bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and Distribution of Fiber Types in Monkey Extraocular Muscle
Authors: Pachter Bruce R.
Published: June 2020

There appears to be a growing debate in the extraocular muscle (EOM) literature as to the number of component fiber populations in monkey and human EOM. Both Durston and Ringel et al., in an attempt to simplify the classification scheme, described three fiber types in these muscles; the orbital-global layers were considered by them to be insignificant in determining fiber classifications. In light of the present study, it now appear that both authors oversimplified and misclassified their fiber populations. In the present investigation, monkey superior rectus EOM was embedded whole in Epon 812 and serially sectioned at 10-15 microns from origin to insertion by a method previously described. Such thick sections can be viewed by phase contrast microscopy and sections of interest can be reembedded and resectioned for EM examination. Initial fiber categories were based on cell diameter, mitochondrial size and distribution, density of sarcoplasmic stain, and the number of neuromuscular junction sites per fiber.
High Resolution X-Ray Contact Microscopy of Hydrated Muscle Filaments
Authors: Panessa B.J. , Dewey M.M. , Brink P. , Gaylinn B. , McCorkle R.A. , Coleman G. , Warren J.B.
Published: June 2020

Soft x-ray contact microscopy is a relatively new method for imaging biological materials at resolutions of 5-30 nm. By using a wet cell, and flash x-ray source to reduce the exposure time, we have been able to obtain high contrast images of hydrated biological specimens at better than 30 nm resolution. This paper describes the technique and results of using this type of “wet” microscopy for the examination of hydrated invertebrate isolated myosin filament, and paramyosin paracrystal,preparations.

Paramyosin paracrystals and myosin filaments were isolated from telson muscle of the horseshoe crab, Limulus polyphemus L. The muscle was homogenized and the cellular debris and filaments segregated by differential centrifugation. Isolated filaments were resuspended in buffer (Tris, pH 7.4-7.5), and dialyzed overnight against buffer containing EDTA at 4°C. Tissue taken from the white adductor muscle of the clam, Mercenaria mercenaria, was similarly homogenized and isolated by standard methods.
Scanning Electron Microscopy of the Polycystic Ovary in Constant Light Rats
Authors: Singh K. B. , Khosho F. K.
Published: June 2020

Exposure of female rats to continuous light eventually induces persistent estrus (PE), a syndrome which in many respects resembles the human polycystic ovary syndrome. Light microscopic studies on the polycystic ovaries of constant light PE rats have been previously reported. In the present study, we have utilized scanning electron microscopy (SEM) to provide information regarding three-dimensional changes occurring on the surface of the polycystic ovary.

Following exposure to continuous light for 50 days, PE was induced in a group of Sprague-Dawley female rats according to the procedure described elsewhere (2). Polycystic ovaries from constant light PE rats and ovaries from controls were fixed in 2.5% glutaraldehyde in 0.1M Na cacodylate buffer, pH 7.3 at room temperature for 24 hours. They were then cut into two equal halves along their long axis and fixed for an additional 3-4 hours. The specimens were processed for SEM studies by a modified OTOTO technique, dehydrated and critical point dried with CO2.
Hepatic Lipid Accumulation and Intracellular Cholesterol Crystal Formation in Hypercholesterolemic Diabetic Rats
Authors: Handley Dean A. , Chien Shu , Arbeeny Cynthia M.
Published: June 2020

Hypercholesterolemia, induced by feeding rats a diet of cholesterol and cholic acid, can be markedly accentuated by diabetes, resulting in serum cholesterol levels greater than 1000 mg/dl and extensive accumulate of cholesterol in the liver. To examine the cellular events of hepatic cholesterol accumulation in cholesterol-fed diabetic rats, we used a fixative containing digitonin and water-soluble embedment to preserve native lipid structures.

