Comparison of protein A-gold and ferritin immunoelectron microscopy of Semliki Forest virus in mouse brain using a rapid processing technique

Processing tissue for transmission electron microscopy by standard laboratory methods can take two to three days. This makes the development of new techniques time consuming and generally restricts the use of the electron microscope in routine diagnostic work. The possibility of viewing tissue with the electron microscope five hours after sampling using rapid processing techniques is presented. The morphology of the tissue appears undamaged with cell and organelle ultrastructures being readily recognized, as is the presence of virus and its replicating stages. When combined with immunoelectron microscopy a rapid labeling protocol is possible. We have used the technique to develop protein A-gold (6 and 16 nm particles) and ferritin immunoelectron microscopic techniques to demonstrate viral antigens in brain cell cultures and brain tissue from mice infected with Semliki Forest virus.     

Morphology and ultrastructure of the earthworm Dendrobaena veneta (Lumbricidae) coelomocytes

Microscope techniques, light microscope (LM), transmission electron microscope (TEM), scanning electron microscope (SEM) were employed to describe and classify coelomocytes of the oligochaete Dendrobaena veneta. Three main cell types were distinguished in the coelomic fluid: eleocytes, amoebocytes and granulocytes. Eleocytes are large, oval cells containing characteristic granules called chloragosomes. Amoebocytes are most numerous coelomocytes and have been divided into two types (I and II). Both amoebocytes of the types I and II often form aggregations of a few to about a dozen cells. Granulocytes are oval cells with spherical nuclei and cytoplasm containing polymorphic, electron dense granules. Contrary to the amoebocytes, the granulocytes do not form aggregations. Morphology and ultrastructure of coelomocytes are presented on micrographs: similarities and differences are compared to coelomocytes of related species.     

Silica in the Mesophyll Cell Walls of Italian Rye Grass (Lolium multiflorum Lam. cv. RvP)

Isolated cell envelopes from the leaf mesophyll of Italian ryegrass were examined in a transmission electron microscope equippedwith an energy-dispersive X-ray spectrometer. Silicon was detectedin these walls at an estimated concentration of 1–2 percent. Samples were also subjected to a range of techniques,for the removal of organic matter, which confirmed the presenceof silica throughout the cell walls. Lolium multiflorum Lam, Italian rye grass, cell walls, silica, electron microscopy, X-ray microanalysis     

Electron microscope tomography of Balbiani Ring hnRNP substructure

Three-dimensional (3-D) reconstructions, by electron microscope tomography, of selectively stained, contrast enhanced Balbiani Ring (BR) hnRNP granules reveal a complex spatial arrangement of RNA-rich domains. This particulate substructure was examined by volume rendering computer graphics. Modeling the arrangement of RNA-rich domains is made difficult by apparent structural flexibility and/or heterogeneity of composition. Formulation of a consensus 3-D arrangement of RNA-rich domains will require an expanded data base of reconstructed BR granules and the development of new image manipulation and analysis techniques. This study demonstrates the potential for ultra-structural cell biology of combining several new techniques: selective nucleic acid staining, electron spectroscopic imaging to enhance contrast, electron microscope tomography and volume rendering computer graphics.Abbreviations BR Balbiani Ring - EMT electron microscope tomography - ESI electron spectroscopic imaging - hnRNP heterogeneous nuclear ribonucleoprotein - OA-B osmium ammine-B - kb kilobases by P.B. Moens     

Techniques for quantitative analyses of calcareous marine phytoplankton

This paper discusses the techniques used to sample and analyse living marine calcareous phytoplankton. The various methods are described and tested within several research projects aimed at the determination of coccolithophore cell densities in seawater. In addition, the potential advantages and drawbacks associated with the application of light and scanning electron microscopic techniques to the quantitative analysis of coccolithophores are discussed. Several tests have been carried out in order to quantify potential errors related to: (1) homogeneity of material distribution on filter membranes; (2) use of different microscopes (scanning electron microscope versus light microscope); (3) use of different filter membranes (cellulose mixed-ester membranes versus polycarbonate membranes); and (4) Utermöhl settling versus filtration method. These tests revealed that major errors in cell density calculations could result from the uneven distribution of coccolithophore specimens on a filter membrane. The error resulting from the use of a light microscope arises from its low resolution, which restricts the identification of species, especially of small coccospheres. The use of different filter membranes does not show a statistically significant difference in cell density calculations, although polycarbonate membranes can be examined much more efficiently with the scanning electron microscopy than cellulose mixed-ester membranes. The Utermöhl method, however, gives lower cell densities consistently (several times) than the filtration method.     

