A general-purpose system for the rapid acquisition and computer processing of optical and scanning electron microscope images—II. Software

The design and implementation of software for the operation of a general-purpose optical and electron microscope image processing system is described. The software is a group of programs, controlled by a command-line interpreter called IPR (Image PRocessing). The interpreter may be used interactively, or groups of commands may be issued indirectly after placing them in files. Programs for specialized image processing applications may obtain the services of the system's memory-resident programs, through the same interprogram communication methods that are used by the command-line interpreter.     

Bipolar budding in yeasts — an electron microscope study

Bud formation in yeasts with bipolar budding was studied by electron microscopy of thin sections.Budding in yeasts of the speciesSaccharomycodes ludwigii, Hanseniaspora valbyensis andWickerhamia fluorescens resulted in concentric rings of scar ridges on the wall of the mother cell. The wall between the ridges consisted of the scar plug left by the former budding and opened up in the formation of the next bud. The wall of the bud arose from under the wall of the mother cell.In the yeasts of the speciesNadsonia elongata more than one bud might be formed from the same plug.InSchizoblastosporion starkeyi-henricii the scar ridges were close together and apparently not separated by the entire plug.In all species a cross wall was formed between mother cell and bud which consisted of an electron-light layer between two layers of more electron-dense material. The cells separated along the light layer.The authors wish to thank Dr J. A. Barnett for corrections of the English text, and Mr J. Cappon for drawing Fig. 1.     

“Chromosome exo-skeleton” in plant cells visualized by scanning electron microscopy

Summary Cytoskeletons surrounding the chromosomes of the root tip cells ofPisum sativum and the generative cells ofAllamanda schottii were visualized using Triton X-100 extraction and scanning electron microscopy. The cytoskeleton surrounding the chromosome consisted of a reticulate network of fibres. This is the first report showing the existence of a chromosome exo-skeleton in plant cells.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid)     

Nuclear lamina—like filaments and nuclear matrix in allium cepa as revealed by scanning electron microscopy

In this study,freeze-fractured specimens of allium cepa root tip meristems were examined under the scanning electron microscope(SEM),This technique permitted the visualization of the outer membrane of the nuclear envelope with nuclear pore complexes and polyribosomes.Some of the cell nuclei prepared with this procedure had fissures of various widths on their nuclear envelopes through which the nuclear lamina-like filaments(LLF) undernearth the nucleoplasmic side of the envelopes were clearly visible.The diameters of these filaments veried between 25 and 125nm.Many of the LLFs showed granular thickenings at places,and were attached to the inner surface of nuclear envelope in some regions .Similar LLFs were also seen at the peripheries of the freeze-fractured faces of nuclei.Meanwhile,the spatial relation between the nuclear matrix filaments(NMF) and other nuclear structures(nucleoli,chromation and peripheral lamina-like filaments) was revealed in these fractured preparations.In addition,the methods and techniques in studying the nuclear lamina morphology and the roles played by NMFs in activities of various nuclear sturctures were discessed in brief.     

SPRING – An image processing package for single-particle based helical reconstruction from electron cryomicrographs

Helical reconstruction from electron cryomicrographs has become a routine technique for macromolecular structure determination of helical assemblies since the first days of Fourier-based three-dimensional image reconstruction. In the past decade, the single-particle technique has had an important impact on the advancement of helical reconstruction. Here, we present the software package SPRING that combines Fourier based symmetry analysis and real-space helical processing into a single workflow. One of the most time-consuming steps in helical reconstruction is the determination of the initial symmetry parameters. First, we propose a class-based helical reconstruction approach that enables the simultaneous exploration and evaluation of many symmetry combinations at low resolution. Second, multiple symmetry solutions can be further assessed and refined by single-particle based helical reconstruction using the correlation of simulated and experimental power spectra. Finally, the 3D structure can be determined to high resolution. In order to validate the procedure, we use the reference specimen Tobacco Mosaic Virus (TMV). After refinement of the helical symmetry, a total of 50,000 asymmetric units from two micrographs are sufficient to reconstruct a subnanometer 3D structure of TMV at 6.4 Å resolution. Furthermore, we introduce the individual programs of the software and discuss enhancements of the helical reconstruction workflow. Thanks to its user-friendly interface and documentation, SPRING can be utilized by the novice as well as the expert user. In addition to the study of well-ordered helical structures, the development of a streamlined workflow for single-particle based helical reconstruction opens new possibilities to analyze specimens that are heterogeneous in symmetries.     

