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。     

原子力显微技术在细胞生物学中的应用

对近年来原子力显微技术(AFM)在细胞生物学中的应用大致归纳为几个方面进行了简单介绍,还指出了细胞表面结构难于识别、细胞内部结构难以原位观察等AFM应用于细胞生物学中的难题,并提出了“形状探针”的概念以及超薄切片的思路以解决这些难题。AFM在细胞生物学中的应用研究还远远不足,需要更多的科学工作者加入其中。     

品制备对超薄切片的影响

电镜超薄切片的质量与组织块的硬度,组织的固定,清洗、脱水、浸透、包埋、包埋块的修整形状,切片机、玻璃刀、架刀角度和收集槽液面等因素都有密切的关系,标准的超薄切片应是厚度适中、均匀、平整、无刀痕、无颤纹和皱折。要想获得理想的超薄切片,作者认为     

保持培养细胞原位、原形的超薄切片制备法

本文介绍先用环氧树脂Epon812包埋剂制成丘状膜,再在膜上培养细胞,直接将膜与细胞一起包埋,制备超薄切片。此种方法不仅能克服以往培养细胞需要离心成团,容易造成细胞破碎、变形的缺点;还能避免琼脂预包埋法悬浮细胞造成的抗原封闭;并能得到为数较多的连续超薄切片。它操作简便,较好地保持了培养细胞生长的原来位置及原有形状,为培养细胞进行免疫电镜的操作和提高制备培养细胞超薄切片的数量和质量提供了一种有效的途径。     

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。     

一种简单而快捷的半薄切片脱树脂新方法

目前,在HE染色、特殊染色或免疫组织化学染色研究方面多采用普通石蜡包埋切片,其切片厚度可薄达4μm,若更薄的切片,切割有一定的难度.对于各种染色而言,越薄的切片,细胞与组织结构越清晰,染色效果越好.环氧树脂(Epon)包埋的组织块不仅用于透射电子显微镜超薄切片的制备,也可用于光镜半薄切片的制备[1].经典的脱树脂的方法是将半薄切片置于氢氧化钠的无水乙醇饱和溶液中浸泡24h,然后再充分水洗.该法耗时长,容易脱片,本实验中摸索出一种简单而快捷的脱树脂的方法,现介绍如下.     

环氧树脂厚切片的染色

供电子显微镜观察的超薄切片,一般用各种环氧树脂作包埋剂。用环氧树脂的包埋头也能切成厚1—2微米的切片,供光学显微镜观察。这种厚度的切片与几百埃计算的超薄切片相对而言,可称之厚切片或半薄切片。在超薄切片之前,用环氧树脂包埋头先切     

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。     

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。     

一种用于DAPI染色的方法--Steedman's wax包埋切片法

以植物的胚珠和子房为实验材料,介绍一种用Steedman's wax 包埋对组织切片中的细胞核进行DAPI染色的方法.Steedman's wax 作为一种低熔点多酯蜡,具有与石蜡相似的性质,切片方法同常规石蜡切片,适合于切成厚度大于5 μm的连续切片.Steedman's wax包埋的切片能成功地进行DAPI染色.与用压片法和Technovit 7100或GMA包埋切片法进行的DAPI染色相比,用Steedman's wax 包埋切片法进行的DAPI染色具有廉价、操作简便、可进行连续切片、图象清晰等优点,特别在植物细胞程序化死亡(PCD)的研究中及细胞核DNA含量测定方面,有着较大的应用价值和潜能.     

The reaction of osteoclasts when releasing osteocytes from osteocytic lacunae in the bone during bone modeling

Osteocytes are released from the osteocytic lacunae when osteoclasts resorb the bone matrix during bone modeling and remodeling. It remains unknown how osteoclasts react when releasing osteocytes during bone modeling, and the fate of these released osteocytes is also unclear. Femoral mid-shafts of 2-day-old kittens were sectioned into serial 0.5 microm-thick semithin or 0.1 microm-thick ultrathin sections, and examined by light microscopy (LM) and transmission electron microscopy (TEM). The sections showed many osteoclasts at the endosteum but there were no osteoblasts. There were many half-released, fully released, half-exposed, and fully exposed osteocytes on the bone surfaces. Many cell-like structures were seen in the cell bodies of osteoclasts by LM, and some semithin sections were re-sectioned into ultrathin sections for re-observation by TEM. By TEM, these were determinated to be mononuclear cells. The serial ultrathin sections showed that the mononuclear cells appeared to be engulfed in osteoclasts on one section but that the cell was connected with the bone surface of the osteocytic lacuna on another section. These results show that the mononuclear cells in the osteoclasts were osteocytes. The present study suggests that osteoclasts engulf some osteocytes but do not engulf others when releasing osteocytes during bone modeling.     