Sprague Dawley male rats were made diabetic by intravenous injection of streptozotocin (45 mg/kg body weight) and fed a powdered diet containing 2% cholesterol, 10% lard and 1% cholic acid (CLC) for 3 wk. Non-diabetic rats were also fed CLC diet. Cholesterol, glucose and insulin levels were monitored for both groups. Livers were fixed by perfusion with 3% glutaraldehyde (30°C) and 0.2% digitonin in cacodylate buffer (1 hr), then by 1% OsO4 in buffer for 2 hr. Staining was in 2% uranyl acetate and embedding in water soluble urea glutaraldehyde. Thin sections were stained with lead citrate and viewed in a Zeiss EM-9S.
An SEM Study of the Filtration System and Mucus Secretion of the Gills of the Oyster Crassostrea Virginica
Authors: Ruby J.R. , Kilgen M.B.
Published: June 2020

The gills of the oyster play a major role in respiration but also serve as a filter feeding device. Although an excellent description of the gills at the light microscope level has been published, there have been very few ultrastructural studies.

The basic morphology of the gills is difficult to ascertain due to the large number of cilia projecting from them. A more complete understanding of these structures can be obtained by using the ethanol cryofracture technique.

Figure 1 depicts a portion of a demibranch which has been cryofractured in transverse, section. It shows the longitudinal folds of the demibranch and the ordinary filaments which form the basic structure of the gills. Also seen in this micrograph is a view from within the water tube which clearly shows the ladder-like arrangement of the chitinous framework which supports the entire gill structure. Many of the cells lining this framework possess cilia (Fig. 2), an observation which has not previously been emphasized.
The Observation of Frozen Hydrated Sections by CTEM and STEM
Authors: Chang J. J. , Walter C. A. , Dubochet J.
Published: June 2020

With the ultimate aim of studying biological structures close to their native state, we are attempting to prepare and examine thin frozen hydrated sections using catalase crystals and skeletal muscle as test objects. There have been a few observations under these conditions.

small droplet of catalase crystals stained in 2% aqueous uranyl acetate and re-suspended in water was placed on a copper specimen holder and plunged into liquid nitrogen. Small pieces of rat femoral muscle were fixed in 2% gluter- aldehyde in 0.05 M cacodylate buffer, and frozen as above after washing in buffer with or without the addition of 1 M sucrose.

Frozen sections for examination in the CTEM were cut with glass knives in a Sorvall MT2B microtome with the LTC2 cryo-attachment operating at ca. -120°C. For examination in the STEM, frozen sections were cut in a Reichert Ultracut with FC4 cryo-attachment operating with the knife and the specimen at -150°C and the chamber at -170°C. Sections that appeared transparent were manipulated onto carbon coated grids with a pre-cooled eyelash probe and then sandwiched in place with a second carbon coated grid.
Uranyl Acetate Preservation of Nucleoplasm in the Sporocytes of Psilotum
Authors: Lee Kit W.
Published: June 2020

As a stain, uranyl acetate (UA) is used on thin sections to enhance the contrast of cellular constituents. It has been shown that the use of uranyl acetate in the fixation process can increase the contrast and preservation of various membrane systems by reducing the extraction of phospholipids during dehydration. Because of its high affinity for nucleic acids, uranyl acetate has also been used as a post fixative to preserve the nuclear region of prokaryotes. This paper compares the nuclei of the eukaryote Psilotum, particularly during meiosis, with and without post fixation with uranyl acetate.

Sporangia at different developmental stages were fixed in cold 3% glutaral-dehyde (GA) in 0.1 M phosphate buffer (pH 7.2) for 4 hours or overnight, and post fixed at room temperature with 2% osmium tetroxide (OsO4) for 2 hours. After washing in distilled water, samples were either (a) dehydrated in a graded series of ethanols and propylene oxide, or (b) placed in 1% aqueous uranyl acetate for 1 hour, followed by 30 minutes washing in distilled water before dehydration.
Visualization of the Adenovirus DNA Terminal Protein, Its Gene, and Messenger RNA
Authors: Broker Thomas R. , Chow Louise T.
Published: June 2020

Studies using biochemical and electron microscopical RNA:DNA heteroduplex methods have shown that the transcription patterns of the human adenoviruses are extremely complicated. Almost all early and late mRNAs are spliced. Each transcriptional unit produces a family of RNAs that share common 5' and 3' ends but have alternative splicing. Most of the genome is expressed as relatively abundant mRNAs, except for the region between coordinates 16 and 27. This segment encodes the RNA leader components for the late rightward-transcribed (r-strand) RNAs. However, DNA-negative temperature-sensitive mutations have been mapped to this interval. Recently, experiments by several laboratories using different techniques have elucidated how these mutations might affect DNA replication.