What is in the intercellular spaces of roots? Evidence from the cryo-analytical-scanning electron microscope

The schizogenous intercellular spaces (i. e. those small spaces formed by cell walls coming apart) in the cortex of the roots of field-grown maize ( Zea mays L.) were studied in planed transverse faces of frozen tissue, very lightly etched and coated with Al. The spaces were mostly filled with either fluid or, in the drier roots, with a flaky deposit. This deposit may have been left behind when water was withdrawn, or may have been debris dislodged by the planing. Even in roots with mostly dry spaces, some wet, fluid-filled spaces remained. X-ray microanalysis of the wet spaces revealed that the fluid contained K (average concentration 230 m M , range 50–750 m M ) and Ca (average concentration 100 m M , range 15 to 550 m M ), and occasionally small amounts of S, P or Cl. No other balancing inorganic anions were detected. Concentrations in the wet intercellular spaces showed considerable variation between one space and the next, and were often quite different from those in the vacuoles of adjacent cells. However, overall the vacuoles of the cells surrounding the spaces showed mean concentrations, and distributions of concentrations, indistinguishable from those of the wet spaces. Analyses of the deposits in the dry spaces were less reliable because of their uneven surface, but the same ions in about the same amounts were found there. The contents of the spaces showed no correlation with either the time of collection of the roots, or with distance from the root tip. Nor was there any change in concentration of these ions in the spaces when the roots were grown for 19 h in distilled water mist. Experiments and evidence are presented suggesting that the observed distribution of ions is probably not an artefact. Pilot experiments showed similar distributions of extracellular ions in roots of barley, Sudan grass and soybean     

Pollen morphology of Sorghum Moench – Sections Eu-sorghum and Para-sorghum

Pollen morphological studies have been carried out by SEM on 23 species of Sorghum (Gramineae) in order to resolve the exine surface patterns in sections Eu-sorghum (subsection Arundinacea-series Spontanea and Sativa and subsection Halepensia), and Para-sorghum. Basically, two exine ornamentation types have been observed viz. granular and insular. In section Eu-sorghum, series Spontanea and Sativa (of subsection Arundinacea) are heterogeneous having both types of exine pattern. In the same section, subsection Halepensia is characterised by having only a granular exine. Section Para-sorghum shows a marked pollen morphological similarity with subsection Halepensia of section Eu-sorghum. Snowden's concept that the two series Spontanea and Sativa are closely related and that the cultivated Sorghum (series Sativa) might have evolved from the wild Sorghum (series Spontanea), is supported by the present observations.     

Environmental scanning electron microscopy (ESEM)—a versatile tool in studying plants

Environmental scanning electron microscopy (ESEM) enables the investigation of hydrated and uncoated plant samples and the in situ observation of dynamic processes. Water vapor in the microscope chamber takes part in secondary electron detection and charge prevention. Two ESEM modes are available and offer a broad spectrum of applications. The environmental or wet mode prevents sample dehydration by the combination of sample cooling (5°C) and a vapor pressure of 4–6 Torr. In the low vacuum mode, the maximum chamber pressure is limited to 1 Torr (corresponding to about 5% relative humidity in the chamber) and allows the simultaneous use of a backscattered electron detector for imaging material contrast. A selection of characteristic plant samples and various applications are presented as a guide to ESEM for plant scientists. Leaf surfaces, trichomes, epicuticular waxes, and inorganic surface layers represent samples being comparatively resistant to dehydration, whereas callus cells and stigmatic tissue are examples for dehydration- and beam-sensitive samples. The potential of investigating dynamic processes in situ is demonstrated by studying anther opening, by tensile testing of leaves, and by performing hydration/dehydration experiments by changing the vapor pressure. Additionally, automated block-face imaging and serial sectioning using in situ ultramicrotomy is presented. The strengths and weaknesses of ESEM are discussed and it is shown that ESEM is a versatile tool in plant science.     

Cell-mediated retraction versus hemodynamic loading – A delicate balance in tissue-engineered heart valves

Preclinical studies of tissue-engineered heart valves (TEHVs) showed retraction of the heart valve leaflets as major failure of function mechanism. This retraction is caused by both passive and active cell stress and passive matrix stress. Cell-mediated retraction induces leaflet shortening that may be counteracted by the hemodynamic loading of the leaflets during diastole. To get insight into this stress balance, the amount and duration of stress generation in engineered heart valve tissue and the stress imposed by physiological hemodynamic loading are quantified via an experimental and a computational approach, respectively.     

