bluuebunny/crossref_metadata_2025 huggingface dataset:
Mucous secretion as observed after ultrarapid freezing and freeze substitution
Authors: Luchtel D.L.
Published: June 2020

Most molecules synthesized for extracellular transport are believed to be secreted from the cell by exocytosis. Exocytosis is also generally accepted as the mechanism of mucous cell secretion. We are studying the control of mucous and fluid secretion in a gastropod mollusc, Agriolimax columbianus, and have shown that it is an excellent model for the study of mucous secretions because of the copiousness and precise control of these secretions and the fact that they are released onto an exposed body surface, rather than within body cavities. Our observations of chemically-fixed material (glutaraldehye followed by osmium) showed “omega figures” typical of mucous vesicle exocytosis. On the other hand, in vivo observations showed that membrane-bound vesicles were shed intact from mucous cells.2 As an alternative to chemical fixation, we used a physical method of fixation, ultrarapid freezing, to determine more accurately the ultrastructural basis of mucous cell secretion.

Intact body wall of the terrestrial slug, A. columbianus, was frozen with a Cryopress™ (Med-Vac Inc., St. Louis, MO) by slamming the tissue against the face of a polished copper block cooled in liquid N2 The frozen tissue was then freeze-substituted in cold (-80°C) osmium-containing acetone for several days, then brought to room temperature and embedded in Epon. Sections for EM were observed in a JEOL-100S TEM.
EM immunocytochemical localization of rhodopsin and IRBP during retinal development
Authors: Ulshafer R. J. , Hauswirth W.W. , van der Langerijt A.
Published: June 2020

Two rod photoreceptor cell-specific proteins, rhodopsin and interphotoreceptor retinoid binding protein (IRBP), were localized during fetal development of the bovine retina using immuncicrytcichemistry. Rhodopsin is the light sensitive protein that, when activated, begins the process of transducing light energy to an electrical response. IRBP is a carrier protein that shuttles light-isomerized vitamin A (retinol) between the rod outer segment and the overlying retinal pigment epithelium where it is recycled to its light-sensitive form. Rhodopsin has been previously imimmocytochemically localized to rod (but not cone) photoreceptor outer segment membranes. IRBP has been localized to the subretinal space using immunocytochemistry.

Retinas were obtained from fetuses at approximately 4, 5, 6, 7, and 8 months of gestation, fixed in 4% paraformaldehyde and 0.5% glutaraldehyde, and embedded at 40°C in epoxy resin. Thin sections were mounted on Ni grids and incubated with antibodies raised against the purified antigens: a mouse monoclonal anti-rhodopsin or a rabbit polyclonal anti-IRBP. A second antibody (Goat-anti-mouse or Goat-anti-rabbit) was conjugated with 15 nm Au particles and reacted with the sections. Control incubations were made using pre-immune rabbit serum and mouse monoclonals made against other tissue or bacterial sources.
Midgut infection of

Heliothis Zea

(Lepidoptera: Noctuidae) by cytoplasmic polyhedrosis virus
Authors: Bong C. F. J.
Published: June 2020

The cytoplasmic polyhedrosis virus (CPV, Family Reoviridae) is an entomopathogen with a very wide host range, affecting mainly lepidopterous insects. The virus often causes chronic infections in insects, infecting mainly the midgut epithelium. Detailed studies of the effects of CPV on silkworm, Bombyx mori, have been done by Japanese workers , but there is very little information on the histopathology of CPV on other insects.

In this study, 5-day old corn earworm (H. zea) larvae were infected per os with CPV. Control larvae were fed sterile water. The midgut was dissected out at 20, 48 and 72h post-treatment for both SEM and TEM studies. In the SEM study the midgut was fixed in 2.5% glutaraldehyde in phosphate buffer at pH 7.2, postfixed in buffered 2% osmium tetroxide at room temperature, dehydrated in ethanol, cryofractured in liquid nitrogen, critical-point dried, mounted, coated with gold-palladium and examined in a Hitachi HHS-2R scanning electron microscope at 20 kV. In the TEM study, the tissues were fixed in half-strength Karnovsky's fixative, postfixed in buffered cold 2% osmium tetroxide, en bloc stained in aqueous 0.5% uranyl acetate, dehydrated in ethanol and embedded in Spurr resin. Ultrathin sections were cut with glass knives, sequentially stained in aqueous uranyl acetate and lead citrate, and observed in a Zeiss 109 transmission electron microscope at 50 kV or a Siemens 101 at 80 kV.
Distribution of K+ and NA+ in lymphocytes suggest binding to nucleic acids and proteins
Authors: Haskin C. , Cameron I. , Hunter K. , Smith N. K. R.
Published: June 2020

