle at the ultrastructural level. Histochemical studies revealed both acidic (sialic acid) and neutral mucin within the tumor cells. Ultrastructural analysis revealed secretory material within membrane-bound vesicles in some cells and within intracytoplasmic lumens in others; some cells contained both membrane-bound vesicles and intracytoplasmic lumens simultaneously. The Golgi derivation of the intracytoplasmic lumens was supported by their presence within or near hyperplastic Golgi complexes. The histochemical characteristics of the secretory material is correlated with their ultrastructural site of accumulation.     

Oncocytoma of the kidney with prominent intracytoplasmic lumina

Oncocytoma is a histologically distinctive neoplasm of the kidney, with a well-recognized cytoarchitectural appearance. On occasion, however, renal oncocytomas are known to exhibit unusual morphologic features that may pose diagnostic difficulties. We present the clinical and pathologic details of an oncocytoma of the kidney with an unusual histologic appearance imparted by the presence of large numbers of prominent intracytoplasmic lumens. Morphologically, the neoplasm was composed of uniform polygonal cells with copious amounts of granular, eosinophilic cytoplasm, round nuclei, and prominent nucleoli, exhibiting an organoid pattern of growth. Intracytoplasmic lumina of varying size were present throughout the tumor. There were no mitotic figures or areas of necrosis present. The diagnosis of oncocytoma was supported by immunohistochemical and ultrastructural studies. By electron microscopy, the intracytoplasmic lumens appeared as membrane bound spaces with associated microvilli. The presence of intracytoplasmic lumina in a significant proportion of cells is an uncommon feature of renal oncocytoma which can generate problems in diagnosis. Awareness of this phenomenon should allow for improved recognition of oncocytomas exhibiting this type of unusual morphology.