少量贴壁培养细胞电镜原位包埋方法的探讨

该文探讨了对少量贴壁培养细胞较易操作且能保存较好超微结构的透射电镜样品包埋的方法。将Hela细胞分为三组:(1)不使用环氧丙烷,将树脂胶囊直接倒扣包埋于塑料培养皿;(2)不使用环氧丙烷,将细胞爬片倒扣包埋于胶囊;(3)使用环氧丙烷并将细胞爬片倒扣包埋于胶囊。将三组带有细胞的树脂胶囊进行超薄切片,电镜观察后发现,第一种方法包埋简便,超薄切片上无细胞缺失孔洞,且超微结构保存较好。     

不同厚度细胞薄切片对 AFM 图像反差的影响

利用原子力显微镜（ AFM ）观察超薄切片的表面,探索表面形貌与切片厚度、朝向等因素的关系以及对图像反差的影响 . 选择三种不同类型的细胞,培养后按电镜超薄切片法固定、包埋并切片后,将不同厚度的切片区分上下表面转移到云母上, AFM 在空气中以接触模式进行观察 . 结果发现,切片表面细胞相对包埋介质的凸起与凹陷与切片本身的厚度密切相关,并随切片厚度的不同呈现有规律的变化 . 实验统计结果显示这种现象可能具有普遍性 .     

AtRabD2b共抑制植株死亡茎段的显微和超微结构观察

采用树脂包埋技术,以AtRabD2b共抑制植株死亡发生茎段为实验材料,制备半薄切片和超薄切片,观察突变体茎段死亡的细胞学特征。结果表明:(1)共抑制植株茎细胞的死亡首先在表皮细胞层中发现,然后向切片圆周两侧以及内侧细胞蔓延。(2)共抑制植株茎细胞出现染色质边缘化、叶绿体内囊体片层膜数目减少、细胞器成分被液泡吞噬等异常现象。这些细胞学特征暗示共抑制植株茎段发生了细胞程序性死亡,由此推断AtRabD基因对拟南芥茎顶端细胞的生长有重要的维持作用。     

用于光学显微镜研究的塑料半薄切片

本文介绍了以环氧树脂为包埋介质的用于光学显微镜的塑料半薄切片的制备技术和部分实验结果。叙述了固定、脱水、渗透、包埋、聚合、切片、染色及封片各程序。作为对石蜡切片技术的补充和发展。塑料半薄切片能充分发挥光学显微镜的分辨能力,能观察到许多在石蜡切片上看不清或看不到的细胞内部结构,如:花粉的外粉壁,萌发孔;细胞的微核,液泡和液泡问的原生质丝等。可用同一包埋材料在半薄切片基础上进行超薄切片,所以半薄切片技术是一种把光学显微镜水平的研究和电子显微镜水平的研究联系在一起的一种过渡性技术。因此,它无论对植物学工作者或其它生物学工作者都是很有用的一项技术。     

神经元单层细胞电镜原位包埋方法的探讨

为探究神经元单层细胞电镜原位包埋方法,该研究以体外培养的大鼠皮层神经元单层细胞为材料,采用了三种不同的方法进行样品制备。结果表明,常规悬浮离心包埋法容易对细胞造成机械损伤,且无法保持原位性;培养在玻璃盖玻片上的细胞,虽能保持较好的原位性,但其超薄切片表层易破碎,遇水溶解,且在透射电镜下可见细胞整体衬度弱和超微结构不完整;而培养在Thermanox塑料薄膜盖玻片上的细胞,可容易观察到相邻细胞间的紧密连接,超微结构清晰,衬度较前组样品好,三维重构之后可很好地显示神经元细胞之间的毗邻关系。因此,相比前两种细胞制样方式,Thermanox塑料薄膜上单层细胞处理过程简便,成功率高。该结果为神经元细胞超微结构的形态学研究进一步提供技术新思路,并有望推动神经元细胞超微结构观察中三维重构技术和光镜/电镜联用技术的开发应用。     

花粉壁的透射电镜标本制备

在透射电镜下观察花粉壁的超薄切片,是当前研究花粉外壁构造的重要方法,也是鉴定花粉的重要依据。但花粉外壁主要由孢粉素组成,因此很坚硬,所以在制备花粉壁的超薄切片时,溶剂较难渗入,给包埋和切片造成一定的困难。本文介绍的方法,是作者多次实验的总结。依照此方法可以得到较为满意的结果。     

大豆根瘤内谷氨酸脱氢酶(GDH)的免疫细胞化学研究

用Lowicryl K_4M树脂包埋大豆根瘤组织块,以蛋白A-胶体金和从羽扇豆根瘤中所提取的GDH作为抗原制备的免疫球蛋白标记上述已包埋的大豆根瘤组织的超薄切片,在大豆根瘤组织内定位GDH。电镜观察与计算机分析结果表明,谷氨酸脱氢酶(GDH)集中分布在靠近大豆根瘤细胞内壁的线粒体上面。     

