眼组织的透射电镜制样方法

介绍一种眼组织透射电子显微镜标本制备方法。组织先用４％多聚甲醛－２．５％戊二醛混合固定液预固定,１％四氧化锇固定液后固定,丙酮逐级脱水,延长Ｅｐｏｎ８１２包埋液的浸透时间并包埋,用玻璃刀或钻石刀作超薄切片,醋酸主枸橼酸铅双重染色,Ｈ６００－Ⅳ型透射电子显微镜观察。此方法的优点在于眼组织各层超微结构保存良好,没有损伤及变性,我们对该方法的操作要点作了讨论。     

New Techniques: A Fast Method for Processing Biopsy Material for Electron Microscopy

A rapid method for fixation and embedding of biopsy material for electron microscopic examination has been developed. The procedure was simplified by fix ing the biopsies with fixative in ready-to-use ampules and hastened by chemical dehydration in 2.2-dimethoxypropane. The procedure takes 4 h and yields excellent results.     

Microcomputer Application: An Image Database System for Electron Microscopy

An electron microscopist in a small electron microscopy laboratory that processes 200 cases per year can generate at least 2000 electron micrographs within this time period. These electron micrographs are stored in filing cabinets, which slowly encroach on existing laboratory space. Related image data are often located in separate filing systems. This type of storage system is inefficient for rapid retrieval of sets of electron micrographs for research, teaching, or retrospective review of patient material. An electron microscopy image base, which is implemented on a microcomputer, can provide flexible and simultaneous access to both digitized electron micrographs and their relevant textual data. In addition to the advantage of more efficient retrieval, electronic storage of textual data and digitized electron micrographs also offers the advantage of decreased storage space for this type of data. Implementation of the initial version of the electron microscopy image base demonstrated that future versions must provide improved resolution of the digitized electron micrograph and sufficient electronic mass storage to support such a large image base. Ongoing developments in database technology and mass storage make it worthwhile to pursue additional refinements of the electron microscopy image base.     

Bronchopulmonary Dysplasia: A Correlative Study by Light, Scanning, and Transmission Electron Microscopy

The sequential stages of bronchopulmonary dysplasia occurring in 18 infants after intensive respiratory therapy supplemented by oxygen in high concentrations were studied by correlative light, scanning, and transmission electron microscopy. Infant survival ranged from 3 to 225 days. The earliest stage was an exudative reaction with a predominance of hyaline membranes. This merged with a subacute reparative response that was replaced by a chronic fibroproliferative stage in infants of longest survival; this stage was complicated by pulmonary fibrosis and emphysema. Correlative scanning and transmission electron microscopy demonstrated that type 2 pneumocytes contributed significantly to the reparative fibroproliferative response by organization of hyaline membranes and reepithelialization of damaged septal walls.     

Fixation/Permeabilization: New Alternative Procedure for Immunofluorescence and mRNA In Situ Hybridization of Vertebrate and Invertebrate Embryos

A new procedure is described to visualize the spatial pattern of expression of proteins and mRNAs in cryosections or whole‐mounted leech, Drosophila, zebrafish, and chick embryos. Our principal contribution is in the use of a nonconventional fixation/permeabilization procedure based on the use of formaldehyde or paraformaldehyde combined with a short C‐chain carboxylic acid. Detergents, methanol, and proteinases were omitted. Hybridization procedures were modified from those of routinely used protocols developed for the same embryos. Results showed that cytoskeletal and other cytoplasmic proteins, as well as different mRNAs, were clearly visualized in the expected regions of the embryos. Our procedure has several advantages over currently used protocols: is simpler, produces better general preservation of cells, yields reliable results, and can be used for embryos of different taxa at different developmental stages. It is hypothesized that short C‐chain aliphatic carboxylic acids modulate the cross‐linking effect of aldehyde fixatives on cell proteins. Developmental Dynamics 242:493–507, 2013. © 2013 Wiley Periodicals, Inc.     

