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)     

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     

Ultrastructure of plant chromosomes by high-resolution scanning electron microscopy

A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen. Depending on the microscope and conditions of preparation, a resolution of a few nanometers is routinely possible. Tilting of the specimen provides a three-dimensional insight into chromosomal structures. Combination of material-dependent signals of backscattered electrons with the secondary electron image allows an unambiguous localization of surface markers.     

A new method of preparing fish chromosomes for scanning electron microscopy

A preparative ion-etching technique has been developed which enhances the images of fish chromosomes obtained by scanning electron microscopy.     

A simplified method for preparing rotifer trophi for scanning electron microscopy

A simple, quick technique for the preparation of rotifer trophi for scanning electron microscopy is described. The method permits visual monitoring of the extraction process and does not require critical point drying of the specimens. Micrographs showing fine, structural detail of the hard parts of the mastax of representatives of the following genera are presented:Asplanchna, Conochilus, Filinia, Hexarthra, Keratella, Proalides, Synchaeta, andTrichocerca.     

荔枝蝽触角化感器的扫描电镜观察

应用扫描电镜技术研究了荔枝蝽(Tessaratoma papillosaD rury)触角化感器的类型、数量和分布。结果表明,毛形感器、刺形感器和锥形感器在荔枝蝽雌雄虫触角上均有分布,其中以毛形感器最多,且毛形感器数量和分布在雌雄间有明显的不同。另外,雄虫触角上还分布有念珠形感器,而腔形感器在雌虫触角上有分布,这2种感器数量相对较少。     

Morphology and ultrastructure of Siberian sturgeon (Acipenser baerii) spermatozoa using scanning and transmission electron microscopy

BACKGROUND INFORMATION: Available data concerning the sperm morphology of teleost fishes demonstrate wide variation. In the present study, the spermatozoa of Siberian sturgeon (Acipenser baerii Brandt, 1869), a chondrostean fish, was investigated. In contrast with teleost fish, chondrostean spermatozoa have a head with a distinct acrosome, whereas other structures, such as a midpiece and a single flagellum, are present in spermatozoa of most species. RESULTS: The average length of the head including the acrosome and the midpiece was 7.01+/-0.83 microm. Ten posterolateral projections derived from the acrosome were present on a subacrosomal region, with mean lengths of 0.94+/-0.15 microm and widths of 0.93+/-0.11 microm. The nucleus consisted of electrodense homogeneous nuclear chromatin. Three intertwining endonuclear canals, bound by membranes, traversed the nucleus longitudinally from the acrosomal end to the basal nuclear fossa region. There were between three and six mitochondria, two types of centrioles (proximal and distal) in the midpiece and two vacuoles composed of lipid droplets. The flagellum (44.75+/-4.93 microm in length), originating from the centriolar apparatus, had a typical 9+2 eukaryotic flagellar organization. In addition, there was an extracellular cytoplasm canal between the cytoplasmic sheath and the flagellum. CONCLUSIONS: A principal components analysis explained the individual morphological variation fairly well. Of the total accumulated variance, 41.45% was accounted for by parameters related to the head and midpiece of the sperm and the length of the flagellum. Comparing the present study with previous studies of morphology of sturgeon spermatozoa, there were large inter- or intra-specific differences that could be valuable taxonomically.     

Multi-resolution correlative focused ion beam scanning electron microscopy: Applications to cell biology

Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB–SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce “keyframe imaging” as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a “target map” of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10⁹ in a single automated experimental run.     