植物材料的冷冻超薄切片制作法

冷冻超薄切片法比常规超薄切片法步骤少、速度快,它不需接触到剧烈的化学试剂及脱水、包埋等,并能良好地保存细胞中的一些水溶性物质,在电镜下所观察到的细胞结构更接近于自然状态。因此,它比较适合于形态学、电镜细胞化学和元素的X-射线微区分析等研究领域。     

生物样品冷冻超薄切片技术简介

电子显微镜超薄切片技术的发展开拓了组织学和细胞学的显微和亚显微结构的研究。但由于常规超薄切片术采用强烈化学囿定,有机溶剂脱水和塑料包埋等步骤所带来的结构损伤和分子活性散失等缺点,人们在光学显微冷冻切片和常规超薄切片基础上,近年来发展了冷冻超薄切片制样技术。七十年代初期,商品冷冻超薄切片装置先后问世,进一步促进了这一技术的发展。     

薄层包埋法的改进

薄层包埋法是北京大学胡适宜教授的实验室最先将其应用到植物材料的制作中（胡适宜和徐丽云,１９９３）。它主要是针对一些植物样品如花粉、花粉管和细胞原生质体等在超薄切片制作中较难定位而设计的。由于该方法能准确定位,因而大大提高了这些样品的超薄切片效率和质量...     

IMPROVED TECHNIQUES FOR THE PREPARATION OF ULTRATHIN FROZEN SECTIONS

Ultrathin frozen sections of biological tissues for electron microscopy provide certain advantages in cytochemical studies in which the penetration of cells by large molecules is necessary and in morphological studies of cellular constituents which are dissolved by the reagents employed in routine plastic embedding. The recent introduction of several types of commercially available cryo-ultramicrotomes makes it possible for many laboratories to employ this valuable tool. This paper summarizes recent improvements in the methods developed in this laboratory for preparing ultrathin frozen sections and reviews some of the inherent problems involved in their use. These procedures may serve as a baseline for other investigators who can then modify or adapt them for their specific purposes.     

Silver enhancement of protein A-gold probes on resin-embedded ultrathin sections. An electron microscopic localization of mouse mammary tumor virus (MMTV) antigens

A simple method is described allowing the enhancement of the visibility of small gold probes for the electron microscopy. This method, which allows the silver intensification of gold directly on epon-embedded ultrathin sections, was used for the electron microscopic localization of Mouse Mammary Tumor Virus (MMTV) antigens in cultured cells derived from GR and BALB/cfRIII mouse mammary tumors. After the immunostaining with the preembedding protein A-gold technique, the ultrathin sections, placed on 200 mesh copper grids, were rehydrated and exposed to a photographic developer containing silver nitrate. During this physical development gold particles are incapsulated in growing shells of metallic silver, which gradually become more and more visible. We were able to obtain a heavy labelling of the viral particles, well visible even at low magnification, with a negligeable background staining. The present technique can be useful whenever it is necessary to use the smallest gold probes today available.     

Silver enhancement of protein A-gold probes on resin-embedded ultrathin sections

Summary A simple method is described allowing the enhancement of the visibility of small gold probes for the electron microscopy.This method, which allows the silver intensification of gold directly on epon-embedded ultrathin sections, was used for the electron microscopic localization of Mouse Mammary Tumor Virus (MMTV) antigens in cultured cells derived from GR and BALB/cfRIII mouse mammary tumors. After the immunostaining with the preembedding protein A-gold technique, the ultrathin sections, placed on 200 mesh copper grids, were rehydrated and exposed to a photographic developer containing silver nitrate. During this physical development gold particles are incapsulated in growing shells of metallic silver, which gradually become more and more visible. We were able to obtain a heavy labelling of the viral particles, well visible even at low magmfication, with a negligeable background staining.The present technique can be useful whenever it is necessary to use the smallest gold probes today available.Supported by contract No. 85.02038.44 from the National Research Council, Rome, Progetto Finalizzato Oncologia     

Electron microscopic and histochemical studies of the mononuclear osteoclast of the mouse.

Osteoclasts collected from the long bones of mice were cultured on dentin slices. To identify osteoclasts, the tartrate-resistant acid phosphatase (TRACPase) activity of cultured cells was histochemically examined by the azo dye method. The TRACPase-positive cells could be distinguished from other cells by light microscopy. The cells were sectioned by alternating semithin and ultrathin sections to observe their ultrastructure and three-dimensional structure. TRACPase activity was detected both in multi-nucleated osteoclasts and in mononuclear cells. Most of the mononuclear TRACPase-positive cells had features similar to preosteoclasts. A mononuclear TRACPase-positive cell was a ruffled border and clear zone was reconstructed three-dimensionally by NIKON COSMOZONE 2SA. The reconstruction showed that this cell possessed a large clear zone and small ruffled border. Under the ruffled border, no lacuna was apparent; but there was disruption of the dentin surface. The results suggest that this cell was a mononuclear osteoclast and that it might have been in the process of making a new lacuna.     