A protein of 55,000 daltons (55KD) M is covalently joined to each 5' terminus of adenovirus DNA isolated from virions. The ends of replicating DNA in infected cells are also bound to proteins. The linkage is via a phosphodiester bond between serine and the terminal deoxy-cytidine.
Periodate Treatment and “All-or-None” Dehydrogenase Staining Phenomena
Authors: Rosa C. G.
Published: June 2020

It has been shown that when suspensions of fresh rodent sperm are incubated in the presence of tetranitro-blue tetrazolium (TNBT) and stained for succinic dehydrogenase (SDH),this enzyme is localized in the mitochondria of the middle-piece. Rat and mouse spermatozoa treated in this manner and postfixed (suspension) in 4°C Millonig's osmium tetroxide for brief periods demonstrate SDH reactivity in mitochondrial profiles that indicate no SDH, almost complete activity, or most commonly, intermediate staining reactions in the internal membranes (Figure 1). In the last instance, the cristal membranes display a heterogeneous formazan (reduced TNBT) staining pattern not in agreement with the Seligman “all-or-none” hypothesis concerning mitochondrial oxidative enzymatic reactivity.

In order to test this hypothesis and circumvent some of the attendant difficulties in these procedures (penetration of reaction mixture components, fixatives), all cytochemical reactions and postfixation were performed on suspensions of sperm from the cauda epididymides of mature rats. An attempt was made to divorce electron densities due to osmium postfixation by employing 1% aqueous sodium periodate (pH 3-4) as a mitochondrial membrane stabilizer through the embedding procedures. Previous use of this treatment allowed preservation of the membranes of these organelles in positive contrast as evidenced by uranyl acetate staining of sectioned material (Figures 2 & 3).
Electron Staining Characteristics of the Inclusion Bodies of Type II Alveolar Epithelial Cells
Authors: Kikkawa Yutaka , Hahn Ho-Soon
Published: June 2020

The inclusion bodies of Type II epithelial cells of the mammalian lung are oval and limited by a unit membrane. They contain highly osmiophilic material. With the standard method of fixation this material is irregularly separated by a number of electron-lucent spaces (Figure 1). Because of this appearance, the inclusion bodies are often referred to as “lamellar inclusions”. Measurable periodic lamellae, however, have never been observed in the inclusions which are located intracellularly.

During the course of the studies to localize acid mucopolysaccharides in the distal air way of the rabbit and rat, it is found that the alveolar surface of the cell membranes of both Type I and II cells and the inclusion bodies within Type II cells satin heavily with colloidal iron at pH 2.0 following the osmication of the tissue with phosphate-buffered solution at pH 7.4 (Figure 2). In addition, the inclusion bodies for the first time show regular periodic lamellae. Each line is granular and measures about 60 Å in width (Figure 3).
Extracellular Connective Tissue Fibrils: A Single Line of Descent
Authors: Low F. N.
Published: June 2020

The fine structure of developing extracellular connective tissue fibrils, is demonstrable in normal chick embryos. Before incubation begins primary fibrils are present in the intermittent boundary membrane (basement membrane, basal lamina) of the basal surface of the epiblast. These give rise to microfibrils which first become free in the tissue space at about 24 hours incubation (Fig. 1). The first noticeable locus of microfibrillar concentration appears between the head process (early notochord) and the dorsal ectoderm. By 40 to 45 hours a heavy tangle of microfibrils occupies the acellular area surrounding the notochord and remains conspicuous during the third day (Fig. 2). At 60 to 72 hours microfibrils begin to appear in close association with free mesenchymal cells (Fig. 3). Periodicity slowly develops in the larger microfibrils and the resultant unit collagen fibrils will aggregate to form the reticular and collagen fibers of light microscopy. During the second and third weeks of incubation microfibrils and unit collagen fibrils contribute to the formation of elastic fibers.
Effects of EDTA, Hyaluronidase and Collagenase on Epiphyseal Cartilage Matrix
Authors: Anderson H. Clarke , Coulter Priscilla R.
Published: June 2020

Epiphyseal cartilage matrix contains fibrils and particles of at least 5 different types: 1. Banded collagen fibrils, present throughout the matrix, but not seen in the lacunae. 2. Non-periodic fine fibrils <100Å in diameter (Fig. 1), which are most notable in the lacunae, and may represent immature collagen. 3. Electron dense matrix granules (Fig. 1) which are often attached to fine fibrils and collagen fibrils, and probably contain protein-polysaccharide although the possibility of a mineral content has not been excluded. 4. Matrix vesicles (Fig. 2) which show a selective distribution throughout the epiphysis, and may play a role in calcification. 5. Needle-like apatite crystals (Fig. 2).

