High-resolution microscopy of cell wall formation in regenerating Mougeotia (Chlorophyceae) protoplasts

Field emission scanning electron microscopy (FESEM) preparation techniques have been successfully adapted for visualization of the internal and external ultrastructure of Mougeotia filaments and protoplasts. FESEM of the innermost layer of cell wall in Mougeotia filaments revealed that microfibrils are deposited parallel to each other in an interconnected mesh and are oriented perpendicular to the direction of elongation. For the first time, the surface of protoplasts at different stages of regeneration has been observed using FESEM. Nascent microfibril deposition occurs between 1 and 2 h after isolation and arrangement of these microfibrils is random for at least 8 h. Observation of the inner surface of the plasma membrane in burst protoplasts showed that microtubules are not strongly attached for at least 3 h after protoplast isolation.     

A proteolytic enzyme, trypsin, for immunofluorescence observation of microtubules in algal cells

The usefulness of a proteolytic enzyme, trypsin, in immunofluorescence microscopy was confirmed in algal ceils. Flagella of Umspora penicilliformis zoo-spores were visible using an anti-β tubulin antibody after trypsin treatment, and the cortical microtubules of vegetative cells could also be clearly detected. Interestingly, centrioles that were not detected in the control observation appeared in gametophyte cells of Acrosiphonia duriuscula and Monostroma angicava using the trypsin treatment.     

The effects of ultrarapid freezing on meiotic and mitotic spindles of mouse oocytes and embryos

Preovulatory mouse oocytes and 2-cell embryos were frozen with dimethyl sulfoxide and propanediol by an ultrarapid method. The survival of frozen oocytes was low (33–34%) compared to that of 2-cell embryos (78–79%) with either cryoprotectant. Development to blastocysts after postthaw culture was about 7–15% for oocytes and 79–80% for the embryos. Ultrarapid freezing preserves cell structure quite well as revealed by electron microscopy, but meiotic oocytes and late 2-cell embryos undergoing mitosis showed evidence of spindle disorganization involving loss or clumping of microtubules resulting in some scattering of chromosomes. Embryos developed from frozen eggs showed clear evidence of micronuclear formation and incomplete incorporation of chromosomal material into main nuclei. These experiments confirm our observations on freezing of human oocytes and show that spindle microtubules are sensitive to freeze-thawing and that cryopreservation could cause chromosomal aberrations during early development. A cautious approach to the introduction of oocyte freezing in human in vitro fertilization (IVF) programs is advocated.     

Imaging of plant microtubules with high resolution scanning electron microscopy

Summary High resolution scanning electron microscopy was used to obtain images of cortical microtubules and associated structures in onion root tips. Specimens were prepared using a modified quick-freeze deep-etch technique utilising cytosolic extraction with saponin and conductive staining with osmium.Abbreviations DMSO dimethylsulfoxide - HRSEM high resolution scanning electron microscope/microscopy - MTSB microtubule stabilising buffer - TEM transmission electron microscope/microscopy     

Field emission scanning electron microscopy of plant cells

Summary Two basic specimen preparation protocols that allow field emission scanning electron microscope imaging of intracellular structures in a wide range of plants are described. Both protocols depend on freeze fracturing to reveal areas of interest and selective removal of cytosol. Removal of cytosol was achieved either by macerating fixed tissues in a dilute solution of osmium tetroxide after freeze fracturing or by permeabilizing the membranes in saponin before fixation and subsequent freeze fracturing. Images of a variety of intracellular structures including all the main organelles as well as cytoskeletal components are presented. The permeabilization protocol can be combined with immunogold labelling to identify specific components such as microtubules. High-resolution three-dimensional imaging was combined with immunogold labelling of microtubules and actin cables in cell-free systems. This approach should be especially valuable for the study of dynamic cellular processes (such as cytoplasmic streaming) in live cells when used in conjunction with modern fluorescence microscopical techniques.Abbreviations DMSO dimethylsulfoxide - FESEM field emission scanning electron microscope (-scopy) - MTSB microtubule-stabilizing buffer - PBS phosphate-buffered saline - SEM scanning electron microscope (-scopy) - TEM transmission electron microscope (-scopy)     

Aspects Physico-chimiques de L’assimilation des Hydrocarbures par Candida lipolytica

The proposed similarity of conformation between α-lactalbumin (α-LA) and hen egg-white lysozyme was tested by the comparison of the thermodynamic parameters obtained from the temperature dependence of denaturation. For the denaturing reaction by guanidine hydrochloride, the value of ΔC_P for α-LA is almost identical with that for lysozyme, which suggests that the amount of the hydrophobic side chains buried in the interior of the molecule is the same in the native state ; the value of ΔH° and ΔS° for α-LA are also close to those for lysozyme, and the small differences are explicable by the proposed molecular model of α-LA, which implies that the somewhat large difference in ΔG° observed previously between the two proteins does not originate from large conformational differences. These results support the conformational similarity between α-LA and lysozyme as represented by the molecular model. The heat-denatured state of α-LA is also characterized by the parameters and discussed.     

A Simple Drying Method for Field Emission Scanning Electron Microscopy for Chromosomes

Hexamethyldisilazane treatment and subsequent air drying of spread plant chromosomes is compared with critical point drying. The two procedures are equivalent for preparing chromosomes for examination by field emission scanning electron microscopy at low voltage.     

Correlative 3D imaging of whole mammalian cells with light and electron microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10–20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.     