The operation of the Mg2+ dependent Na+K+ ATPase “pump” located in the plasma membrane has generally been assumed to be responsible for maintaining a major portion of the cell’s asymmetric ion distribution by pumping cellular Na+ out and extracellular K+ in. This model assumes the “pump” is the primary determinant of K+ content, that the mM concentrations of Na+ and K+ exist in a free state and are therefore immediately available to the pump. If this is the case, analysis of ion content within the cytoplasm and nuclear compartments should show a Na+ content that is negatively correlated to the K+ content and multiple regression analysis of ion content should show Na+ concentration to be a major predictor of K+ content. However, recent experiments are beginning to show that K+ is not necessarily in a free state in the cell but may instead be bound to protein and nucleic acids, von Zglinicki et al. (1986) found no significant concentration differences between the decondensed chromatin within the nuclei and the cytoplasm, but found K+ concentrations in condensed chromatin to be higher than was consistent for ions free in solution. Furthermore, Kellermayer et al. (1984) found that disruption of lymphocyte membrane integrity did not result in the immediate loss of K+ from the nuclei as expected if K+ ions were free in solution. The results suggest that K+ content is associated with an essentially immobile component such as proteins or nucleic acids. We were interested in determining what the state of the K+ ion was in the nucleolar and condensed chromatin compartments of the cell. We wished to specifically test if the K+ ion concentration were negatively correlated with Na+ ion concentration with Na+ being the major predictor for K+ content as the “pump” model would require, or if, P and S content would be the major predictors of K+ were K+ bound to proteins (the major source of total cellular sulfur) or to nucleic acids (the major source of cellular phosphorus).
Validation of convection-limited cooling for Electron Microscopy
Authors: Bailey Stuart M.
Published: June 2020

Rapid freezing is a universal method of sample fixation for electron microscopy. Freeze-fracture replication, low temperature embedment and cryo-sectioning, and cryo-microscopy all depend on freezing specimens rapidly enough so that structure can be preserved unaltered. The most common method of rapid freezing is plunging specimens directly into liquid cryogens. In an earlier study, convection from the cryogen to the specimen was predicted to be the limiting step in rapid freezing of small samples for electron microscopy when the Biot modulus, (h V /kA) which determines the ratio of convective to conductive heat transfer, is less than 1. For convection-limited cooling, cooling rates are proportional to the surface area/volume ratio (A/V), independent of the sample thermal conductivity (k), linearly dependent on the heat transfer coefficient between the liquid cryogen and the specimen (h), and inversely proportional to the product of sample density and heat capacity (pCp):

Ts and Tc are the sample temperature and the cryogen temperature, respectively.
The effect of 2,3,4-trimethylpentane on the ultrastructure of proximal tubular cells in primary cell culture
Authors: Mattie D.R. , Maslanka J.J. , Del Raso N.J. , Chase M.R.
Published: June 2020

To aid in the assessment of the risk of Air Force personnel working with hydrocarbon fuels and compounds, an attempt was made to further characterize the nephropathy that results from exposure to hydrocarbons. The purpose of this study was to isolate and establish purified primary cultures of male rat proximal tubular cells suitable for experimental exposure to sublethal concentrations of solubilized 2,3, 4-trimethylpentane (TMP), a model hydrocarbon. Experiments were conducted to evaluate the cytotoxicity and metabolism of solubilized TMP in media containing or lacking the protein albumin.

Proximal tubule cells in primary suspension culture were exposed to one of the following levels of TMP: 7.9, 12.0, 15.7, 19.1 or 25.5 mM. After 4 hours of exposure, pelleted cells were fixed for transmission electron microscopy by resuspension in 2% glutaraldehyde and 2.5% paraformaldehyde in 0.1M cacodylate buffer at pH 7.4. After a minimum fixation of at least 24 hours, the cells were post-fixed with 2% osmium tetroxide in 0,1M cacodylate buffer at pH 7.4. Cells were processed into Polybed 812 plastic capsules. Sections one micron thick, were cut in order to verify that cells were intact and suitable for thin sectioning. Thin sections (60- 90 nm) were cut on an ultramicrotome using a diamond knife. Thin sections, stained with uranyl acetate and lead citrate, were examined with a transmission electron microscope at 60 kV. Photographs of representative proximal tubule cells were taken at three levels of magnification.
High-resolution SEM of kidney tissue using cryo-techniques
Authors: Walther P , Herter P , Hentschel J , Hentschel H
Published: June 2020

The kidney is a complex zonated organ with a variety of different cell types. For the study of the functional and morphological features, the precise localization in the zones is relevant, which requires the evaluation of rather large portions of tissue. Transmission electron microscopy of replicas of tissue is limited by difficulties to obtain sufficiently large specimens. In order to overcome this problem cryopreparation methods and high resolution field emission scanning electron microscopy (SEM) were used.

1 mm3 cubes of perfusion fixed rabbit kidneys cryoprotected with glycerol were frozen by plunging into liquid propane. For further preparation two different methods were employed.

1: Samples were fractured in liquid nitrogen with a scalpel, freeze substituted using methanol with glutaraldehyde and osmiumtetroxid, warmed to room temperature, critical point dried, and coated by electron gun evaporation with 2 nm of platinum at an angle of 45°, and 10 nm of carbon perpendicularly.
Immunolabeling of vitellogenin in the oocyte of viviparous fish, Poecilia reticulata
Authors: Hori Reiji , Phang Violet , Toong Jin Lam
Published: June 2020

The guppy is viviparous and fertilization is internal. The spermatozoa are introduced by gonopodium of the male and the fertilization may occur upon the completion of maturation. The maturation of the primary oocyte probably starts immediately after puturition. The present study is to observe the changes of the follicular layer and egg cortex in the oocyte of the guppy and also to observe the accumulation of vitellogenin in the oocyte by use of immunolabeling method.