Metachromatic staining and electron dense reaction of glycosaminoglycans by means of Cuprolinic Blue

Summary The cationic phthalocyanin-like dye Cuprolinic Blue, unlike phthalocyanin dyes such as Alcian Blue or Astra Blue, can definitely exhibit a clear metachromatic reaction with appropriate substrates, The application of Cuprolinic Blue to epoxy-embedded semithin sections revealed that mast cell cytoplasmic granules, goblet cell mucin and cartilage matrix stained in violet shades (metachromatic), whereas nuclear chromatin presented a bright blue coloration (orthochromatic). The metachromatic structures showed a high degree of contrast when ultrathin sections treated with Cuprolinic Blue were examined by electron microscopy.Cytophotometric measurements of stained components from the large intestine showed different absorption maxima: at 580 nm for mucin and at 640 nm for nuclei. The spectroscopical analysis revealed a clear-cut metachromatic shift when the dye was in the presence of chondroitin—4-sulphate. The addition of aluminium metal to Cuprolinic Blue solutions resulted in a striking spectral change; under such conditions the dye showed absorption maximum at 530 nm.     

A novel strategy to obtain a hyaluronan monolayer on solid substrates

The aim of this study was to find a novel simple method to obtain polysaccharide ultrathin layers on solid substrates to investigate the interaction between the surface and the biological environment. A Hyaluronan (Hyal) monolayer with a well-defined chemistry was obtained by exploiting the capability of organosilanes to spontaneously adhere onto glass surfaces. A silane alkylic chain was conjugated with Hyal, and the derivatized polysaccharide was allowed to spontaneously adhere onto a glass surface. The elemental analysis of the modified polysaccharide demonstrated that one out of five disaccharide units was conjugated with the alkyl silane chain, corresponding to a substitution degree of the carboxylate groups of approximately 20%. The film of the modified polysaccharide was characterized by means of X-ray photoelectron spectroscopy (XPS), water contact angle, and atomic force microscopy (AFM) measurements. XPS analysis demonstrated that we obtained a Hyal layer with a thickness of about 2.0 nm corresponding to a Hyal monolayer. The Hyal-coated surfaces appeared to be rather smooth and highly hydrophilic and showed significant resistance to nonspecific cell adhesion.     

Short and long range order of the morphology of silk from Latrodectus hesperus (Black Widow) as characterized by atomic force microscopy.

The surfaces of both stretched and unstretched silk threads from the cobweb weaver, Latrodectus hesperus (Black Widow) have been examined by atomic force microscopy (AFM). AFM images of cobweb scaffolding threads show both unordered and highly ordered regions. Two types of fibers within the threads were observed: thicker (approximately 300 nm in diameter) fibers oriented parallel to the thread axis and thinner (10-100 nm) fibrils oriented across the thread axis. While regions which lacked parallel fibers or fibrils were observed on threads at all strain values, the probability of observing fibers and/or fibrils increased with strain. High-resolution AFM images show that with increasing strain, both mean fiber and fibril diameters decrease and that fibrils align themselves more closely with the thread axis. The observation of fibers and fibrils within the cobweb threads has implications for current models of the secondary and tertiary structure and organization of spider silk.     

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.     

The bacteriophage phi29 head-tail connector imaged at high resolution with the atomic force microscope in buffer solution.

The surfaces of two- and three-dimensional phi29 connector crystals were imaged in buffer solution by atomic force microscopy (AFM). Both topographies show a rectangular unit cell with dimensions of 16.5 nm x 16.5 nm. High resolution images of connectors from the two-dimensional crystal surface show two connectors per unit cell confirming the p42(1)2 symmetry. The height of the connector was estimated to be at least 7.6 nm, a value close to that found in previous studies using different techniques. The 12 subunits of the wide connector domain were clearly resolved and showed a right-handed vorticity. The channel running along the connector had a diameter of 3.7 nm in the wide domain, while it was 1.7 nm in the narrow domain end, thus suggesting a tronco-conical channel shape. Moreover, the narrow connector end appears to be rather flexible. When the force applied to the stylus was between 50 and 100 pN, the connector end was fully extended. At forces of approximately 150 pN, these ends were pushed towards the crystal surface. The complementation of the AFM data with the three-dimensional reconstruction obtained from electron microscopy not only confirmed the model proposed, but also offers new insights that may help to explain the role of the connector in DNA packing.     

Model films from native cellulose nanofibrils. Preparation, swelling, and surface interactions

