Modification of a Scanning Transmission Electron Microscope Specimen Holder for Large Section Viewing

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter “giant” grids is explained and the procedure for sample preparation is outlined The modification aids the microscopist in his evaluation of tissue structural relationship by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm² electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

Ultrastructural localization of silver deposits in the receptacle cells of carnation flowers

Carnations were treated with a silver thiosulphate complex to prevent wilting of the flowers. The ultrastructural localization of silver and sulphur in the receptacle tissue was investigated by electron microscopy. Electron-dense deposits were present in the receptacle tissue. Coarse-grained deposts (diam. 60–100 nm) were predominantly observed at the inner side of the cell wall, whereas fine-grained deposits (diam. 20–60 nm) were predominantly present inside the cell-wall region and in the intercellular spaces. These particles were analyzed for chemical elements by X-ray analytical electron microscopy (Philips EM 400 plus Edax energy dispersive analyzer, type 711). In both types of deposits, the presence of silver and sulphur was verified. Point analysis revealed that in both precipitates the S/Ag ratio was of the same order.Abbreviations CTEM conventional transmission electron microscope - STEM scanning transmission electron microscope The AEM unit is a joint unit of the Erasmus University of Rotterdam, the University of Leyden, and the Health Organization TNO. The analytical microscope was purchased with a grant from the Dutch Organization for Pure Scientific Research (ZWO) through BION     

Electrospun sodium alginate/poly(ethylene oxide) core-shell nanofibers scaffolds potential for tissue engineering applications

Core-shell structure nanofibers of sodium alginate/poly(ethylene oxide) were prepared via electrospinning their dispersions in water solution. The core-shell structure morphology of the obtained nanofibers was viewed under scanning electron microscope (SEM) and transmission electron microscope (TEM), and X-ray photoelectron spectroscopy (XPS) analysis was used to further quantify the chemical composition of the core-shell composite SA/PEO nanofibers surface in detail. Furthermore, one-step cross-linking method through being immersed in CaCl₂ solution was investigated to improve the anti-water property of the electrospun nanofibers mats in order to facilitate their practical applications as tissue engineering scaffolds, and the changes of the structural of nanofibers before and after cross-linking was characterized by Fourier transform infrared (FT-IR). Indirect cytotoxicity assessment indicated that SA/PEO nanofibers membrane was nontoxic to the fibroblasts cells, and cell culture suggested that SA/PEO nanofibers tended to promote fibroblasts cells attachment and proliferation. It was assumed that the nanofibers membrane of electrospun SA/PEO could be used for tissue engineering scaffolds.     

Selection for Electron Microscopy of Specific Areas in Large Epoxy Tissue Sections

Tissue blocks 2 × 2 × 0.4 cm were fixed 6-24 hr in phosphate-buffered 6% glutaraldehyde then sliced to 2 × 2 × 0.1 cm and rinsed in phosphate buffer for at least 12 hr. Fixation was continued for 2 hr in phosphate-buffered 1-2% OsO₄. The slices were dehydrated, infiltrated with Araldite, and embedded in flat-bottomed plastic molds. Sectioning at 4-8 μ with a sliding microtome was facilitated by addition of 10% dibutylphalate to the standard epoxy mixture. The sections were spread on water and attached to coverslips by drying, then heating to 80 C for 1 min. Staining 2 min with 1-3% KMnO₄ and temporary mounting in glycerol on a slide allowed the desired area for electron microscopy to be selected and marked. This area was then cemented to the facet of a conventional epoxy casting with a drop of epoxy resin (without added dibutylphthalate). After polymerization, the coverslip was removed by quick cooling leaving a flat re-embedded portion of the original section. This portion was viewed by transillumination in a dissecting microscope and trimmed of surplus tissue. Ultrathin sections for electron microscopy were obtained in the usual manner.     

Freeze-fracture electron microscopy

The freeze-fracture technique consists of physically breaking apart (fracturing) a frozen biological sample; structural detail exposed by the fracture plane is then visualized by vacuum-deposition of platinum-carbon to make a replica for examination in the transmission electron microscope. The four key steps in making a freeze-fracture replica are (i) rapid freezing, (ii) fracturing, (iii) replication and (iv) replica cleaning. In routine protocols, a pretreatment step is carried out before freezing, typically comprising fixation in glutaraldehyde followed by cryoprotection with glycerol. An optional etching step, involving vacuum sublimation of ice, may be carried out after fracturing. Freeze fracture is unique among electron microscopic techniques in providing planar views of the internal organization of membranes. Deep etching of ultrarapidly frozen samples permits visualization of the surface structure of cells and their components. Images provided by freeze fracture and related techniques have profoundly shaped our understanding of the functional morphology of the cell.     