Highly purified vitellogenin from the fish was used as immunogen to obtain rabbit anti-vitel 1ogenin antibody. Immunoglobulin (IgG) fraction was prepared by precipitation of the antiserum with 50% ammonium sulfate followed by gel filtration through Toyopearl HW55S column chromatography.

The oocyte of the guppy, Poecilia reticulata were obtained by dissecting the female. Pre-embedding technigue was applied to immunolabeling of vitellogenin. The oocytes fixed for five hours in chille d 1% g l utaraldehyde and 2 % paraformaldehyde solution diluted with 0.1 M phosphate buffer (pH: 7.0)
Cytochemical studies of the multivesicular bodies
Authors: Tang Xueming
Published: June 2020

Multivesicular bodies were observed frequently in electron microscope photographs of Leydig cells from normal adult rat testes. Their formation, evolution and fate were analyzed morphologically in preparations treated to show cytidine monophosphatase ( CMPase ) activity and in animals sacrificed at various time intervals ranging from 5 minutes to 2 hours after a single intratesticular injection of cationic ferritin. The CMPase medium contains 2mM cerium chloride, 2mM cytidine monophosphate, 5mM manganese chloride and 40mM sodium acetate buffer ( pH 5.0 ) with 4% sucrose. Cationic ferritin was used as a tracer for demonstrating the endocytic activity of Leydig cells.

Cytochemical experiments showed that, in contrast to lysosomes which were CMPase positive, the premultivesicular bodies and the pale multivesicular bodies were CMPase negative. The dense multivesicular bodies were frequently apposed to strongly reactive lysosomes and they aquire their hydrolytic enzymes by fusion with lysosomes and showed CMPase activity.
Localization of DNA in chromatin using ESI and osmium ammine staining
Authors: Woodcock C.L. , Horowitz R.A. , Bazett-Jones D. P. , Olins A.L.
Published: June 2020

In the eukaryotic nucleus, DNA is packaged into nucleosomes, and the nucleosome chain folded into ‘30nm’ chromatin fibers. A number of different model structures, each with a specific location of nucleosomal and linker DNA have been proposed for the arrangment of nucleosomes within the fiber. We are exploring two strategies for testing the models by localizing DNA within chromatin: electron spectroscopic imaging (ESI) of phosphorus atoms, and osmium ammine (OSAM) staining, a method based on the DNA-specific Feulgen reaction.

Sperm were obtained from Patiria miniata (starfish), fixed in 2% GA in 150mM NaCl, 15mM HEPES pH 8.0, and embedded In Lowiciyl K11M at -55C. For OSAM staining, sections 100nm to 150nm thick were treated as described, and stereo pairs recorded at 40,000x and 100KV using a Philips CM10 TEM. (The new osmium ammine-B stain is available from Polysciences Inc). Uranyl-lead (U-Pb) staining was as described. ESI was carried out on unstained, very thin (<30 nm) beveled sections at 80KV using a Zeiss EM902. Images were recorded at 20,000x and 30,000x with median energy losses of 110eV, 120eV and 160eV, and a window of 20eV.
Purification and Ultrastructural Analysis of Terminal Sequences from the Cross-Linked, Linear Plasmids of Borrelia Burgdorferi
Authors: Lubke Lori L. , Garon Claude F.
Published: June 2020

Lyme borreliosis is now the most common arthropod-borne disease in the United States. Borrelia burgdorferi, the causative agent, has been isolated from humans, mammals, birds and arthropods and is cultivable in the laboratory. Careful structural analysis of the DNA content of several isolates has revealed, in addition to a 900 kb linear genome, a unique collection of crosslinked linear and covalently closed, circular DNA molecules ranging from 2-50 kb in length. While all of the linear species were shown to rapidly reanneal to linear duplexes after alkaline denaturation, single-stranded circular molecules measuring twice the length of the linears could be produced by treatment with methyl mercury, glyoxal and urea prior to mounting for electron microscopy. This rapid reannealing mediated by the terminal sequences of the molecule was used in a unique ion exchange column chromatography system to purify terminal, restriction fragments from any which originate internally. Purified terminal fragments could then be compared and characterized further by conventional methods.
Scanning Tunneling Microscopy and Atomic Force Microscopy of Enzymes and Enzyme Complexes
Authors: Edstrom Ronald D. , Yang Xiuru , Gurnack Mary E. , Miller Marcia A. , Yang Rui , Lee Gil , Evans D. Fennell
Published: June 2020

Many of the questions in biochemistry and cell biology are concerned with the relationships of proteins and other macromolecules in complex arrays which are responsible for carrying out metabolic sequences. The simplistic notion that the enzymes we isolate in soluble form from the cytoplasm were also soluble in vivo is being replaced by the concept that these enzymes occur in organized systems within the cell. In this newer view, the cytoplasm is organized and the “soluble enzymes” are in fact fixed in the cellular space and the only soluble components of the cell are small metabolites, inorganic ions etc. Further support for the concept of metabolic organization is provided by the evidence of metabolic channeling. It has been shown that for some metabolic pathways, the intermediates are not in free diffusion equilibrium with the bulk liquid in the cell but are passed along, more or less directly, from one enzyme to the next.
Some Ultrastructural Aspects of Chronic Gastritis
Authors: Bartel Hieronim , Orkisz Stanislaw , Chojnacki Jan
Published: June 2020

Despite numerous observations and publications on the aethiology and pathogenesis of chronic gastritis many questions remain unanswered (1,2,3,4,6). Views on the aethiolcgy of chronic gastritis changed when in 1982 Campylobacter pyloridis (later named Campylobacter pylori CP) was first isolated in Western Australia from the stomach of patients with gastritis lesions and peptic ulceration (5).

