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.     

Differential Staining of Tannin in Sections of Epoxy-Embedded Plant Cells

A staining procedure is described for the light microscopic localization of ergastic tannins in epoxy sections of plant cells embedded for study by transmission electron microscopy. Callus and cell suspensions of Pseudotsuga menziesii and Pinus taeda fixed in glutaraldehyde:acrolein and then OsO₄, followed by epoxy embedding, were sectioned 0.5 μn thick, stained on a glass slide with ethanolic Sudan black B at 60 C as described by Bronner, and then mounted in Karo syrup. Tannin deposits stained brownish-orange and were easily distinguished from lipid bodies of similar size, which stained dark blue to black, and from starch grains, which were unstained. The significance of this differential polychromasia was confirmed by transmission electron microscopy. This staining proadure should prove valuable in the cytoplasmic evaluation of the plant cell ergastics (especially tannins) via light microscopy whether or not electron microscopic examination is intended.     

Revision of the genus Cryptomonas (Cryptophyceae): a combination of molecular phylogeny and morphology provides insights into a long-hidden dimorphism

Seventy-three strains of cryptophytes assigned to the genera Cryptomonas, Campylomonas or Chilomonas were studied by light microscopy, spectrophotometry and whole-mount electron microscopy. Twelve groups of strains were distinguished by light and whole mount electron microscopy using a combination of characters, mainly cell size, type of periplast and presence/absence and number of pyrenoids. However, characters previously used to distinguish Cryptomonas from Campylomonas (e.g. the type of periplast: polygonal periplast plates vs. a continuous periplast sheet) were found to occur together in dimorphic strains, indicating that periplast types relate to different life-history stages of a single taxon. To evaluate the taxonomic significance of the type of periplast and other characters previously used to distinguish genera and species, representatives of each strain group were subjected to molecular phylogenetic analyses using two nuclear ribosomal DNA regions (ITS2, partial LSU rDNA) and a nucleomorph ribosomal gene (SSU rDNA). The results of the phylogenetic study provide molecular evidence for a life history-dependent dimorphism in the genus Cryptomonas: the genus Campylomonas represents the alternate morph of Cryptomonas. Campylomonas and Chilomonas are reduced to synonyms of Cryptomonas, the genus Cryptomonas is revised and typified, two new species are described and six species are emended.     

A Simple Method For The Preparation Of Sperm Of External Fertilizers For Scanning Electron Microscopy

A simple technique is described for the study of sperm of some external fertilizers (sea urchin, Parechinus angulosus; teleost, Liza dumerili; anurans, Xenopus laevis and Bufo rangeri) by means of scanning electron microscopy. The technique involves: (i) dilution of sperm with water of the natural environment of the animals or artificial media resembling their natural environment; (ii) spreading of sperm suspension on 10-mm square Perspex plates; (iii) fixation of diluted sperm with 2.5 pH 7.4 Millonig phosphate buffered glutaraldehyde (1 hour); (iv) repeated washing in phosphate buffer, air drying and coating with palladium-gold in a vacuum evaporator. Postfixation with 1% osmium tetroxide did not improve the results and dehydration with an alcohol series was not necessary.     

Aedes aegypti: Sensilla trichodea and stimulus-conducting structures

Much of the morphology of the olfactory sensilla on the antennae of the mosquito Aedes aegypti (L.) has been described, however little is known about the fate of odour molecules once they have been adsorbed onto the surfaces of sensilla. A stimulus-conducting system of pores, pore kettles, and pore tubules has been described for the sensilla trichodea (olfactory hairs) of several insects but not mosquitoes. Scanning electron microscopy was used to identify the s. trichodea of Ae. aegypti and to attempt visualization of their pore openings. Chemical fixation, cryopreparation, freeze drying, and negative staining, with high resolution transmission electron microscopy (TEM), were used to locate putative stimulus-conducting structures associated with the pores. TEM sections using Dalton's fixative or freeze drying showed pores and pore tubules, whereas pore tubules were poorly preserved in cryoprepared sections. The putative stimulus-conducting structures were clearly demonstrated by negative staining of whole sensilla which was quick and easy. The current hypothesis of olfactory stimulus conduction is extended to include Ae. aegypti.     

The Use of Backscattered Electrons to Examine Selectively Stained Nerve Fibers in the Scanning Electron Microscope

Selective staining of neuronal tissues using standard light microscopic techniques has been combined with backscattered electron scanning electron microscopy. This technique allows neurons to be readily distinguished from their surrounding tissues and examined at high resolution. The technique overcomes some of the problems involved in scanning electron microscopy of nervous tissue in situ.     