Blocks of formalin-fixed epiphysis from weanling mice were digested with the following agents in 0.1M phosphate buffer: a) 5% ethylenediaminetetraacetate (EDTA) at pH 8.3, b) 0.015% bovine testicular hyaluronidase (Sigma, type IV, 750 units/mg) at pH 5.5, and c) 0.1% collagenase (Worthington, chromatograhically pure, 200 units/mg) at pH 7.4. All digestions were carried out at 37°C overnight. Following digestion tissues were examined by light and electron microscopy to determine changes in the various fibrils and particles of the matrix.
Intercellular Substances of the Uterine Cervix: Electron Microscopic Histochemical Studies of Hormone-Dependent Changes in the Mouse
Authors: Leppi T. J. , Kinnison P. A. , Gaffney S. P.
Published: June 2020

Earlier study of the connective tissue ground substance in uterine cervical walls of ovariectomized mice established the presence of a histochemically stainable extracellular material which varied in amount depending on hormonal conditions. The administration of estrogen singly or in combination with relaxin resulted in the appearance within the stroma of colloidal iron-positive material visible as Prussian blue. No iron staining was present in cervices from mice treated with relaxin alone or the hormone vehicles. The stainable material was tentatively identified as acid muco-polysaccharide(s) on the basis of its lability toward testicular hyaluroni-dase. The present ultrastructural histo- and cytochemical study of uterine cervices from mice in similar hormonal states was undertaken to: 1) identify clearly the connective tissue ground substance in this site; 2) establish a correlation with the previous light microscopic results; and 3) characterize further the histochemical staining properties of the intercellular material.
Mesosome-like Structures in Cryptococcus neoformans Studied by Chemical Fixation and by the Freeze-etching Technique
Authors: Edwards M. R. , Holt S. C.
Published: June 2020

The general features of Cryptococcus neoformans, a pathogenic yeast, have been studied with the electron microscope. In the course of such a study it was noted that the plasma membrane of C. neoformans, occasionally invaginated into the cytoplasm and formed membranous organelles which resembled bacterial mesosomes. The present investigation was undertaken in order to examine such structures in detail and to compare the results from chemical fixation with those of freeze-etching.

Cells were grown in Sabouraud's agar at 25-27° C for 24-48 hr and fixed with 4% glutaraldehyde in 0.15 M phosphate (Sbrensen's) buffer, at room temperature, for 2 hr; after being thoroughly washed in the buffer and post-fixed in osmium tetroxide, in the same buffer, they were dehydrated in ethyl alcohol and embedded in Epon. Thin sections were cut in a LKB microtome, double stained with uranyl and lead salts and examined in the Siemens Elmiskop IA.
Mitochondrial Changes in Choline-Deficient Rat Myocardium
Authors: Zaki F. G.
Published: June 2020

Choline-deficiency was induced in Holtzman young rats of both sexes by feeding them a high fat - low protein diet.

Preliminary studies of the ultrastructural changes in the myocardium of these animals have been recently reported from this laboratory. Myocardial lesions first appeared in the form of intraventricular mural thrombi, loss of cross striation of muscle fibers and focal necrosis of muscle cells associated with interstitial myocarditis. Prolonged choline-deficiency induced cardiomegaly associated with pericardial edema.