The aim of this study, carried out with cooperation with gastroenterologist, has been to investigate the ultrastructural changes in gastric surface epithelium and lamina propria in selected patients endoscopicaUy diagnosed gastritis.

Observations have been carried out on 20 patients (10 men and 10 women). Biopsy material was obtained during endoscopy. In cases when the presence of bacteria has been confirmed further E M investigations have been introduced. Semithin sections for LM observations and ultrathin sections for EM were prepared according to routine EM technique.

CP was found both on the airface of gastric epithelium cells, and what has not been reported so far, inside the lumen of the fundic glands (Fig. 1).
Effects of prenatal ethanol exposure on primate cardiac mitochondria
Authors: Pomphrey M. , Uphoff C. , Nyquist-Battie C.
Published: June 2020

Fetal alcohol syndrome affects the development of the cardiac tissue in children, causing ventral septal defects and ventricular hypertrophy. Initial ultrastructural studies using rodents revealed mitochondria as the primary organelle affected by ethanol. Mitochondria from these animals displayed the classic pattern of mitochondrial degradation: condensation, crystolysis, and vacuolization. However an inherent problem existed in these models: the rodent heart is still immature at birth and full cardiac development does not occur until 18 days post-partem. This study uses the primate Macaca nemestrina in order to more closely parallel the cardiac development of a human infant.Furthermore, alcohol was administered in periodic doses to simulate binge drinking.

Mothers were given weekly doses of one of the following isocaloric treatments: 0.0, 0.3, 0.6. 1.2, 1.8. or 4.1 g ethanol per kg body weight of the dam from week one of pregnancy until full-term. At birth the animals were removed from their mothers and hand-reared with appropriate peer contact.
Oxygen radicals participate in alloxan-induced pulmonary EDEMA
Authors: Kawada Tomoo , Arakawa Michio , Kambara Kenjiro , Segawa Takashi , Ando Fumio , Oda Hiroshi , Nakahara Koji , Hirakawa Senri , Emura Shoichi , Shoumura Shizuko , Isono Hideo
Published: June 2020

We know that alloxan causes increased-permeability pulmonary edema and that alloxan generates oxygen radicals (H2O2, O2−, ·OH) in blood. Therefore, we hypothesize that alloxan-generated oxygen radicals damage pulmonary capillary endothelial cells, and, possibly, alveolar epithelial cells as well. We examined whether oxygen radical scavengers, such as catalase or dimethylsulfoxide (DMSO), protected against alloxaninduced pulmonary edema.

Five dogs in each following group were anesthetized: control group: physiological saline (20ml/kg/h); alloxan group: physiological saline + alloxan (75mg/kg) bolus injection at the beginning of the experiment; catalase group: physiological saline + catalase (150,000u/kg) bolus injection before injection of alloxan; DMSO group: physiological saline + DMSO (0.4mg/kg) bolus injection before alloxan. All dogs had 30-min baseline period and 3-h intervention period. Hemodynamics and circulating substances were measured at the specific points of time. At the end of intervention period, the dogs were killed and had the lungs removed for electron microscopic study and lung water measurement with direct destructive method.
Phagocytosis and hydrogen peroxide production of Eosinophils in the rats spontaneously infected with Mycoplasma pulmonis
Authors: Moriguchi Keiichi , Hirai Kei-Ichi
Published: June 2020

The ability of hydrogen peroxide (H2O2) production was cytochemically compared in eosinophils (EP) between specific pathogen free (SPF) and spontaneously Mycoplasma pulmonis-infected male rats (Wistar strain).

Peritoneal cells including EP (PEP) were harvested with a cold Hanks' balanced salt solution containing 0.1% glucose (HBSSG). Simultaneously, blood granulocytes with eosinophils (BEP) were isolated from the same animals by a Ficoll-Hypaque method. Cells were incubated with latex particles in HBSSG for 60-90 min. Thereafter, cells were washed and transferred into a cerium (Ce) medium consisting of 0.1 M tris - maleate buffer, pH 7.5, 1mM CeCl3, 10 mM sodium azide, 0.1. glucose and 0.25 M sucrose. The incubation was carried out for 20 min at 37°C with occasional stirring. Some cells were incubated for 60 min at 37 °C in a DAB medium containing 0.1% 3.3’-diaminobenzidine 4HCl with particles in 0.1 M phosphate buffer, pH 7.4. All cells were then fixed with glutaraldehyde and osmium tetroxide before processing for electron microscopy, x-ray microanalysis of the subcellular electron-dense reaction deposits was performed under a Tracor-Northern EDX attached to a JEM-1200EX STEM system.
Light and electron microscopic studies on red leg disease of the prawn (penaeus ortientalis kishinouye)
Authors: Lu Meiling Wu Youlu , Guoxing Zheng
Published: June 2020