Biological ultrastructure research; the first 50 years

The second half of the 20th century has witnessed the birth and growth of biological ultrastructure research--a branch of cell biology in which electron microscopy plays an important role. After a humble start in around 1950, when only a limited arsenal of instrumentation was available, a wealth of auxiliary methodologies were developed and gradually put in use. Here we review these techniques: ultramicrotomy of "optimally" fixed and prepared samples, histochemical methods such as immuno-electron microscopy and electron microscope autoradiography, negative staining techniques, freeze-fracturing and other techniques. Closer to the millennium shift, various cryotechniques have gradually developed. Together with computer-based reconstruction methods they are likely to play increasingly more important roles in the future.     

Studying intracellular transport using high-pressure freezing and Correlative Light Electron Microscopy

Correlative Light Electron Microscopy (CLEM) aims at combining the best of light and electron microscopy in one experiment. Light microscopy (LM) is especially suited for providing a general overview with data from lots of different cells and by using live cell imaging it can show the history or sequence of events between or inside cells. Electron microscopy (EM) on the other hand can provide a much higher resolution image of a particular event and provide additional spatial information, the so-called reference space. CLEM thus has certain strengths over the application of both LM and EM techniques separately. But combining both modalities however generally also means making compromises in one or both of the techniques. Most often the preservation of ultrastructure for the electron microscopy part is sacrificed. Ideally samples should be visualized in its most native state both in the light microscope as well as the electron microscope. For electron microscopy this currently means that the sample will have to be cryo-fixed instead of the standard chemical fixation. In this paper we will discuss the rationale for using cryofixation for CLEM experiments. In particular we will highlight a CLEM technique using high-pressure freezing in combination with live cell imaging. In addition we examine some of the EM analysis tools that may be useful in combination with CLEM techniques.     

A light and electron microscope study of intra-epithelial putative mechanoreceptors in squid suckers

Putative mechanoreceptor neurons in the cuticularized epithelium of the suckers of the squid Lolliguncula brevis, were investigated using light and electron microscope techniques. These neurons were found to have a multipolar shape, thick unbranched axon, glial cell ensheathment, and accessory nerve fiber innervation. The need for electrophysiological and/or behavioral studies on these putative mechanoreceptors is emphasized.     

Scanning Electron Microscopic Study of Cytoskeleton in Isolated Generative Cells of Lily

Isolated generative cells of lily were extracted with Triton X-100, ammonium sulphate and RNase. The exposed contents were then viewed in the scanning electron microscope after critical point drying. The treatments revealed that in the cytoplasm of the generative cell there was a reticulate network of cytoskeleton. This reticulate network of cytoskeletal scaffold had two layers: (1) an outer layer (near the membrane) consisting of long and thick fibres that were tightly knitted together, and (2) an inner layer (near the nucleus) consisting of thin and short fibres that were loosely knitted together. Indirect evidence using immunofluorescence techniques for labelling microtubules and TRITC-phalloidin staining of actin microfilaments indicated that the cytoskeleton seen in the scanning electron microscope appeared likely to be a microtubule cytoskeleton rather than a microfilament cytoskeleton.     

百合离体生殖细胞骨架的扫描电镜观察

从百合的花粉管分离出来的生殖细胞,经表面活化剂Ｔｒｉｔｏｎ Ｘ－１００ 及核糖核酸酶、硫酸铵离析处理。离析后的细胞经临界点干燥,扫描电镜观察显示：在离体生殖细胞的胞质内有一个非常复杂的支架网络系统。这一网络系统有内外两层：外层（靠近细胞膜）网络结构紧密,纤维束粗长;内层（靠近核）网络结构疏松,纤维束短细。一些间接证据显示,这一支架网络系统可能为微管骨架     

伊曲康唑对皮肤癣菌形态学影响研究

目的观察皮肤癣菌在伊曲康唑作用下的形态学变化。方法应用美国CLSI制订的标准M38-A方案进行伊曲康唑对皮肤癣菌的体外药敏试验,测定最小抑菌浓度（MIC）,将伊曲康唑作用前后的皮肤癣菌分别制成标本,在光学显微镜、扫描电子显微镜和透射电子显微镜下观察形态学变化。结果伊曲康唑作用于皮肤癣菌后,在光学显微镜下菌丝变得弯曲、短粗,顶端和局部出现膨大;扫描电子显微镜下菌丝变得弯曲、短粗、干瘪,顶端和局部出现膨大,有不规则分支,表面粗糙,有大小不等的凹陷;透射电子显微镜下菌丝变得皱缩,有凹陷,双层细胞壁结构消失或不完整,细胞膜不连续,皱缩细胞膜和细胞壁之间及胞浆内出现许多小的高电子密度颗粒,细胞器也变得不清晰。结论伊曲康唑使皮肤癣菌的形态发生明显变化。     

Towards native-state imaging in biological context in the electron microscope

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context.     