During the early phase of this nutritional disorder, heart mitochondria - despite of not showing any swelling similar to that usually encountered in liver mitochondria of the same animal - ware the most ubiquitous site of marked structural abnormalities. Early changes in mitochondria appeared as vacuolation, disorganization, disruption and loss of cristae. Degenerating mitochondria were often seen quite enlarged and their matrix was replaced by whorls of myelin figures resembling lysosomal structures especially where muscle fibers were undergoing necrosis. In some areas, mitochondria appeared to be unusually clumped together where some contained membranelined vacuoles and others enclosed dense bodies and granular inclusions.
Replicas of Freeze-Cleaved Tubular Myelin Figures
Authors: Weinstein Ronald S. , Ivanetich Leonida , Nash Gerald
Published: June 2020

Freeze-cleave technics have now been used to examine the surface topography of a variety of biological membranes. With these technics, membranes appear as thin sheets and their outer surfaces are frequently associated with a distinctive particulate component (Figure 1). Areas not covered with such particles are flat and substructure can usually be detected in these flat regions with high quality replicas. Speculation on the functional significance of the membrane-associated particles has centered on the possibility that they may represent multi-enzyme complexes and specialized sites possibly associated with transport functions. The suggestion that some particles may represent a particulate contaminant adsorbed onto the cold fracture face has also been considered. To further explore the contamination theory, myelin figures were examined for the presence of a small particulate component similar to that normally seen on many biologically active membranes.
Mitochondrial Inclusions
Authors: Hruban Z.
Published: June 2020

Two types of inclusions in the matrix of mitochondria were observed in our comparative studies on the fine structure of hepatocytes and renal proximal tubular cells of apparently healthy animals. Mitochondrial inclusions occuring in various cell types in healthy and diseased tissues have been recently reviewed by Haust ( Exp. Molec. Pathol. 8: 123, 1968).

Multiple small inclusions were seen in the proximal tubular cells of deer mice, Peromyscus boylei (Fig. 1) and P. californicus. These inclusions had an electron lucid center and a narrow dense periphery. Large electron dense inclusions with one or more lucid areas were seen in proximal tubular cells of the slow loris, Nicticebus coucang (Fig. 2). The dense portion of the inclusion was composed of dense laminae about 65 Å thick (Fig. 3). These Inclusions, seen in the deer mice and in the slow loris, are not limited by a membrane and do not show a regular spatial relationship to mitochondrial cristae. The term “lipoid inclusions” is proposed for these structures. Osmiophil intramitochondrial granules are usually absent in mitochondria containing lipoid inclusions. Transitions are found between osmiophil granules and lipoid inclusions.
Evidence for Golgi Origin of Melanosomes
Authors: Maul Gerd G.
Published: June 2020

Electron microscopy has provided evidence that the melanosome evolves as a membrane bound structure with a highly complex internal architecture. The premelanosomes are found in close proximity to the golgi apparatus. Therefore, it was generally agreed that the melanosomes originate from the golgi apparatus.

Vesicles have been described to pinch off the cysternae of the golgi apparatus. The vesicles would then grow and acquire a dense material. This material is aggregating to form the characteristic helical strands onto which melanin is deposited. Cloned human malignant melanoma lines were used to reinvestigate the problem of melanosome formation. The reconstruction of serial sections revealed the arrangement of premelanosomes and melanosomes in relation to the golgi complex. This study demonstrated that premelanosomes and melanosomes are continuous with the golgi complex by a smooth-surfaced tubular endoplasmic reticulum (SER) (Fig. la-d). The continuity of membranes of the SER and the premelanosome is depicted in Fig. 2. In this early premelanosome the protein strands have not yet coiled up into a helix. Rough-surfaced endoplasmic reticulum (RER) was also observed to be continuous with the golgi apparatus and melanosomes. After melanogenesis has started (Fig. 3) small vesicles appear inside the premelanosomes.
Scanning Electron Microscopy and Electron Microprobe Analysis of Malignant Cell Cultures
Authors: Johnsson-Hegyeli R. I. , Hegyeli A. F. , Landstrom D. K. , Lane W. C.
Published: June 2020

Last year we reported on the use of reflected light interference microscopy (RLIM) for the direct color photography of the surfaces of living normal and malignant cell cultures without the use of replicas, fixatives, or stains. The surface topography of living cells was found to follow underlying cellular structures such as nuceloli, nuclear membranes, and cytoplasmic organelles, making possible the study of their three-dimensional relationships in time. The technique makes possible the direct examination of cells grown on opaque as well as transparent surfaces. The successful in situ electron microprobe analysis of the elemental composition and distribution within single tissue culture cells was also reported.