The red leg disease of the prawn (Penaeus orientalis Kishinouye) is a new kind of disease which occurred and prevailed in many maricuiture farm in East China in recent years. The article reports the pathological changes in the microstructure of some tissues from the diseased prawn, which are studied by light microscopy (LM) and transmission electron micros copy(TEM). The result shows that small number of bacteria exist in the haemolymph of the diseased prawn and that the number of haemocytes decreases obviously. Lots of tree-like or radiation-like orange-red pigment granules are seen int the derma layer of the shell of the swimming feet by LM. In the ultrathin section of the swimming feet many parasitic bacteria can be observed assembling or scattering in the cytoplasm as well as in the cytoplasm of midgut, heart and gill fillarnents(Fig 4-6). Its proved that the bacteria separated from the haemlymph of the diseased prawn are the pathogen of the red leg disease known as Vibrio angullarum through the tests of the manual infection and bacteriological identifications. Its a short bacillus without germ, but with a single-ended flagella which has sheath.
Localization of lipopolysaccharides in reconstituted lipid-porin membranes
Authors: Hoenger A. , Aebi U. , Engel A.
Published: June 2020

The E.coli outer membrane is a highly organized asymmetric structure with densely interspersed transmembrane proteins, lipopolysaccharides (LPS) exclusively on the extracellular side and the phospholipids as well as lipoproteins on the periplasmic side. A major fraction of these outer membrane proteins (omp) act as channels, allowing for the passage of nutrients and metabolites up to 600 D. In their functional state these so called porins are trimers tightly associated with LPS molecules and exhibiting three channels at the extracellular side that appear to merge into one exit at the periplasmic side. LPS is a unique constituent of the bacterial outer membrane, containing a lipid A region and a strainspecific sugar moiety. It is essential for proper integration of omps in the outer membrane and is required for voltage induced channel opening of ompF trimers. From this point of view the localization of LPS molecules is of significant interest. Here we report on the direct visualization of the LPS sugar moiety protruding from the regular membrane reconstituted from ompF porin and phospholipids.
Comparative microscopic features of ant-garden fungi cultivated by leafcutter ants
Authors: Moninger Thomas O. , Cazin John , Walker Donald H.
Published: June 2020

Leafcutter ants, the most destructive herbivore in the tropical New World, have earned pest statusin the geographical area extending from the United States to Argentina. To meet nutritional needs andto provide a culture medium for their ant-fungus garden, which is utilized as food for their larvae, the ants attack a wide variety of plant species. They also can utilize various stored dry foodstuff such as cereal grains, flour, beans, and cattle meal. Where they are prevalent, the ants may occupy portions of pasture land, and old collapsed nests may produce holes dangerous for cattle. Nest collapsealso has resulted in sinking or collapse of building foundations, as well as paved roads. Estimates made 65 years ago suggested that yearly losses due to these insects was in the range of one billion dollars.
Immunogold localization of calmodulin in root caps of the maize Cultivar Merit
Authors: Nguyen P.T. , Uphoff C. , Stinemetz C.L.
Published: June 2020

Considerable evidence suggest that the calcium-binding protein calmodulin (CaM) may mediate calcium action and/or transport important in the gravity response of plants. Calmodulin is present in both shoots and roots and is capable of regulating calcium transport in plant vesicles. In roots calmodulin is concentrated in the tip, the gravisensing region of the root; and is reported to be closely associated with amyloplasts, organelles suggested to play a primary role in gravi-perception. Inhibitors of CaM such as chlorpromazine, calmidazolium, and compound 48/80 interfere with the gravitropic response of both snoots and roots. The magnitude of the inhibition corresponded well with the extent to which the drug binds to endogenous CaM. Compound 48/80 and calmidazolium block gravi-induced changes in electrical currents across root tips, a phenomenon thought to be associated with the sensing of the gravity stimulus.

In this study, we have investigated the subcellular distribution of CaM in graviresponsive and non-graviresponsive root caps of the maize cultivar Merit.
Some observations on various tissues of mouse with the environmental Scanning Electron Microscopy
Authors: Fujimaki Noboru , Kano Yumiko , Ishikawa Harunori , Ohmori Atsuo , Kawata Shintaro
Published: June 2020

The environmental scanning electron microscope (ESEM) have recently been developed to permit the observation on wet specimens under environmental gas conditions by improving the differential vacuum system. ESEM has been successfully applied to some fields of material science. However, its application to biological samples is still limited. Hence, we tried to examine the availability of ESEM for the fine structural observation on mammalian soft tissues under saturated water pressure without drying.