An Image Analysis Workstation Designed for Multiple Users: Application of Quantitative Digital Imaging Techniques to Electron Microscopy

The purpose of the present study is to describe the setup of an image analysis workstation designed for multiple users, and to show the application of digital imaging techniques to the analysis of electron microscopic images. The image analysis system consists of a conventional light microscope mounted on a table-top, vibration-free platform, a light box for viewing negatives, two separate video cameras, a switch box, a video monitor, a digitizing tablet, a computer, and mor-phometric software packages. The system can quantitate the amount that each of the 256 gray levels contributes to the image, perform morpho-metric analysis (eg, shape and size) on individual gray level-defined subimages, and perform statistical analysis. Each operator has access to his or her own data and program setups through the use of 21.4-Mb removable Bernoulli cartridges. This setup for multiple users prevents the cluttering of the hard drive of the computer and avoids the possibility of accidentally removing the stored data of another user. The quantitative capabilities of the digital imaging system is demonstrated using an image of a normal lymphocyte and an apoptotic cell (ie, a cell which has undergone programmed cell death), both captured on the same electron microscopic negative. A comparison of the histograms of nuclear densities determined for these two cells reveals subtleties in gray level distribution not appreciated by the naked eye.     

Correlative Light Optical,Scanning Electron,and Transmission Electron Microscopy of Skeletal Muscle in Muscular Dystrophy and Muscular Atrophy: A Pilot Study

Biopsies of skeletal muscle from three different cases of muscular dystrophy and one case of spinal muscular atrophy that had been fixed with Karnovsky's fluid were either routinely prepared for scanning electron microscopy (SEM) or were frozen to-20°C and sectioned on a steel knife in a cryostat at 5-10 μm. The sections were coverslipped and examined using a light microscope equipped with polarizing optics (Pol). After areas were selected, the sections were prepared for SEM and thereby examined. The tissues on the slides that had been observed with light microscopy (LM) and SEM were prepared further for transmission electron microscopy (TEM) by infiltrating them with Epon and cutting sections at approximately 100 nm on an ultramicrotome. It is shown that the stage of contraction in one pathologic myofiber may vary along its length. The following advantages may be realized by using correlative (Pol → SEM → TEM) microscopy on skeletal muscle biopsies: 1) lesions can be differentiated from “normal” surrounding tissue; 2) doubtful structures can be reexamined with the SEM and TEM; and 3) the SEM image of different states of muscle contraction can be reinterpreted in the light of the Pol or TEM image.     

Correlative Light Optical, Scanning Electron, and Transmission Electron Microscopy of Skeletal Muscle in Muscular Dystrophy and Muscular Atrophy: A Pilot Study

Biopsies of skeletal muscle from three different cases of muscular dystrophy and one case of spinal muscular atrophy that had been fixed with Karnovsky's fluid were either routinely prepared for scanning electron microscopy (SEM) or were frozen to-20°C and sectioned on a steel knife in a cryostat at 5-10 μm. The sections were coverslipped and examined using a light microscope equipped with polarizing optics (Pol). After areas were selected, the sections were prepared for SEM and thereby examined. The tissues on the slides that had been observed with light microscopy (LM) and SEM were prepared further for transmission electron microscopy (TEM) by infiltrating them with Epon and cutting sections at approximately 100 nm on an ultramicrotome. It is shown that the stage of contraction in one pathologic myofiber may vary along its length. The following advantages may be realized by using correlative (Pol → SEM → TEM) microscopy on skeletal muscle biopsies: 1) lesions can be differentiated from “normal” surrounding tissue; 2) doubtful structures can be reexamined with the SEM and TEM; and 3) the SEM image of different states of muscle contraction can be reinterpreted in the light of the Pol or TEM image.     

Electron Microscopy Renders the Diagnostic Capabilities of Cytopathology More Precise: An Approach to Everyday Practice

Cytology is a powerful diagnostic tool but to make definitive diagnoses, the use of ancillary techniques is imperative. By combining immunohistochemistry (IHC) and electron microscopy (EM), cytologic diagnoses can be as precise as those of surgical pathology. In the authors' daily practice of cytopathology they use all ancillary techniques available to them: histochemistry, IHC, EM, flow cytometry, and molecular pathology. IHC is frequently used as an ancillary technique in their daily practice but EM is many times their technique of choice. By the use of EM the authors can make specific final diagnoses, make the diagnosis more definitive, narrow the differential diagnosis, or determine the origin of a neoplasm with unknown primary site. Specimens obtained by fine-needle aspiration as well as all body fluids are suitable for EM. The limiting factor is to obtain the appropriate material with the diagnostic cells for ultrastructural examination. The common diagnostic dilemmas in the everyday practice of cytology are the following: mesothelioma vs. adenocarcinoma, neuroendocrine differentiation or not, the distinction of melanoma from adenocarcinoma and sarcoma, hepatocellular carcinoma vs. adenocarcinoma, and the origin of adenocarcinomas of unknown primary. The authors discuss how they approach these diagnostic problems in their everyday practice and how they incorporate EM in solving them.     