Some factors determining protein aggregation during ultrafiltration

The factors contributing to protein aggregation in albumin ultrafiltration were investigated as a function of operation conditions. The nature of protein deposits was examined by electron microscopy. Protein aggregation appears to occur as a result of rapid supersaturation of protein molecules and high solvent velocity (shear) in the concentrated layer near the membrane surface. The shear occurring in the solvent flow on the membrane surface probably unfolds protein molecules and thus promotes flocculation due to collision between particles. (c) 1993 John Wiley & Sons, Inc.     

Microtubules Beneath the Pellicles of Two Ciliate Protozoa As Seen With the Sem

SYNOPSIS. Chemical procedures remove some of the outer 3 limiting membranes of 2 ciliate protozoa, Euplotes eurystomus and Tetrahymena pyriformis , and reveal sheets of microtubules in their ectoplasm for SEM study. This greatly enhances the analysis of the 3-dimensional geometry of these sheets, as is shown especially for E. eurystomus. In this organism, sheets of microtubules can readily be observed and described as they course through or around parts of the oral apparatus and other 3-dimensionally complex regions.     

Correlative Light and Scanning Electron Microscopy for Observing the Three-Dimensional Ultrastructure of Membranous Cell Organelles in Relation to Their Molecular Components

Although the osmium maceration method has been used to observe three-dimensional (3D) structures of membranous cell organelles with scanning electron microscopy (SEM), the use of osmium tetroxide for membrane fixation and the removal of cytosolic soluble proteins largely impairs the antigenicity of molecules in the specimens. In the present study, we developed a novel method to combine cryosectioning with the maceration method for correlative immunocytochemical analysis. We first immunocytochemically stained a semi-thin cryosection cut from a pituitary tissue block with a cryo-ultramicrotome, according to the Tokuyasu method, before preparing an osmium-macerated specimen from the remaining tissue block. Correlative microscopy was performed by observing the same area between the immunostained section and the adjacent face of the tissue block. Using this correlative method, we could accurately identify the gonadotropes of pituitary glands in various experimental conditions with SEM. At 4 weeks after castration, dilated cisternae of rough endoplasmic reticulum (RER) were distributed throughout the cytoplasm. On the other hand, an extremely dilated cisterna of the RER occupied the large region of the cytoplasm at 12 weeks after castration. This novel method has the potential to analyze the relationship between the distribution of functional molecules and the 3D ultrastructure in different composite tissues.     

Translation Initiation Factor eIF3 Probably Binds with Microtubules in Mammalian Cells

Association of the translation apparatus with the cytoskeleton is essential for its transportation within the cell and probably also for translation regulation. Very little is known about the involvement of particular proteins of this association. A polypeptide homologous with the heavy chain of translation initiation factor eIF3 p170 was found earlier in a microtubule preparation from adrenal cells. Antibody A167 directed against the recombinant fragment of p170 has been generated to study eIF3 interaction with microtubules in mammalian cells. This antibody was shown to recognize a single 170-kDa polypeptide in eIF3 preparations as well as in homogenates of various cell types. A167 allowed detection of the 170-kDa polypeptide in microtubule preparation from bovine brain and confirmation of its presence in microtubule preparations from adrenal cells. As shown by immunofluorescence microscopy using A167, the 170-kDa polypeptide is mainly located in the endoplasm within numerous small and some large granules. Cell treatment with cycloheximide resulted in growth and clustering of the large granules, and partial antigen redistribution along intracellular microtubules. These new experimental data indicate that mammalian translation factor eIF3 may bind with microtubules.     

免疫脂质体对白血病细胞杀伤的扫描电镜观察

采用脂质体技术包裹中华眼镜蛇膜毒素(CT)并与抗人T细胞单克隆抗体结合制备成pH敏感型免疫脂质体.它在pH8.0～7.0间十分稳定,但在接近细胞浆微酸性环境(pH<6.0)时很容易破裂而释出包裹之内容物.用这一免疫脂质体处理人白血病T淋巴细胞系CEM细胞并用扫描电镜观察脂质体对靶细胞的作用,显示这一免疫脂质体可特异性杀灭靶细胞而对抗原阴性细胞影响不大．     

Adhesion of intermediate filaments and lipid droplets in adrenal cells studied by field emission scanning electron microscopy

High-resolution field emission scanning electron microscopy was used to study the organisation of intermediate filaments around lipid droplets and their binding to these droplets, in primary culture of bovine adrenal cells. Whole-mount preparations of intermediate filaments and bound lipid droplets were prepared from cells grown on Formvar-coated grids and processed by freeze-drying. Intermediate filaments were seen as an interconnected network enveloping the entire droplet. The bound filaments appear to be directly adherent to the surface of the droplet and hence take on its curved contour. The binding of the filaments to the droplets was determined by means of tilting. This study provides a new approach to investigate the cytoskeleton and its associated structures with high-resolution three-dimensional images.     

粗茎鳞毛蕨原叶体细胞有丝分裂过程中微管列阵的变化

应用Steedman‘s wax切片法,间接免疫荧光标记技术和激光共聚焦扫描显微镜技术研究了粗茎鳞毛蕨（Dryopteris crassirhizoma Nakai)原叶体大液泡化细胞和分生组织细胞有丝分裂过程中微管列阵的变化。结果显示：应用高浓度的多聚甲醛（8%）可以很好地保持大液泡化细胞的结构和微管的抗原性。结果也显示Steedman‘s wax切片法和间接免疫荧光标记技术的优点;（1）避免在微管标记过程中酶解细胞壁;（2）在乙醇脱水过程中样品中叶绿素的自发荧光被减到最小;（3）能够详细观察到有丝分裂过程中微管骨架的变化。因此,这种方法可以被广泛用来调查简单植物体和复杂植物体中细胞的有丝分裂过程以及发育过程中微管骨架的变化。