Native cellulose model films containing both amorphous and crystalline cellulose I regions were prepared by spin-coating aqueous cellulose nanofibril dispersions onto silica substrates. Nanofibrils from wood pulp with low and high charge density were used to prepare the model films. Because the low charged nanofibrils did not fully cover the silica substrates, an anchoring substance was selected to improve the coverage. The model surfaces were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The effect of nanofibril charge density, electrolyte concentration, and pH on swelling and surface interactions of the model film was studied by quartz crystal microbalance with dissipation (QCM-D) and AFM force measurements. The results showed that the best coverage for the low charged fibrils was achieved by using 3-aminopropyltrimethoxysilane (APTS) as an anchoring substance and hence it was chosen as the anchor. The AFM and XPS measurements showed that the fibrils are covering the substrates. Charge density of the fibrils affected the morphology of the model surfaces. The low charged fibrils formed a network structure while the highly charged fibrils formed denser film structure. The average thickness of the films corresponded to a monolayer of fibrils, and the average rms roughness of the films was 4 and 2 nm for the low and high charged nanofibril films, respectively. The model surfaces were stable in QCM-D swelling experiments, and the behavior of the nanofibril surfaces at different electrolyte concentrations and pHs correlated with other studies and the theories of Donnan. The AFM force measurements with the model surfaces showed well reproducible results, and the swelling results correlated with the swelling observed by QCM-D. Both steric and electrostatic forces were observed and the influence of steric forces increased as the films were swelling due to changes in pH and electrolyte concentration. These films differ from previous model cellulose films due to their chemical composition (crystalline cellulose I and amorphous regions) and fibrillar structure and hence serve as excellent models for the pulp fiber surface.     

Atomic-force microscopy imaging of plasma membranes purified from spinach leaves

Summary Plasma membranes purified from spinach leaves by aqueous two-phase partitioning were examined by atomic-force microscopy (AFM) in phosphate buffer, and details on their structure were reported at nanometric scale. Examination of the fresh membrane preparation deposited on mica revealed a complex organization of the surface. It appeared composed of a first layer of material, about 8 nm in thickness, that practically covered all the mica surface and on which stand structures highly heterogeneous in shape and size. High-resolution imaging showed that the surface of the first layer appeared relatively smooth in some regions, whereas different characteristic features were observed in other regions. They consisted of globular-to-elliptical protruding particles of various sizes, from 4–5 nm x-y size for the smallest to 40–70 nm for the largest, and of channel-like structures 25–30 nm in diameter with a central hole. Macromolecular assemblies of protruding particles of various shapes were imaged. Addition of the proteolytic enzyme pronase led to a net roughness decrease in regions covered with particles, indicating their proteinaceous nature. The results open fascinating perspectives in the investigation of membrane surfaces in plant cells with the possibility to get structural information at the nanometric range.Abbreviations AFM atomic-force microscopy - EM electron microscopy - TMAFM tapping-mode atomic-force microscopy     

Changes in the hyperelastic properties of endothelial cells induced by tumor necrosis factor-alpha

Mechanical properties of living cells can be determined using atomic force microscopy (AFM). In this study, a novel analysis was developed to determine the mechanical properties of adherent monolayers of pulmonary microvascular endothelial cells (ECs) using AFM and finite element modeling, which considers both the finite thickness of ECs and their nonlinear elastic properties, as well as the large strain induced by AFM. Comparison of this model with the more traditional Hertzian model, which assumes linear elastic behavior, small strains, and infinite cell thickness, suggests that the new analysis can predict the mechanical response of ECs during AFM indentation better than Hertz's model, especially when using force-displacement data obtained from large indentations (>100 nm). The shear moduli and distensibility of ECs were greater when using small indentations (<100 nm) compared to large indentations (>100 nm). Tumor necrosis factor-α induced changes in the mechanical properties of ECs, which included a decrease in the average shear moduli that occurred in all regions of the ECs and an increase in distensibility in the central regions when measured using small indentations. These changes can be modeled as changes in a chain network structure within the ECs.     

The nucleolonema changes its three-dimensional conformation during interphase in root-tip meristems of onion

Summary The three-dimensional architecture of a filamentous nucleolar structure, called the “nucleolonema”, was investigated in onion root-tip cells by applying a silver impregnation technique to air-dried cells and serial ultrathin sections. The entire configuration of the nucleolonema was revealed when silver staining was applied to air-dried cells. The nucleolonema was knobbly or segmented along its entire length and showed great variation in thickness. Three categories of nucleolonema were discriminated depending on thickness; each had an average value of 0.5, 1.0, and 1.3 μm, respectively. Some root tips were embedded in Lowicryl K4M resin and cut into serial ultrathin sections about 100 nm thick. When these sections were subjected to silver impregnation, segments of nucleolonema were visualized. Most of them were found to contain achromatic holes. These holes apparently corresponded to the fibrillar centres seen with the electron microscope. According to the profiles of the holes, nucleolonema structures were classified into three types: (1) nucleolonema with no distinct holes, (2) those with beaded holes, and (3) those with cylindrical holes. The thicknesses were 0.7–0.8, 0.9–1.2, or 1.2–1.4 μm for nucleolonemata with no holes, beaded holes, or cylindrical holes, respectively. The argyrophilic wall of nucleolonemata with holes was about 0.4 μm thick, roughly compatible with the thinnest nucleolonema seen in air-dried specimens. The crescent-shaped segments were sometimes observed when the nucleolonema was sectioned transversely, suggesting that the achromatic holes are exposed to the nucleoplasm, in other words, the nucleolonema is partially degraded. Thus, the nucleolonema was not always structurally stable during interphase. The results suggest that the nucleolonemata gradually become knobbly and increase their thickness, with concomitant expansion of the fibrillar centres sometimes degrading into approximately 0.5 μm thick strands.