The application of scanning electron microscopy to the systematics of the neotropical Peripatidae (Onychophora)

The taxonomy of the New World Peripatidae is poorly known both because of their rarity and difficulty in the determination of specimens. The distribution of papillae on the dorsal integument is an important taxonomic; character but is difficult to interpret under the light microscope. In this study the integument of a range of New World onychophoran species was studied using scanning electron microscopy. Twenty-one species were examined. Three species of Peripatopsidae were also studied for comparison. This technique revealed (a) the pattern of distribution of papillae on the integument, and (b) the morphology of the papillae. A pattern of three smaller papillae between two large was common to many species. Differences observed in some species were probably modifications of this basic pattern. There was interspecific variation in the number of scales in the apical and basal pieces of the papillae. The specimens examined fell into two main groups: (1) species with more than three scale ranks in the apical piece ( Oroperipatus and Peripatus ) and (2) species with three or fewer scale ranks ( Epiperipatus, Macroperipatus and others). The results thus support a distinction between Epiperipatus and Peripatus . However Macroperipatus is shown not to be a natural group. The relationships of some species which did not fit into this pattern are discussed. This paper shows scanning electron microscopy to be a potentially useful technique in a revision of the New World Peripatidae.     

Light microscopy and scanning electron microscopy of the In vitro evagination process of Echinococcus multilocularis protoscolices

Marchiondo A. A. and Andersen F. L. 1984. Light microscopy and scanning electron microscopy of the in vitro evagination process of Echinococcus multilocularis protoscolices. International Journal for Parasitotogy14:151–157. During histogenesis of the protoscolices of Echinococcus multilocularis, the apical portion of the protoscolex consisting of the suckers, rostellum and hook region develops as an introversion and invagination within the tissue of the basal portion. In vitro incubation of protoscolices in evagination fluid stimulates the emergence of the apical portion. The initiation of evagination is first detected by a surface change in the basal portion. The smooth contour of this surface which lacks microtriches becomes transformed into tegumental indentations that form transverse and longitudinal furrows within the basal tegument as the protoscolices contract and expand, respectively. An orifice formed at the site or junction where the apical portion is invaginated begins to expand laterally in order to allow emergence of the suckers. The hooks are arranged within the invaginated protoscolex with blades directed towards the basal orifice, the handles directed towards the peduncle and the guards directed laterally. This arrangement persists throughout the evagination of the suckers and rostellum until the apical dome of the hook region emerges, thereby rotating the blades laterally in the direction of the peduncle and rotating the handles and guards medially to assume a coronal arrangement. Evagination is an asynchronous event and therefore allows observation of individual protoscolices in various stages of emergence.     

Field emission scanning electron microscopy of microtubule arrays in higher plant cells

Summary Specimen preparation protocols that allow field emission scanning electron microscope imaging of microtubules in plant cells were developed, involving simultaneous permeabilization with saponin and stabilization of microtubules with taxol. All categories of microtubule array were observed in onion root tip cells and in tobacco BY-2 cells grown in suspension culture and synchronized to provide high frequencies of mitotic stages. Cortical arrays consist of overlapping microtubules with free ends; individual microtubules directly overlie individual microfibrils in the cell wall. Preprophase bands and spindle microtubule bundles were also imaged. Phragmoplasts revealed early stages of wall deposition in the included cell plates and features interpreted as relating to high rates of microtubule turnover at the growing margins. It was possible to combine high resolution three-dimensional imaging with immunogold labelling of microtubules. Individual gold particles were readily distinguished decorating microtubules in the preparations; the method should be vaulable for studying many features of plant cell microtubules and their associated macromolecules.Abbreviations FESEM field emission gun scanning electron microscope - MTSB microtubule stabilising buffer Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Microscopy electron scanning of fossilized sickle-cells

In a microscopica electronic scanning follow-up study on human skeletons from the Hellenistic Period in the Arab-Persian Gulf, fossilized sickle-cells were found in the bone marrow of one of the excavated skeletons. The ultrastructure of these cells was compared to that of fossilized normal red blood cells of other skeletons from the same site, and to similar pseudo-pathological structures. The finding of fossilized evidence of sickle-cell anemia of that date is withoutprecedent, and makes an evaluation of related gross-anatomical skeletal changes possible.     

Focussed ion beam milling and scanning electron microscopy of brain tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.     