Focused ion beam (FIB)/scanning electron microscopy (SEM) in tissue structural research

The focused ion beam (FIB) and scanning electron microscope (SEM) are commonly used in material sciences for imaging and analysis of materials. Over the last decade, the combined FIB/SEM system has proven to be also applicable in the life sciences. We have examined the potential of the focused ion beam/scanning electron microscope system for the investigation of biological tissues of the model organism Porcellio scaber (Crustacea: Isopoda). Tissue from digestive glands was prepared as for conventional SEM or as for transmission electron microscopy (TEM). The samples were transferred into FIB/SEM for FIB milling and an imaging operation. FIB-milled regions were secondary electron imaged, back-scattered electron imaged, or energy dispersive X-ray (EDX) analyzed. Our results demonstrated that FIB/SEM enables simultaneous investigation of sample gross morphology, cell surface characteristics, and subsurface structures. The same FIB-exposed regions were analyzed by EDX to provide basic compositional data. When samples were prepared as for TEM, the information obtained with FIB/SEM is comparable, though at limited magnification, to that obtained from TEM. A combination of imaging, micro-manipulation, and compositional analysis appears of particular interest in the investigation of epithelial tissues, which are subjected to various endogenous and exogenous conditions affecting their structure and function. The FIB/SEM is a promising tool for an overall examination of epithelial tissue under normal, stressed, or pathological conditions.     

Histopathological effect ofClostridium perfringens 8-6 enterotoxin on rabbit intestine

Intestinal damage caused by an enterotoxin from a coatless spore mutant ofClostridium perfringens type A (8-6) was identified by both light and scanning electron microscopy. Under the light microscope, damage to the epithelial layer of the villus and to the lamina propria was evident. Whole tissue viewed under the scanning electron microscope confirmed the two distinct forms of damage seen by light microscopy and showed that the action of the enterotoxin on an individual villus appears to occur in a specific sequence. The gross tissue damage observed contrasts with that found in previous studies of the action ofClostridium perfringens enterotoxin on rabbit ileal tissue; this suggests that the 8-6 enterotoxin may have a different mode of action on the cell, which subsequently leads to death and lysis.     

Preparation of chromosome spreads for electron (TEM,SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactionsAbbreviations CLSM confocal laser scanning microscope - EM electron microscopy - kV kilovolt(s) - LM light microscope - SEM scanning electron microscope - STEM scanning-transmission electron microscope - TEM transmission electron microscope     

Ruthenium Red-Mediated Osmium Binding for Examining Uncoated Biological Material Under the Scanning Electron Microscope

A simple technique for examining uncoated soft biological material under the scanning electron microscope is described. Rat tissues were initially fixed in 2.5% glutaraldehyde either by intravascular perfusion or by immersion. The samples were placed in buffered 2.5% glutaraldehyde containing 0.05% ruthenium red and postfixed in buffered 1% osmium tetroxide containing 0.05% ruthenium red. The samples were alternately incubated 3 to 5 times in 1% O₃O₄ and 0.1% ruthenium red solutions with continuous shaking at room temperature. The specimens were dehydrated, critical point dried, mounted and examined under the scanning electron microscope. Contour details were clearly defined at both the external and cut surfaces of the tissues. The specimens could be observed for more than 30 minutes without excessive charging or glow effects and the material remained stable under the beam at 20-25 kV and at various magnifications.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

Transmission and scanning electron microscope preparations of the same cell culture.

A technique has been developed which allows transmission electron microscopy and scanning electron microscopy to be performed on the same cell culture sample. The technique uses the Costar 3,393 Leighton Tube containing a plastic insert, which does not stick to epoxy, for transmission electron microscopy. A cut piece of the plastic insert can be critical point dried, sputter coated and viewed under high vacuum with the scanning electron microscope.     

Membrane turnover in crab photoreceptors studied by high-resolution scanning electron microscopy and by a new technique of thick-section transmission electron microscopy

Summary Crab photoreceptors were examined after treatment by the osmium-DMSO-osmium method for high-resolution scanning electron microscopy. This technique of specimen preparation was also adapted for transmission electron microscopy, enabling sections up to 1 urn thick to be viewed in a conventional microscope at 75 kV. With appropriate pretreatment, some cytoskeletal elements can be visualised by both techniques. The methods were then used to investigate some of the daily changes known to occur in photoreceptor cell structure. Striking differences were found in the structure of Golgi bodies present in retinula cells during the synthesis and breakdown phases of the daily cycle of photoreceptor membrane turnover. Cyclic changes were also noticed in the mitochondria of retinula cells, and additional evidence was found for a previously proposed model of rhabdomeral microvillus formation.     