An emended description of Amphidiniopsis arenaria Hoppenrath 2000, based on material from the Sea of Japan

Cells of a taxon similar to the type species of Amphidiniopsis, Amphidiniopsis kofoidii, were found during the course of a study of marine sand-dwelling dinoflagellates in Peter the Great Bay, Sea of Japan, Russia. These specimens were examined in detail using light and scanning electron microscopy and were identified as Amphidiniopsis arenaria, a species known so far only from the North Sea, Germany. Morphological variability within the species and details of its sulcal construction are described here for the first time.     

光合电子流对光响应的机理模型及其应用

光合电子流对光响应的机理可以揭示植物光合电子流与光强、植物捕光色素分子物理特性之间的关系。该文讨论了光合电子流对光响应的机理模型的特性以及捕光色素分子的物理性质, 并利用此模型拟合了山莴苣(Lagedium sibiricum)、一年蓬(Erigeron annuus)和紫菀(Aster tataricus)的光合电子流对光响应的曲线。由此模型不仅可以得到植物的最大光合电子流、饱和光强、初始斜率等参数, 还可以获得捕光色素分子有效光能吸收截面和处于最低激发态的捕光色素分子数对光的响应关系。结果表明: 随光强的增加, 山莴苣的捕光色素分子的有效光能吸收截面下降最快, 紫苑的下降速度最慢; 山莴苣处于最低激发态的捕光色素分子数增长速度最快, 紫苑的增长速度最小。捕光色素分子的有效光能吸收截面随光强增加而下降、处于最低激发态的捕光色素分子数随光强增加而增加的特性将减少其光能的吸收和激子的传递, 因而有利于减少强光对植物产生的光伤害。     

Ultrastructure and histochemistry of the metacercarial cyst of Spelotrema nicolli (Microphallidae: Trematoda)

Based on electron microscopy and histochemical tests, four layers are described for the metacercarial cyst of Spelotrema nicolli. The outermost layer I is made up of host connective tissue. Layer II, which may contain lipid, is a narrow band varying in electron density. Layer III is composed of mucoprotein with a granular fine structure. The innermost layer is made up of columnar, proteinaceous crystals, which radiate out from the inner edge of the cyst. Observations on the epidermis of Spelotrema nicolli contrast previous work on the mechanism of cyst formation in microphallids.     

X-ray microanalysis of biological specimens by high voltage electron microscopy

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200–400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory.

X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/B ratios at 300 kV.

From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300–400 kV was very useful resulting in high P/B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.     

Superficial Warming of Epoxy Blocks for Cutting of 25-150 μm Sections to be Resectioned in the 40-90 nm Range

An improved method is described in which tissue areas can be initially identified in thick sections by light microscopy and isolated for subsequent ultrathin sections and observation by electron microscopy. This is achieved by embedding in hard Epon which can be sectioned at 25-150 μm on a sliding microtome after softening the blockface by applying a 60-70 C tacking iron to its surface immediately before each section is taken. The thick sections are then mounted on plastic slides to enable light microscopic selection of areas to be observed by electron microscopy. The selected areas are remounted on faced Epon blanks and resectioned at less than 50 nm. This technique makes it possible to obtain thick sections while maintaining an Epon hard enough for good serial ultrathin sections.     

The flagellates of the termite Hodotermopsis sjoestedti: Immunological and ultrastructural characterization of four new species in the genera Spirotrichonympha, Spironympha and Microjoenia

Four species of spirotrichonymphids representing three genera Spirotrichonympha, Spironympha and Microjoenia symbiotic in the termite Hodotermopsis sjoestedti, have been studied by light and immunofluorescence microscopy and transmission electron microscopy. The genus Spirotrichonympha represented by Spirotrichonympha cincta n. sp. is characterized by a compound axostyle composed of several fibers or subaxostyles, and this species has peculiar undescribed structures associated with the flagellar lines. The genus Spironympha is characterized by flagellar lines restricted to the anterior area and a simple tubular axostyle limited by 1–3 layers of microtubules. The two new Spironympha species Spironympha obtusa and Spironympha oblonga are distinguished by peculiarities in the flagellar lines and the axostyle, as revealed by immunofluorescence and electron microscopy. The genus Microjoenia represented by Microjoenia minuta n. sp. has radiating flagellar lines and an axostyle with two microtubular rows originating from the pelta-axostyle rows covering the anterior cap. Four new species were named and one was assigned to the SSU rRNA sequences provided by the molecular phylogenetic studies by Ohkuma et al. [2000. Phylogenetic identification of hypermastigotes, Pseudotrichonympha, Spirotrichonympha, Holomastigotes, and parabasalian symbionts in the hindgut of termites. J. Eukaryot. Microbiol. 47, 249–259; 2005. Molecular phylogeny of parabasalids inferred from small subunit rRNA sequences, with emphasis on the Hypermastigea. Mol. Phylogenet. Evol. 35, 646–655].     