Thickening of Glomerular Capillary Walls: A Guide to Differential Diagnosis by Electron Microscopy

Thickening of capillary walls is a feature of many glomerular diseases. Widening of the wall may be produced by deposits and other changes affecting either subepithelial and subendothelial regions or the glomerular basement membrane itself. Careful light microscopic examination using special stains can distinguish some patterns of capillary wall thickening, but electron microscopy is needed to demonstrate most lesions clearly. In this brief review, a guide to the major causes for capillary wall thickening is discussed, using a simple classification, and some of the patterns are illustrated. Precise delineation of the capillary wall changes in glomerular diseases is important to insure accurate classification and a clear understanding of pathogenetic mechanisms.     

Thickening of Glomerular Capillary Walls: A Guide to Differential Diagnosis by Electron Microscopy

Thickening of capillary walls is a feature of many glomerular diseases. Widening of the wall may be produced by deposits and other changes affecting either subepithelial and subendothelial regions or the glomerular basement membrane itself. Careful light microscopic examination using special stains can distinguish some patterns of capillary wall thickening, but electron microscopy is needed to demonstrate most lesions clearly. In this brief review, a guide to the major causes for capillary wall thickening is discussed, using a simple classification, and some of the patterns are illustrated. Precise delineation of the capillary wall changes in glomerular diseases is important to insure accurate classification and a clear understanding of pathogenetic mechanisms.     

Cessation of Gonadotropin-Releasing Hormone Antagonist on Triggering Day: An Alternative Method for Flexible Multiple-Dose Protocol

This study was performed to analyze retrospectively outcomes of stimulated in vitro fertilization (IVF) cycles where the gonadotropin-releasing hormone (GnRH) antagonist was omitted on ovulation triggering day. A total of 92 consecutive IVF cycles were included in 65 women who are undergoing ovarian stimulation with recombinant FSH. A GnRH antagonist, cetrorelix 0.25 mg/day, was started when leading follicle reached 14 mm in diameter until the day of hCG administration (Group A, 66 cycles) or until the day before hCG administration (Group B, 26 cycles). The duration of ovarian stimulation, total dose of gonadotropins, serum estradiol levels on hCG administration day, and the number of oocytes retrieved were not significantly different between the two groups. The total dose of GnRH antagonist was significantly lower in Group B compared to Group A (2.7±0.8 vs. 3.2±0.9 ampoules). There was no premature luteinization in the subjects. The proportion of mature oocytes (71.4% vs. 61.7%) and fertilization rate of mature (86.3±19.7% vs. 71.8±31.7%) was significantly higher in Group B. There were no significant differences in embryo quality and clinical pregnancy rates. Our results suggest that cessation of the GnRH antagonist on the day of hCG administration during a flexible multiple-dose protocol could reduce the total dose of GnRH antagonist without compromising IVF results.     

A Cross‐Talk of Decidual Stromal Cells,Trophoblast, and Immune Cells: A Prerequisite for the Success of Pregnancy

Embryo implantation and formation of a functional placenta are complex processes that require a plethora of regulatory mechanisms involving both mother and embryo cells. Recently, an important role in this complicated cells and factors network was assigned to the decidual stromal cells (DSC) and trophoblast cells. Decidualization includes biochemical changes that trigger DSC to produce a number of factors required for the implantation and induction of immunotolerance in maternal immune system. Immunotolerance is achieved by a cascade of strictly controlled events starting with selective homing of immune cells to the feto‐maternal site, regulated proliferation, and predominant differentiation into a regulatory type of immune cells. Furthermore, cytotoxic effector functions are reduced owing to the influence of steroid hormones, factors, cytokines, and inhibitory receptors. Altogether the entire immune system of the mother is switched to tolerogenic functional state which is a prerequisite for the successful maintenance of pregnancy.     