Molecular mechanisms of cardiac pathology in diabetes – Experimental insights

Diabetic cardiomyopathy is a distinct pathology independent of co-morbidities such as coronary artery disease and hypertension. Diminished glucose uptake due to impaired insulin signaling and decreased expression of glucose transporters is associated with a shift towards increased reliance on fatty acid oxidation and reduced cardiac efficiency in diabetic hearts. The cardiac metabolic profile in diabetes is influenced by disturbances in circulating glucose, insulin and fatty acids, and alterations in cardiomyocyte signaling. In this review, we focus on recent preclinical advances in understanding the molecular mechanisms of diabetic cardiomyopathy. Genetic manipulation of cardiomyocyte insulin signaling intermediates has demonstrated that partial cardiac functional rescue can be achieved by upregulation of the insulin signaling pathway in diabetic hearts. Inconsistent findings have been reported relating to the role of cardiac AMPK and β-adrenergic signaling in diabetes, and systemic administration of agents targeting these pathways appear to elicit some cardiac benefit, but whether these effects are related to direct cardiac actions is uncertain. Overload of cardiomyocyte fuel storage is evident in the diabetic heart, with accumulation of glycogen and lipid droplets. Cardiac metabolic dysregulation in diabetes has been linked with oxidative stress and autophagy disturbance, which may lead to cell death induction, fibrotic ‘backfill’ and cardiac dysfunction. This review examines the weight of evidence relating to the molecular mechanisms of diabetic cardiomyopathy, with a particular focus on metabolic and signaling pathways. Areas of uncertainty in the field are highlighted and important knowledge gaps for further investigation are identified. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

A stereologic electron microscope study of “tubular myelin figures” in alveolar fluids of rat lungs

Summary The three-dimensional structure of a composite material found in alveolar exudate of oxygen poisoned lungs but also present in normal lungs is stereologically analysed. It is composed of tubules of 450 Å diameter which are tightly packed in a quadratic lattice. The wall of the tub vile is formed by four-winged osmiophilic filaments which are located in the corners of the quadratic lattice; their interior is made up of a hydrophilic substance which contains either a tubule or a filament of moderate electron density. The osmiophilic substance of the walls is continuous with associated myelin figures which can be resolved into lamellae with a periodicity of 42 Å and can thus be considered to be water crystals of phospholipids. The nature of the content of the tubules, which presumably exerts the formative force on the phospholipid lamellae to form tubules, remains undetermined.Dedicated to Prof. W. Bargmann in honor of his 60th birthday.The research reported here has been sponsored by the Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung (Nr. 2569); by the Stiftung für wissenschaftliche Forschung an der Universität Zürich; by the National Institutes of Health, USPHS, through grant RF-57; and the 6570th Aerospace Medical Research Laboratories under contract AF 61(052)-784 through the European Office of Aerospace Research (OAR), United States Air Force.     

The gastrointestinal tract of Adélie penguins – morphology and function

The aim of this study was to provide data on the morphology of the gastrointestinal tract of Adélie penguins (Pygoscelis adeliae). It was found to consist of a long oesophagus, a two-chambered stomach, a small intestine measuring only 5.22body length, two rudimentary caeca and a short colon, typical of carnivorous birds. The stomach comprised a glandular proventriculus and a muscular gizzard that frequently contained grit. An acidic pH was recorded in both chambers. Ultrastructural studies of the small intestinal mucosal membrane revealed epithelial cells with elongated, irregular microvilli and high affinity for toluidine blue, absorptive intestinal epithelial cells and goblet cells. Numerous large lymphocyte-like cells were observed close to the brush border of the epithelium, and empty spaces on the epithelial surface reflected normal cell loss in the small intestine. The rudimentary caeca and colon provide relatively little volume and time for symbiotic bacteria to aid the digestion of crustacean chitin.     

The strategy of “Useful Sun” improves physical endurance and adaptation of cardiac morphology

The action of solar light transformed by special screens has been studied on CD-1 male mice. In the active control group, mice were irradiated through screens absorbing the UV-component. In the experimental group, screens transforming the UV-component into the orange-red light were used. In the active control, changes in the swimming activity, as compared to the same parameter before irradiation, were manifested much less than in animals of the experimental group. A morphological analysis showed changes in the structure of all cardiomyocyte organelles studied: the relative area of mitochondria in the experimental mice increased by more than 20% compared to intact animals (p < 0.05). A significant increase in the area of the sarcoplasmic reticulum, by 23.4% (p < 0.05), and in the volume of the myofibrillar apparatus, by 19.4% (p < 0.05), was detected. The results of our experiment show that the irradiation with using an additional orangered component improves the physical endurance 1.5 times and initiates morphogenetic processes in cardiac muscle cells.     

Butterfly morphology in a molecular age – Does it still matter in butterfly systematics?

We review morphological characters considered important for understanding butterfly phylogeny and evolution in the light of recent large-scale molecular phylogenies of the group. A number of the most important morphological works from the past half century are reviewed and morphological character evolution is reassessed based on the most recent phylogenetic results. In particular, higher level butterfly morphology is evaluated based on a very recent study combining an elaborate morphological dataset with a similar molecular one. Special attention is also given to the families Papilionidae, Nymphalidae and Hesperiidae which have all seen morphological and molecular efforts come together in large, combined works in recent years. In all of the examined cases the synergistic effect of combining elaborate morphological datasets with ditto molecular clearly outweigh the merits of either data type analysed on its own (even for ‘genome size’ molecular datasets). It is evident that morphology, far from being obsolete or arcane, still has an immensely important role to play in butterfly (and insect) phylogenetics. Not least because understanding morphology is essential for understanding and evaluating the evolutionary scenarios phylogenetic trees are supposed to illustrate.     