Transmission And Scanning Electron Microscope Preparations Of The Same Cell Culture

A technique has been developed which allows transmission electron microscopy and scanning electron microscopy to be performed on the same cell culture sample. The technique uses the Costar 3393 Leighton Tube containing a plastic insert, which does not stick to epoxy, for transmission electron microscopy. A cut piece of the plastic insert can be critical point dried, sputter coated and viewed under high vacuum with the scanning electron microscope.     

Ultrastructure of Cells Cultured on Polycarbonate Membranes

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 pm or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.     

Scanning Electron Microscopy of Selected Members of the Streptomyces hygroscopicus Group

Distinctive variations in whole spore morphology and spore surface morphology of Streptomyces hygroscopicus strains, which had been observed previously by the authors by the preshadowed carbon replica technique, were confirmed by observations with the scanning electron microscope.     

PRELIMINARY ANALYSIS OF QUANTITATIVE GENETICS AND PHENOTYPIC PLASTICITY IN AULACOSEIRA SUBARCTICA (BACILLAR-IOPHYTA)

Several clones of Aulacoseira subarctica were isolated from Yellowstone, Lewis, and East Rosebud Lakes (Montana, Wyoming). Two to four clones from each lake were grown in batch cultures under three light intensities, 2, 11.4 and 115 μE m⁻² s⁻¹. Clones were conditioned to their light environment for a three-week period. Inoculants from the conditioned clones taken during log phase of growth, were grown until in log phase, then samples were collected. Five randomly chosen valves for 2 replicates of each clone were examined using a scanning electron microscope and captured on film at a magnification of 20,000x. Each image was digitized and quantitative morphometric characters were measured. A preliminary quantitative genetic analysis was performed on selected characters within each light environment. Plasticity of characters within clones across the three light regimes were also examined. The amount of variability found within characters in A. subarctica will be discussed in terms of environmental, genetic, and microenvironmental sources.     

Examination of endopelic and epilithic algal community structure employing scanning electron microscopy

1. Biological community structure within a stream periphyton mat and sediment core was examined using scanning electron microscopy. Samples were fixed, freeze-fractured and viewed under normal conditions with a scanning electron microscope.
2. This technique enabled the analysis of orientation and spatial distribution of algal cells within the three-dimensional algal communities.
3. Fractured samples from a periphyton mat and sediment core at the Procter & Gamble Experimental Stream Facility showed layering of some dominant diatom species, such as Nitzschia dissipata , Melosira varians and Gyrosigma attenuatum . The spatial relationship of nematodes as potential algal predators was also revealed.
4. Advantages, disadvantages and potential future applications of this freeze fracture technique are discussed.     

Pigment epithelial ensheathment and phagocytosis of extrafoveal cones in human retina.

The association between extrafoveal cone outer segments and pigment epithelial cells was studied by transmission electron microscopy in three human retinas; ages 5,45 and 60. The pigment epithelial apical surface from a fourth human retina, age 38,was viewed in the scanning electron microscope. Multiple villous-like apical processes protrude from the pigment epithelium into the space above each cone. Sometimes one or more of these processes is sheet-like in form and contains a wealth of intracellular organelles, including mitochondria. One or more of the villous-like procesess reaches the cone and expands to ensheath the upper one-third of the outer segment. Llike vertebrate rods, extrafoveal human cones shed their terminal disks in packets and these packets are phagocytosed by the ensheathing apical processes. The phagosomes then ascend in the processes toward the pigment epithelia soma. Digestion of phagosomes appears to begin in the apical processes.     

In Situ Multiple Sampling of Attached Bacteria for Scanning and Transmission Electron Microscopy

An in situ electron microscope sampling technique for characterizing cells attached to smooth surfaces is demonstrated with an ultraviolet-induced mutant of Streptococcus mutans. The sterilized sampling unit consists of a 9 cm plastic Petri dish containing a glass slide, a 12 mm round coverglass, and a coverglass with Formvar-carbon coated copper grids. After the bacterial culture in a liquid medium is incubated in the Petri dish, the slide with attached bacteria is washed in double-distilled water, air-dried, coated with platinum and carbon, and processed for replicas and shadowed specimens for transmission electron microscopy. The coverglass is similarly washed, fixed in 2% glutaraldehyde, air- or freeze-dried, coated with palladium/gold, and examined in the scanning electron microscope. The coverglass with grids is rinsed in double distilled water, the grids are transferred to a filter paper and stained with a loopful of 2% phosphotungstic acid at pH 5.5. The bacteria growing on the surface of the plastic Petri dish are fixed, dehydrated, and embedded in situ with Epon. Sectioned and stained specimens are then examined in the transmission electron microscope. This procedure also appears useful with such other attached systems as normal or infected tissue culture cells.     

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。