The Effects of Rearing Light Level and Duration Differences on the Optic Nerve,Brain, and Associated Structures in Developing Zebrafish Larvae: A Light and Transmission Electron Microscope Study

The ultrastructure of the optic nerve, brain, and some associated structures of larval zebrafish, grown under three different light regimens were studied. Fish grown under cyclic light (control), constant dark (CD), and constant light (CL) were studied for 4 and 8 days postfertilization (dpf). We also studied the control and CD fish at 15 dpf. The brains of the control and CL fish were larger at 4 dpf than at 8 dpf. In all 4 dpf fish, the brain occupied the entire expanse between the two retinas and the optic nerve extended the shortest distance between the retina and the brain. The 15 dpf zebrafish had the smallest brain size. Groups of skeletal muscle cells associated with the optic nerves became visible in all older larvae. In the 15 dpf larvae, bulges and dilations in the optic nerve occurred as it reached the brain and optic chiasms occurred proximal to the brain. Electron microscopy yielded information about myelinated and unmyelinated axons in the optic nerve, the dimensions of neurotubules, neurofilaments, and myofilaments, including a unique variation in actin myofilaments, and a confirmation of reported myosin myofilament changes (but with dimensions). We also describe the ultrastructure of a sheath‐like structure that is confluent over the optic nerve and the brain, which has not been described before in zebrafish. Also presented are images of associated fibroblasts, epithelial cells lining the mouth, cartilage plates, blood vessels, nerve bundles, and skeletal muscle cells, most of which have not been previously described in the literature. Anat Rec,, 2012. © 2012 Wiley Periodicals, Inc.     

Electron Microscopy Remains the Gold Standard for the Diagnosis of Epithelial Malignant Mesothelioma: A Case Study

This is a case of idiopathic epithelial malignant mesothelioma in a 47-year-old mechanic. The advent of a large battery of immunochemical markers has provided new tools for the diagnosis of mesothelioma in recent years; however, immunostaining can often be misleading or inconsistent, as demonstrated in this case. This report highlights the lasting utility of electron microscopy in the diagnosis of mesothelioma. Ultrastructural features of epithelial mesothelioma were discernable using electron microscopy even on somewhat poorly preserved chest wall biopsy specimens from paraffin blocks. These images, combined with immunostains and a fiber analysis from the lungs, allowed for a final diagnosis of a non-asbestos-related malignant epithelial mesothelioma in this patient.     

Contribution of Electron Microscopy to Understanding Cellular Differentiation in Mesenchymal Tumors of the Gastrointestinal Tract: A Study of 82 Tumors

Eighty-two mesenchymal tumors of the gastrointestinal tract were examined by electron microscopy for the purposes of subtyping for diagnostic precision and of understanding cellular differentiation. Tumors were subclassified into leiomyoma/leiomyosarcoma, tumors of the interstitial cell of Cajal (equivalent to traditionally defined GISTs [Miettinen et al. Hum Pathol. 1999; 30:1213-1220; Mod Pathol. 2000; 13:1134-1142]), gastrointestinal autonomic nerve tumors (GANTs), and fibroblastic and myofibroblastic tumors, using criteria from the literature. Leiomyoma/leiomyosarcoma were diagnosed by myofilaments, attachment plaques, plasmalemmal caveolae, and lamina; GIST by processes or cell bodies full of intermediate filaments, solitary focal densities amid intermediate filaments, attachment plaques with incomplete lamina, scarce myofilaments, and smooth endoplasmic reticulum; GANTs by neuroendocrine granules, cell bodies/processes full of intermediate filaments (more rarely microtubules), and smooth endoplasmic reticulum; fibroblastic/myofibroblastic tumors by abundant rough endoplasmic reticulum, myofilaments, and fibronexuses. Seventy-three tumors (89%) were successfully subclassified, as 5 leiomyoma/leiomyosarcoma (6%), 36 GISTs (44%), 22 GANTs (27%), 10 fibroblastic and myofibroblastic tumors (12%). Results indicated overlap between poorly differentiated leiomyosarcoma and GIST, and between GIST and GANT. GANT is emphasized as a neuronal tumor identifiable by electron microscopy, and thereby distinguishable from GIST.     