Adult male DDN mice were used for materials. Fresh tissues without fixation were excised under barbital anesthesia. The tubular viscera were opened and washed with saline after excision and mounted on the ice-chilled specimen holder and examined under ESEM. Internal structures of the parenchymal organs were examined on the exposed surfaces by cutting with razor blades after perfusion fixation with ½ Karnovsky fixative. Some specimens were postfixed with 1 % OsO4. After fixation, the specimens were rinsed with saline and examined under wet condition.
The effect of chemical treatment on thin sections for post-embedding labeling of carbohydrate antigenic sites
Authors: McCaul T. F. , Williams J. C.
Published: June 2020

Pretreatment of thin sections with Na-m-periodate, an efficient oxidising agent of carbohydrates, has been shown to enhance antigenic recognition by unmasking protein antigenic sites. Such a treatment was not suitable for detecting carbohydrate determinants of phase I lipopolysaccharide (LPS-I) of Coxiella burnetii since the quantity of labelling was reduced. Recent study has demonstrated that immunolabelling of an immunogenic surface protein of similar pathogenic bacterium, C. burnetii, was greatly enhanced after exposure of thin sections at 100°C to a solubilization buffer containing sodium dodecyl sulphate (SDS) and sodium deoxycholate (DOC). In the present study, we report the effect of the solubilization buffer, and proteinase K, which effectively removes peptides and native proteins, on immunolabelling of carbohydrate determinants.

C. burnetii (9MIC7), which was purifed from infected yolk sac material of hen eggs by Renografin gradient centrifugation, was fixed for 3 h in 1.5% glutaraldehyde and 0.2% picric acid in 66 mM Na-cacodylate buffer, pH 6.8; pre-embedded in 2% Difco Nobel Agar, and then rinsed once (5 min) in the same buffer. Dehydration was first carried out in 50% methanol (15 min), then in 70% (two changes, 1h each); followed by an intermediate step (66% LR White resin in 70% methanol for 30 min).
Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)
Authors: Hully J. R. , Luehrsen K. R. , Aoyagi K. , Shoemaker C. , Abramson R.
Published: June 2020

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.
Fluorescent Molecules as Direct and Indirect Labels for in situ Hybridization
Authors: Durrant Ian
Published: June 2020

In situ hybridization has traditionally been performed with radioactive probes. Over recent years effort has been spent in elucidating non-radioactive alternatives for a variety of molecular biology systems. Non-radioactive in situ hybridization is possible, but to date the systems available have, in general, delivered lower sensitivity and higher backgrounds than traditional methods. We have studied a variety of alternative labelling and detection processes; different systems may be required for different applications.

Fluorescein has been developed as a new label for all aspects of non-radioactive in situ hybridization. Separate systems have been investigated for DNA, RNA and oligonucleotide probes (see Figure 1). These probes can be applied to frozen sections, paraffin sections, whole mounts, cells and chromosomes. The standard non-radioactive detection system utilizes alkaline phosphatase as an end point to produce a coloured reaction product precipitated at the point of hybridization. This gives both good resolution and sensitivity. Other options becoming available, based on different enzymes and other formats of detection, include peroxidase and gold in conjunction with colour, chemiluminescence and direct and indirect fluorescence. These alternatives have been investigated using the fluorescein based probe labelling systems.
Differential Contrast Imaging of High Resolution TEM Micrographs of Rotary Shadowed Heterodouplex Protein Complexes
Authors: Moretz R.C. , Crute James J. , Peters K.-R.
Published: June 2020

The resolution attainable from rotary shadowed molecules is limited by sample preparation method, the distribution of the metal through the shadowing procedure and the grain structure of the metal film. Utilizing very thin metal coats (less than a few 0.1 nm measured perpendicular to the substrate) and metals that form small grain sizes improves the resolution. Differential hysteresis (DH) processing of the micrograph data offers the opportunity to utilize the full range of contrast information present in the images and to improve the structural analysis and interpretation of high resolution rotary shadowed macromolecules. A protein complex of interest that shows varying structures in the presence or absence of ATP was prepared by glycerol spray onto freshly cleaved mica. The mica was placed on the stage of a Cressington CFE-50 and dried in vacuo for 5 min. Samples were rotary shadowed with 0.2 nm W/Ta (measured on the support) at 5° incident angle, followed by stabilization with 2.5 to 3.0 nm C. Replicas were examined in a Hitachi H-7000 with a LaB6gun at 100 kV at direct magnifications of 100,000x. Micrographs were taken on Kodak type 4489 EM film at one f-stop higher than for standard silver halide printing to provide a full gray scale data file during digitization.
High-pressure freezing and freeze substitution fixation of the plant pathogenic fungus Exobasidium vaccinii
Authors: Richardson Elizabeth A. , Mims Charles W.
Published: June 2020

Members of the small fungal genus Exobasidium are all plant pathogens. Most species are noted for their ability to produce large fleshy galls on leaves and flower parts. They produce their hyphae and specialized absorbing structures known as haustoria inside these galls and their sexual reproductive structures (basidia) on gall surfaces. The objective of this study was to examine the feasibility of using high pressure freezing (HPF) followed by freeze-substitution (FS) fixation to prepare Exobasidium induced galls on Rhododendron sp. for study with transmission electron microscopy (TEM). HPF fixation followed by FS fixation is considered to be the best method for preserving large samples of plant tissue for TEM. This approach has shown promise for the examination of host-pathogen relationships in fungal diseases of plants.
Monte-Carlo Estimations for the Precision of Iterative Structure Refinement in QHREM
Authors: Möbus G.
Published: June 2020

1.