Tubular spinae are long-distance connectors between bacteria

The marine pseudomonad D71 (NCMB 2018) ['Spinomonas maritima'] can be induced to produce long tubular surface appendages (spinae) in a growth medium of low osmolarity. In general, spina-carrying cells show these appendages with open distal ends. We examined cultured cells by scanning and transmission electron microscopy, using both critical-point drying and thin sectioning after embedding with agarose protection. By scanning electron microscopy, spinae were observed that connected cells over distances of several micrometers. Ultrathin sections often revealed an additional layer outside the outer membrane, resembling an S-layer. The inner and outer cell membranes were often joined at spina-insertion areas. Furthermore, evidence was found in ultrathin sections for uninterrupted tubes connecting two cells over a distance of up to 7 microns. We propose, therefore, that spinae form the framework for wide open cell clusters; we hypothesize that these spinae might also permit an exchange of cell-to-cell signals.     

Studies on the ultrastructure of some cyanolichen haustoria

Summary Four fruticose lichens of different genera, all belonging to the cyanolichen familyLichinaceae were studied by ultrathin sectioning and freeze-fracturing/-etching in order to see details in the structure of the photobiont-mycobiont interface. Within the haustorial region, the fibrillar sheath of the photobiont was almost absent and the thickness of the fungal cell wall was strongly reduced.The wavy outline of the cytoplasmic membrane in haustorial cells, which is so obvious in ultrathin sections, was found to be an artifact,i.e., originating during specimen preparation, it was not found in freeze-fractured samples.Invaginations of the fungal cytoplasmic membrane that were 25–125nm in width and 50–800 nm in length occurred in ultrathin sections and freeze-fractured samples. The invaginations were located within the cytoplasmic membrane of haustorial and non-haustorial cells.No differences between freshly collected and rewetted dry herbarium specimens could be detected by means of transmission electron microscopy.     

A novel flat-embedding method to prepare ultrathin cryosections from cultured cells in their in situ orientation.

Immunogold labeling of ultrathin cryosections provides a sensitive and quantitative method to localize proteins at the ultrastructural level. An obligatory step in the routine preparation of cryosections from cultured cells is the detachment of cells from their substrate and subsequent pelleting. This procedure precludes visualization of cells in their in situ orientation and hampers the study of polarized cells. Here we describe a method to sample cultured cells from a petri dish or coverslip by embedding them in a 12% gelatin slab. Subsequently, sections can be prepared in parallel or perpendicular to the plane of growth. Our method extends the cryosectioning technique to applications in studying polarized cells and correlative light-electron microscopy.     

Ultrastructure of Lymphocystis Virus

Lymphocystis virus obtained from bluegills (Lepomis macrochirus) was cultured in the permanent bluegill cell line BF-2 and examined by electron microscopy in ultrathin sections of cell cultures and in negative-contrast preparations from cells and from centrifuged culture medium. According to negative-contrast preparations, the icosahedral virions have an overall diameter close to but not exceeding 300 mμ. Delicate filaments seem to issue from the vertices. In collapsed virions, an ordered array of morphological units was seen. Positively contrasted virions in ultrathin sections show a shell with three dark (heavy metal-stained) layers alternating with and separated by two clear layers. The acquisition of an additional outer membrane during release from the cell, as found in African swine fever virus, was never seen. Morphologically, lymphocystis virus is considered to be closely related to Tipula iridescent virus.     

Three-dimensional ultrastructure of anionic sites of the glomerular basement membrane by a quick-freezing and deep-etching method using a cationic tracer

The ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18-21 particles/1000 nm of LRE) with fairly regular interspacing (45-65 nm) as reported previously.(ABSTRACT TRUNCATED AT 250 WORDS)     

Connexin 43 and the glucose transporter,GLUT1, in the ciliary body of the rat

To investigate the relationship between the gap junction protein connexin 43 and the glucose transporter GLUT1, their localization was visualized by double-immunofluorescence microscopy using frozen sections as well as immunogold staining of ultrathin frozen sections. In pigmented epithelial cells, most of the GLUT1 was localized along the plasma membrane facing the blood vessels, whereas in non-pigmented epithelial cells. it was present along the plasma membrane facing the aqueous humor. Connexin 43 was abundant in the ciliary body and localized mainly in the gap junctions connecting the pigmented and non-pigmented epithelial cells. Localization of GLUT1 and connexin 43 in the blood-aqueous barrier suggests that GLUT1, connexin 43, and GLUT1 disposed in this order could be a machinery responsible for the transport of glucose across the blood-aqueous barrier.