Attachment of hepatitis C virus to cultured cells: A novel predictive factor for successful interferon therapy

In order to identify reliable predictive factors for the response to interferon (IFN) therapy, we examined 52 patients with chronic hepatitis C virus (HCV) infection who underwent IFN therapy. Titers of free virions that were not complexed with antibodies were determined by immunoprecipitation of the pretreatment sera with antihuman immunoglobulin. Cell attachment titers were determined by inoculating HPB-Ma cells, a human T-cell line, with the patients' pretreatment sera. A sustained remission was achieved in 9 out of 28 patients with genotype 1b infection and in 18 out of 24 patients with genotype 2 infection. The free virion titers and the cell attachment titers correlated well with the serum HCV RNA levels in patients with genotype 1b infection (r = 0.817, P < 0.001; r = 0.802, P < 0.001, respectively), but not in those with genotype 2 infection. They exhibited a high correlation with the responsiveness to IFN in all patients combined (P < 0.001). A multiple logistic regression analysis was performed to identify the factor most significantly associated with the response to IFN among all confounding factors. The analysis indicated that the cell attachment is the most reliable predictive factor regarding a favorable response to IFN therapy in patients with chronic HCV infection. J. Med. Virol. 56:25–32, 1998. © 1998 Wiley-Liss, Inc.     

Epithelial Cell Rests of Malassez and OX6‐Immunopositive Cells in the Periodontal Ligament of Rat Molars: A Light and Transmission Electron Microscope Study

It has been shown that human and cat epithelial cell rests of Malassez (ERM) consist of heterogeneous cell populations. Immunohistochemical and immunoelectron microscopic analyses have verified the presence of neuroendocrine and Merkel‐like cells in both of these epithelia. During experimental orthodontic tooth movement, immunocompetent cells have also been found in the vicinity of ERM in rat periodontal ligament (PDL), but have not been characterized in normal rat PDL. The aim of this study was to investigate the presence and distribution of MHC class II antigen presenting cells by using OX6 antibody in ERM of rat molars by light and transmission electron microscopy. Immunohistochemical and immunoelectron microscopic observations of rat maxillary molars confirmed the presence of OX6‐positive cells in contact with ERM. Some immunopositive cytoplasmic processes containing vesicles interdigitated with cells of the Malassez epithelial clusters. Based on these findings it can be concluded that immunocompetent cells are localized close to Malassez epithelial clusters in normal rat PDL. Furthermore, the ultrastructural evidences indicate a possible interaction between the epithelial and immunocompent cells and suggest morphological and functional properties for ERM. Anat Rec, 291:242–253, 2008. © 2008 Wiley‐Liss, Inc.     

Induction of nitric oxide release from the human alveolar epithelial cell line A549: an in vitro correlate of innate immune response to Mycobacterium tuberculosis

In view of the presence of a large number of epithelial cells in the alveoli of the lung and their ability to produce various cytokines and chemokines, the possible role of alveolar epithelial cells in the innate immune response to tuberculosis was examined. The human alveolar epithelial cell line A549 was used as a model. The ability of A549 cells to induce nitric oxide (NO) in response to Mycobacterium tuberculosis infection was taken as an in vitro correlate of innate immunity. M. tuberculosis infection induced A549 cells to produce significant levels of NO and to express inducible nitric oxide synthase mRNA at 48 hr of infection. However, the amount of NO released at this point was not mycobactericidal. Cytokine stimulation (interferon-gamma, tumour necrosis factor-alpha, interleukin-1beta, alone or in combination) of the infected A549 cells induced a higher concentration of NO. The study of colony-forming units (CFU) as a measure of the mycobactericidal capacity of A549 cells revealed a reduction in CFU of M. tuberculosis by 39.29% (from 10.62 +/- 0.48 - 6.392 +/- 0.54) following cytokine stimulation of the infected cells. Interestingly gamma-irradiated M. tuberculosis H37Rv could also induce higher than basal level of NO. Therefore we examined mycobacterial antigenic components for their possible role in NO production. We observed that A549 cells produced significantly higher amounts of NO at 48 hr when treated with mycobacterial whole cell lysates, cell wall or cell membrane preparations. The release of NO and the resultant mycobactericidal activity could be further enhanced by simultaneously conditioning the M. tuberculosis infected A549 cells with cytokine and mycobacterial components. These results suggest that alveolar epithelial cells respond to their microenvironment, which is constituted of various cytokines and macrophage-processed antigens and may contribute to the innate immune response to tuberculosis.