Plant Protoplasts Immobilized in Calcium Alginate: A Simple Method of Preparing Fragile Cells for Transmission Electron Microscopy

A simple method for electron microscopic preparation of plant protoplasts is described. The main problems in preparing these fragile protoplasts for electron microscopy have been cell collapse due to steep gradients between protoplasts and fixatives and unacceptable loss of material during the many steps of the procedure. These problems may be solved by immobilization of the protoplasts in calcium alginate beads. The free diffusion properties of this gel prevent steep gradients. The beads also simplify handling and prevent loss of material. Protoplasts isolated from hypocotyls of rape, Brassica napus (var. Niklas), have been used as a model system. Transmission electron microscopy of the immobilized protoplasts osmotically stabilized with glucose demonstrated adequate structural and ultrastructural preservation.     

Global diversity and biogeography of Skeletonema species (bacillariophyta)

Recent studies have shown that the cosmopolitan diatom Skeletonema costatum sensu lato is composed of several morphologically and genetically distinct species. To assess whether the separate species have a cosmopolitan distribution, we analysed 184 strains from marine and estuarine sites worldwide. We identified the strains using light and electron microscopy, and we sequenced the hyper-variable region of nuclear LSU rDNA. All recently described species were genetically distinct, and all but two were morphologically distinct. Variability was found for the only ultrastructural character used to distinguish Skeletonema dohrnii and S. marinoi, which cannot be identified based on morphology alone. Furthermore, multiple genetically distinct taxa, which may represent cryptic species, were found within the S. menzelii and S. tropicum clades. We found that all currently recognized species of Skeletonema are widespread, however, gaps seem to occur in their geographical ranges. For example, some species are found in both the northern and southern temperate latitudes whereas other species appear to have only subtropical to tropical ranges. Skeletonema pseudocostatum and S. grethae seem to have more restricted geographical ranges because the former was not found along American coasts and the latter was encountered only in US waters. A taxonomic update is provided for Skeletonema strains currently available in several culture collections, which could aid reinterpretation of results obtained in comparative studies using these strains.     

A Simple Apparatus for the Freeze-Drying of Biological Specimens Preparatory to Scanning Electron Microscopy

In this paper an apparatus is described which facilitates the freeze-drying of specimens prior to their examination by scanning electron microscopy (SEM). The apparatus has been constructed following the principle of a Dewar flask. It enables the operator to keep his SEM specimens free from contamination until immediately prior to coating and examination in the microscope. Examples of a human dental plaque and of a pure colony of Bacterionema matruchotii as prepared by this method are illustrated.     

Electron Microscopic Identification and Morphologic Preservation of Enriched Populations of Lung Cells Isolated by Laser Flow Cytometry and Cell Sorting: A New Technique

There is increasing need to verify the identities of cell subpopulations enriched by laser flow cytometry and fluorescence-activated cell sorting (FACS). When cell subpopulations isolated from whole organs or tissues have similar characteristics (e.g., size, granularity, staining), light, phase contrast or fluorescence microscopy may not provide sufficient resolution to identify isolated cells accurately and many flow cytometric parameters (e.g., viability, fluorescence) require the cells to be live at the point of analysis where the cell transects the laser beam. In some studies, cells identified by fluorescence microscopy as a highly enriched subpopulation were found by electron microscopy to contain significant populations of other cell types. A technique, fixation-in-flow (FIF), has been developed to increase ability to correlate morphological and laser analyses of cell subpopulations. Sheath fluid is replaced by fixative, permitting fixation to be initiated immediately after laser beam analysis of live cells. This new procedure yields improved cytoarchitectural preservation of recovered cell subpopulation(s) for evaluation by transmission or scanning electron microscopy. This report presents results from applying the methodology to identify more accurately cell subpopulations of the distal lung, specifically type II pneumocytes, Clara cells and pulmonary macrophages. A modification of this procedure was employed to isolate fibroblast subpopulations from murine lung fibroblasts grown in vitro and the procedure is being used to determine the responses of cultured fibroblasts to other permutations (e.g., X-irradiation, cytokines).     

Isolation of Onchocerca gibsoni tissue from frozen nodules for biochemical use

A new procedure is described which enables gram quantities of adult Onchocerca tissue to be isolated from frozen connective tissue nodules, thus minimizing the risk of enzymatic degradation. Bovine connective tissue nodules containing adult Onchocerca gibsoni worms were obtained from Australia frozen at −70°C and sectioned while still frozen into 3 mm thick slabs. The sections were thawed immediately before use, worm segments removed, rinsed, pelleted, and flash frozen in liquid nitrogen. Quality of the isolated material was demonstrated by the presence of an intact adult epicuticle as determined by electron microscopy, and by the presence of viable uterine larvae and cells. This procedure is applicable to other nodule-forming worms such as Onchocerca volvulus and is suitable for investigations which require the isolation of labile molecules or those present in minute quantities.