An electron microscope study of spermiogenesis in Spirorbis (Laeospira) mörchi Levinsen (Polychaeta)

Summary 1. In the hermaphroditic polychaete Spirorbis (Laeospira) mörchi, early spermatids develop in clusters within the coelom of the male segments. The cells within a single cluster are all in the same stage of development and are connected by an extensive cytoplasmic bridge system. 2. The acrosome forms in a single lamella of the Golgi apparatus which bears a close association to the plasma membrane. The final position of the acrosome is at a point considerably removed from the site of formation. 3. The nuclear changes culminating in condensation and elongation of the head are described. A rearrangement of cytoplasmic microtubules occurs simultaneously with nuclear elongation. 4. Redundant nuclear envelope, resulting from nuclear volume reduction, is pinched off in the form of four vesicles. The latter structures are lost with the residual cytoplasm. 5. Pour spherical mitochondria elongate to become incorporated into the middle-piece. A rearrangement of microtubules also occurs simultaneously with mitochondrial elongation. Cytoplasmic microtubules are absent from the fully formed sperm.This investigation was made possible through a Postdoctoral Fellowship from the National Science Foundation, 44150. — I wish to express my gratitude to Dr. John Luft for the training I received in the techniques of electron microscopy. Dr. James Koehler and Dr. Daniel Szollosi are thanked for their many helpful suggestions.     

A freeze-fracture electron microscope study of Trichomonas vaginalis Donné and Tritrichomonas foetus (Riedmüller)

Two strains of Trichomonas vaginalis, JH162A , with low pathogenicity, and Balt 44, with high pathogenicity, as well as one highly pathogenic strain, KV-1, of Tritrichomonas foetus were studied by freeze-fracture electron microscopy. The protoplasmic faces ( PFs ) of the cell membranes of all three strains of both species had similar numbers of intramembranous particles (IMPs); however, the particles in the external faces (EFs) of these membranes were least abundant in Trichomonas vaginalis strain Balt 44 and most numerous in those of strain JH162A of this species. In Tritrichomonas foetus strain KV-1 the number of IMPs in the EF was close to but somewhat lower than that in the mild strain of the human urogenital trichomonad . In both species, the anterior, but not the recurrent, flagella had rosette-like formations, consisting of approximately 9 to 12 IMPs on both the PFs and EFs. The numbers and distribution of the rosettes appeared to vary among different flagella and in different areas of individual flagella of a single organism belonging to either species. The freeze-fracture electron micrographs provided a more complete understanding of the fine structure of undulating membranes of Trichomonadinae , as represented by Trichomonas vaginalis, and of Tritrichomonadinae (the Tritrichomonas augusta -type), as exemplified by Tritrichomonas foetus, than was gained from previous transmission and scanning electron microscope studies. Typically three longitudinal rows of IMPs on the PF of the recurrent flagellum of Trichomonas vaginalis were noted in the area of attachment of this flagellum to the undulating membrane. The functional aspects of the various structures and differences between certain organelles revealed in the two trichomonad species by the freeze-fracture method are discussed.     

Uptake and processing of DNA by Acinetobacter calcoaceticus – a review

In natural transformation, DNA in the form of macromolecular fragments can be translocated across the cell envelope of prokaryotic microorganisms. During the past two decades, several, largely mutually contradictory, hypotheses have been forwarded to explain the molecular mechanism and bioenergetics of this translocation process. Other biomacromolecules are translocated across the bacterial cell envelope as well, such as polysaccharides and proteins, the latter for instance in the process of the assembly of type-IV pili. This brings up the question whether or not common components are involved.Here, we review analyses of DNA translocation in Acinetobacter calcoaceticus, a Gram-negative eubacterium that is able to migrate through twitching motility, and also shows a high frequency of natural transformation. DNA uptake in this organism is an energy-dependent process. Upon entry into the cells, the DNA fragments are integrated into the resident chromosome when a sufficiently large region of mutual homology is available (200 to 400 bp). However, this process is rather inefficient, and on the average 500 bp of each incoming fragment is degraded through exonuclease activity. Upon covalent attachment of a bulky protein molecule to the transforming DNA, the DNA-translocation machinery becomes blocked in further translocation activity.Since A. calcoaceticus is not well suited for transposon mutagenesis, a random mutagenesis procedure has been developed, based on the ligation of an antibiotic-resistance marker to random fragments of chromosomal DNA. This method was used to generate several mutants impaired in the natural transformation process. Three of these have been characterized in detail. No components, common to the translocation of macromolecules through the cell envelope of Acinetobacter, have been detected in this screen.