Structure and composition of cyanophycin granules in the cyanobacterium Aphanocapsa 6308

Cyanophycin granules in the unicellular cyanobacterium Aphanocapsa 6308 were examined with the electron microscope in both thin section and by freeze-fracture techniques. Purified granules were examined with the electron microscope, by arginine determinations, by chromatography, and by elemental analysis. They are similar in ultrastructure and composition to those isolated from the nitrogen-fixing cyanobacterium Anabaena cylindrica, consisting of equal molar quantities of L-arginine and L-aspartic acid.     

Possibilities for the histochemical study of the nuclei of normal brain cells and in pathology

Possibilities of application of some histochemical methods to studying cell nuclei of brain are reviewed considering the following techniques: hybridization histochemistry on the light and electron microscope levels, immunohistochemistry and immunoelectron microscopy, absorbtion and fluorescence histochemistry of nucleic acids, histones, non-histone proteins of chromatin, and of cell nucleus lipids, electron histochemistry. Besides, some physicochemical and molecular-biological methods are considered. Data on human brain research in the norm and upon various brain disorders are particularly provided.     

Three-dimensional imaging of biological complexity

Over the past 5 years, thanks to advances in both instrumentation and computational speed, three-dimensional imaging techniques using the electron microscope have been greatly improved in two areas: electron tomography of cell organelles or cell sections and reconstruction of macromolecules from single particles. Ice embedment has brought a breakthrough in the degree of preservation of specimens under close-to-native conditions. The current challenge is to push the resolution of electron tomographic imaging to a point where macromolecular signatures can be recognized within the cellular context. We show first progress toward this goal by examples in two areas of application: the structure of the muscle triad junction and the architecture and fine structure of mitochondria. As techniques of cryo-microtomy are perfected, we hope to be able to apply tomography to high-pressure frozen sections of tissue.     

Freeze substitution of fungi for cytological analysis

The preparatory techniques of freeze substitution for electron microscopy are given in detail. Included is a simple, reliable method for the selection of individual freeze-substituted cells from flat embedments via light microscopy for subsequent electron microscope analysis. These mthods are intended for studying cells in monolayer cultures, tissues, suspensions of cells, or cell fractions and are widely applicable for cell biologists in any field. An historical background and review of the relevant literature is included so that the reader will be fully prepared to use of modify the techniques and to troubleshoot results as required. All materials that are needed are thoroughly discussed so that any laboratory can adopt freeze substitution as a routine procedure.     

Ultrastructure ofAspergillus fumigatus conidia development and maturation

Summary The development and maturation of conidia of the pathogenic fungus,Aspergillus fumigatus, were studied with the electron microscope. Thin sectioning and freeze-etching techniques were employed to examine young and mature phialides, as well as developing and mature conidia. The morphological changes observed during the process of conidia formation and their maturation centered primarily around the cell wall layers and the inclusions in the cytoplasm.This investigation was supported in part by Grant #A108119 from the National Institutes of Health.     

Comparison of two direct-count techniques for enumerating aquatic bacteria.

Planktonic bacteria from an estuary were concentrated on membrane filters and counted with both a scanning electron microscope and an epi-illuminated fluorescent microscope. Counts on 0.2 micron Nuclepore filters (polycarbonate) were significantly higher (P less than 0.001) than counts on 0.2-micron Sartorius filters (cellulose). In contrast, there was not a statistically significant difference between the two techniques when Nuclepore filters were used (0.5 less than P less than 0.9). The average cell volume from this study area was 0.047 micron3. The estimated number of bacteria ranged from 10(6) to 10(7) bacteria per ml, representing from 4 to 40 mg of C per m3.     

Comparison of two direct-count techniques for enumerating aquatic bacteria.

Planktonic bacteria from an estuary were concentrated on membrane filters and counted with both a scanning electron microscope and an epi-illuminated fluorescent microscope. Counts on 0.2 micron Nuclepore filters (polycarbonate) were significantly higher (P less than 0.001) than counts on 0.2-micron Sartorius filters (cellulose). In contrast, there was not a statistically significant difference between the two techniques when Nuclepore filters were used (0.5 less than P less than 0.9). The average cell volume from this study area was 0.047 micron3. The estimated number of bacteria ranged from 10(6) to 10(7) bacteria per ml, representing from 4 to 40 mg of C per m3.     

ELECTRON MICROSCOPY OF THE NUCLEAR MEMBRANE OF AMOEBA PROTEUS

An electron microscope study of the nuclear membrane of Amoeba proteus by thin sectioning techniques has revealed an ultrastructure in the outer layer of the membrane that is homologous to the pores and annuli observed in the nuclear membranes of many other cell types studied by these techniques. An inner honeycombed layer apparently unique to Amoeba proteus is also described.