微红梢斑螟(鳞翅目:螟蛾科)成虫触角感器的扫描电镜观察

通过扫描电镜对微红梢斑螟雌、雄成虫触角的外部形态及感器进行观察,结果表明,微红梢斑螟雌、雄成虫触角共观察到10类感器.其中,毛形感器(Ⅰ、Ⅱ型)、刺形感器(Ⅰ、Ⅱ型)、耳性感器、腔锥形感器、Bǒhm氏鬃毛共7类感器在雌、雄虫触角上均有分布;而栓锥形感器仅在雌虫触角上被发现,钟形感器和鳞形感器这2类感器仅在雄虫触角上被发...     

Microstructural characteristics of thin biofilms through optical and scanning electron microscopy

The combination of a conventional optical microscope with a specially designed glass flow cell was used to visualize in situ biofilms formed on opaque thin biomaterials through a simple non-invasive way (optical microscopy of thin biofilms, OMTB). Comparisons of OMTB with scanning electron microscopy (SEM) images were made. Thin metallic dental biomaterials were used as substrata. They were immersed in a synthetic saliva and in a modified Mitis–Salivarius medium inoculated with a consortium of oral microorganisms. To study the effect of bacterial motility, Pseudomonas fluorescens cultures were also used. The processes which give rise to the formation of the biofilm were monitored through OMTB. Biofilm microstructures like pores, water channels, streamers and chains of Streptococci, attached to the surface or floating in the viscous interfacial environment, could be distinguished. Thickness and roughness of the biofilms formed on thin substrata could also be evaluated. Distortions introduced by pretreatments carried out to prepare biological materials for SEM observations could be detected by comparing OMTB and SEM images. SEM images (obtained at high magnification but ex situ, not in real time and with pretreatment of the samples) and OMTB images (obtained in situ, without pretreatments, in real time but at low magnification) in combination provided complementary information to study biofilm processes on thin substrata.     

Scanning electron microscopy of experimental adiaspiromycosis

Adiaspiromycotic granulomas of mice experimentally inoculated with fungusEmmonsia crescens Emmons et Jellison 1960 were examined by scanning electron microscopy. Their morphology, surface structures, and germinating adiaspores isolated from granulomas are described.     

Visualization of the cytostome in Trypanosoma cruzi by high resolution field emission scanning electron microscopy using secondary and backscattered electron imaging

High resolution scanning electron microscopy was used to analyze the surface of epimastigote, amastigote and trypomastigote forms of Trypanosoma cruzi. Significant differences were observed between these forms and in different areas of the same cell. The cytostome found in amastigote and epimastigote forms could be easily visualized in images, which resemble those obtained only using the freeze-fracture technique. In contrast to other areas of the cell surface, the region of the cytostome, localized close to the flagellar pocket, showed a rugous surface and an opening with a diameter of 90 nm. Gold-labeled concanavalin A binds to the whole cell surface. However, the extent of binding was much higher in the region of the cytostome. The results obtained show that high resolution scanning electron microscopy is a powerful technique for analyzing the surface of protozoa.     

Observation of Woronin bodies in Arthrinium aureum by scanning electron microscopy

Woronin bodies are cytoplasmic organelles of filamentous fungi that can be observed on one, or both sides of each septum. The goal of this paper is to illustrate the presence of them in hyphae of Arthrinium aureum by means of scanning electron microscopy and to show that they act as a safety plug to close septa pores in hypha. Results show that Woronin bodies as an immediate response to prevent a cytoplasm loss. Results support hypothesis proposed previously in literature. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sequential scanning electron microscopy of a growing plant meristem

Summary A two-step replica technique has been developed for sequential study of the epidermal cell pattern of a living plant by scanning electron microscopy. This method is nondestructive, allows periodic high resolution observation of the same developing tissue, and can precede use of any destructive technique, such as transmission electron microscopy. The replicas can be trimmed allowing observation of occluded surfaces, such as the areas between leaves, which are inaccessible in continuousin vivo studies. Here we study the developing leaf primordium ofGraptopetalum and discuss potential uses of the technique.