Introduction:

Quantitative high-resolution TEM (QHREM) serves for the determination of structures of crystal defects. The structure retrieval problem is usually considered to be solved when a simulated image calculated from a model structure matches with high quality to the experimental image. Often, however, surprisingly large (and continuous) shifts of individual atomic columns are possible before the simulation looses its similarity to the experiment. Therefore, we propose printing structure retrieval results as “atomic ball pictures” with the radius of the ball as a circle of confidence (replacing the chemical atomic radius otherwise used for these pictures). Statistical methods are necessary to derive the proper radius of the circles which are element and site specific : Monte-Carlo based strategies are highly suitable for this purpose. As a model system we use two bulk sections from a micrograph of a Cu/Sapphire interface (Fig. 1). For interface properties see.
Techniques for quantitative HREM analysis of structure and displacements at interfacial steps, facets and facet junctions
Authors: Dahmen U. , Paciomik S. , Michel D. , Hÿtch M.
Published: June 2020

Quantitative High Resolution Electron Microscopy (QHREM) has made considerable recent advances in the analysis of localized atomic structure, especially in flat, periodic interfaces. This is largely due to the increased speed and sophistication of computer algorithms that compare experimental and simulated images, and to strategies for refining this comparison. However, defects such as dislocations, disclinations or steps often lead to elastic distortion fields in the experimental image that are not included in the simulations of an idealized interface. In the presence of such defects, it is difficult to separate the short-range atomic structure immediately at the core of the interface from the Ionger-range displacement fields due to steps and dislocations.

In this work we describe quantitative techniques to measure atomic structure and displacement fields in these two different regimes, and to refine the structure of the core when defects lead to deviations from ideal periodic conditions. For the regime of the localized structure, we have introduced a template matching technique which measures the precise location and similarity of structural units characteristic for an interface structure. In the regime of longer-range displacement fields, the moiré technique and the closely related geometrical phase technique have been shown to be useful quantitative tools.
Quantitative structure determination of interfaces through Z-contrast imaging and electron energy loss spectroscopy
Authors: Pennycook S. J. , Nellist P. D. , Browning N. D. , Langjahr P. A. , Rühle M.
Published: June 2020

The simultaneous use of Z-contrast imaging with parallel detection EELS in the STEM provides a powerful means for determining the atomic structure of grain boundaries. The incoherent Z-contrast image of the high atomic number columns can be directly inverted to their real space arrangement, without the use of preconceived structure models. Positions and intensities may be accurately quantified through a maximum entropy analysis. Light elements that are not visible in the Z-contrast image can be studied through EELS; their coordination polyhedra determined from the spectral fine structure. It even appears feasible to contemplate 3D structure refinement through multiple scattering calculations.

The power of this approach is illustrated by the recent study of a series of SrTiC>3 bicrystals, which has provided significant insight into some of the basic issues of grain boundaries in ceramics. Figure 1 shows the structural units deduced from a set of 24°, 36° and 65° symmetric boundaries, and 24° and 45° asymmetric boundaries. It can be seen that apart from unit cells and fragments from the perfect crystal, only three units are needed to construct any arbitrary tilt boundary. For symmetric boundaries, only two units are required, each having the same Burgers, vector of a<100>. Both units are pentagons, on either the Sr or Ti sublattice, and both contain two columns of the other sublattice, imaging in positions too close for the atoms in each column to be coplanar. Each column was therefore assumed to be half full, with the pair forming a single zig-zag column. For asymmetric boundaries, crystal geometry requires two types of dislocations; the additional unit was found to have a Burgers’ vector of a<110>. Such a unit is a larger source of strain, and is especially important to the transport characteristics of cuprate superconductors. These zig-zag columns avoid the problem of like-ion repulsion; they have also been seen in TiO2 and YBa2Cu3O7-x and may be a general feature of ionic materials.
Simulated Chemical Imaging of InGaAs/InAlAs Interfaces
Authors: Mountjoy G. , Crozier P.A. , Fejes P.L. , Tsui R.K. , Kramer G.D.
Published: June 2020

For many semiconductor devices, a detailed knowledge of interface composition on an atomic scale is important for understanding the device performance. One technique for obtaining such knowledge is “chemical imaging”. This technique has previously been applied to Si/Ge and GaAs/AlAs interfaces. Recently, there has been interest in constructing quantum wells using the (In0.532Ga0.468)As/(In0.522Al0.478)As system (hereafter InGaAs/InAlAs), which is lattice-matched to InP (lattice constant of 5:869Å).

The chemical imaging technique uses high resolution electron microscopy (HREM) images taken under special conditions of microscope defocus df, and specimen thickness t. Under the correct conditions, the images contain features which are linearly dependent on composition x. For heterogeneous structures (e.g. InGaAs/InAlAs), a change in x affects <200> beams preferentially. In this case, imaging in the <100> orientation is favorable, because four <200> beams occur. It is necessary to define a function of the image intensity h(I) which is proportional to x for some values of t and df. Here we follow the approach of Thoma and Cerva and use h(I) equal to the <200> frequency component of the image intensity (I200) normalized relative to I200 for x=0 and x=1. The resolution of h(I) is limited to one <200> period, or 1/2 unit cell (2.93Å).
Use of low-temperature scanning electron microscopy to observe icicles, ice fabric, rime and frost
Authors: Wergin William P. , Rango Albert , Erbe Eric F.
Published: June 2020

Previous studies showed that low temperature scanning electron microscopy (SEM) can be used to record images of precipitated snow crystals, which collectively form structures that are commonly known as snowflakes. Information about the structure of snow crystals can be used to improve models that estimate the water equivalent of the winter snowpack. These models, which are based on satellite microwave data, have practical use in approximating the quantity of water that will be available for crop irrigation and hydroelectric power. Our previous success of using low temperature SEM to image snow crystals has encouraged us to utilize this technique for other types of frozen aqueous specimens.
Facet Topography and Dislocation Structure as a Possible Source for Electrical Heterogeneity in [001] TILT Bicrystals of YBa2Cu3O7-δ
Authors: Tsu I-Fei , Kaiser D.L. , Babcock S.E.
Published: June 2020

A current theme in the study of the critical current density behavior of YBa2Cu3O7-δ (YBCO) grain boundaries is that their electromagnetic properties are heterogeneous on various length scales ranging from 10s of microns to ˜ 1 Å. Recently, combined electromagnetic and TEM studies on four flux-grown bicrystals have demonstrated a direct correlation between the length scale of the boundaries’ saw-tooth facet configurations and the apparent length scale of the electrical heterogeneity. In that work, enhanced critical current densities are observed at applied fields where the facet period is commensurate with the spacing of the Abrikosov flux vortices which must be pinned if higher critical current density values are recorded. To understand the microstructural origin of the flux pinning, the grain boundary topography and grain boundary dislocation (GBD) network structure of [001] tilt YBCO bicrystals were studied by TEM and HRTEM.
Practical Correction of Three Fold Astigmatism in the Philips CM TEM
Authors: Bleeker Arno J. , Overwijk Mark H.F. , Otten Max T.
Published: June 2020

With the improvement of the optical properties of the modern TEM objective lenses the point resolution is pushed beyond 0.2 nm. The objective lens of the CM300 UltraTwin combines a Cs of 0. 65 mm with a Cc of 1.4 mm. At 300 kV this results in a point resolution of 0.17 nm. Together with a high-brightness field-emission gun with an energy spread of 0.8 eV the information limit is pushed down to 0.1 nm. The rotationally symmetric part of the phase contrast transfer function (pctf), whose first zero at Scherzer focus determines the point resolution, is mainly determined by the Cs and defocus. Apart from the rotationally symmetric part there is also the non-rotationally symmetric part of the pctf. Here the main contributors are not only two-fold astigmatism and beam tilt but also three-fold astigmatism. The two-fold astigmatism together with the beam tilt can be corrected in a straight-forward way using the coma-free alignment and the objective stigmator. However, this only works well when the coefficient of three-fold astigmatism is negligible compared to the other aberration coefficients. Unfortunately this is not generally the case with the modern high-resolution objective lenses. Measurements done at a CM300 SuperTwin FEG showed a three fold-astigmatism of 1100 nm which is consistent with measurements done by others. A three-fold astigmatism of 1000 nm already sinificantly influences the image at a spatial frequency corresponding to 0.2 nm which is even above the point resolution of the objective lens. In principle it is possible to correct for the three-fold astigmatism a posteriori when through-focus series are taken or when off-axis holography is employed. This is, however not possible for single images. The only possibility is then to correct for the three-fold astigmatism in the microscope by the addition of a hexapole corrector near the objective lens.
Gapless Single-Pole Magnetic Lens for Low-Voltage SEM
Authors: Tsai F.C. , Crewe A.V.
Published: June 2020

An unconventional single pole lens design has attracted attention because it promises a highly confined strong field distribution which in turns results in smaller third aberrations than that of the conventional lenses. However in this type of lens heat dissipation becomes crucial. In the low electron energy range (less than 5 KV), however, we can keep all the benefits from the single pole piece without being concerned about the cooling requirement.

The key to the design is to avoid the unnecessary strong fields in any region away from the axis, which can absorb a lot of applied magnetomotive force (MMF) if this off-axis region happens to be in the air. This implies that a reasonable design is a gapless geometry. In our design (Fig. 1), a hemisphere tip sits on the top of a central pole. At the rim between the boundary of the central tapered pole and the tip is a 45° semi-angle funnel-shaped exterior pole. These three parts physically contact each other and form a gapless geometry. The central and exterior poles also contact each other at the bottom of the lens to form the high permeability magnetic circuit loop. The cross section of each part of the lens has been chosen in such a way that the region close to the tip will saturate first.
Procedures for x-ray microanalysis of layered structures: accuracy and limits
Authors: Pouchou J. L. , Thiot J. F.
Published: June 2020

The procedures presently available for X-ray microanalysis (XRMA) of layered structures derive more or less directly from the semi-empirical ϕ(ρz) models proposed in the 80's. In the last years, the spreading of advanced computer programs such as Strata made more people aware than these methods were effective, and could be used in many cases as a complement or a substitute to near-surface analysis methods. New approaches for ϕ(ρz) reconstruction, based on the angular and energetic distributions of the electrons, are emerging (e.g. IntriX model). They should enable to describe more accurately structures with strong atomic number variations. Monte-Carlo simulations (M-C) are useful to assess ϕ(ρz) models, but for daily work their effectiveness seems presently restricted to the elaboration of calibration charts to be used for repetitive situations.