This paper deals with the parallel and combined use of scanning electron microscopy (SEM) and the two previous techniques in a study of living and fixed cancer cells. All three studies can be carried out consecutively on the same experimental specimens without disturbing the cells or their structural relationships to each other and the surface on which they are grown. KB carcinoma cells were grown on glass coverslips in closed Leighto tubes as previously described. The cultures were photographed alive by means of RLIM, then fixed with a fixative modified from Sabatini, et al (1963).
Ultrastructure of Cardiac Myocytes from The Inner and Outer Zones of Fibrillating Bovine Hearts
Authors: Ghidoni J. J. , Thomas H.
Published: June 2020

Ventricular fibrillation is a clinical event which probably accounts for untold numbers of sudden deaths. The very nature of this arrhythmia, which prevents effective pumping of blood, has precluded the morphologic study of hearts that have fibrillated for appreciable periods. Current cardiac assist devices now provide the means of studying prolonged ventricular fibrillation. The circulation of 150 to 200-pound calves was maintained with left ventricular bypass pumps for as long as 40 hours of continuous ventricular fibrillation. Seven calves were studied histologically; one preparation that fibrillated for 24 hours was suitably preserved for ultrastructural study. Tissue was fixed in 5 % cacodylate buffered glutaraldehyde, rinsed in buffer for 48 hours, post-fixed in OsO4, and embedded in a mixture of Maraglas, D.E.R. 732, and DDSA.
Oncogenesis and the Lucké Virus
Authors: Vatter A. E. , Zambernard J.
Published: June 2020

Oncogenic viruses, like viruses in general, can be divided into two classes, those that contain deoxyribonucleic acid (DNA) and those that contain ribonucleic acid (RNA). The RNA viruses have been recovered readily from the tumors which they cause whereas, the DNA-virus induced tumors have not yielded the virus. Since DNA viruses cannot be recovered, the bulk of present day investigations have been concerned with RNA viruses.

The Lucké renal adenocarcinoma is a spontaneous tumor which occurs in northern leopard frogs (Rana pipiens) and has received increased attention in recent years because of its probable viral etiology. This hypothesis was first advanced by Lucké after he observed intranuclear inclusions in some of the tumor cells. Tumors with inclusions were examined at the fine structural level by Fawcett who showed that they contained immature and mature virus˗like particles.

The use of this system in the study of oncogenic tumors offers several unique features, the virus has been shown to contain DNA and it can be recovered from the tumor, also, it is temperature sensitive. This latter feature is of importance because the virus can be transformed from a latent to a vegetative state by lowering or elevating the environmental temperature.
Changes in Hepatocytes Following Decapitation of Chick Embryos
Authors: Dossel William E.
Published: June 2020

Deposition of glycogen in embryonic chick livers begins during the 6th day of incubation, and continues until day 10 when the stored glycogen begins to disappear; depletion of reserves is most evident on day 12. On day 14, glycogen reappears and increases up to hatching. Hypophysectomy, by means of decapitation of the embryo at 33 hours, eliminates the period of depletion and prevents the onset of function in the embryonic endocrine organs as well. Glycogen deposition in hepatocytes of mammalian embryos occurs throughout intrauterine development, but can be interrupted by decapitation.

This EM-based investigation provides morphological evidence supporting conclusions reached in earlier studies wherein glycogen content of livers from normal and decapitated 12 day-old embryos was assayed chemically (2). In livers of normal embryos, glycogen fields (i.e. accumulations of alpha and beta glycogen particles, related to agranular reticulum), appeared infrequently in hepatocytes throughout the liver (fig. 2) while in those of decapitated embryos, glycogen fields were abundant and characteristically composed of heavy accumulations of glycogen (fig. 3).
An Embedding Mixture for Electron Microscopic Technique Composed Solely of Maraglas, Araldite and Catalyst
Authors: McClendon Dale E. , Soloff Bernard L.
Published: June 2020

We have found the following embedding mixture to be useful: Maraglas 655 - 70 ml., Araldite 502 - 30 ml., benzyldimethylamine - 3 ml. The tissue blocks are processed routinely through a 1:3 mixture of propylene oxide: complete embedding mixture. After overnight soaking in this mixture, the tissue blocks are allowed to sink for two hours in capsules loaded with fresh embedding mixture. The capsules are then processed four hours in a 37 - 40° C. oven, followed by three days in a 65° C. oven. This treatment yields a hard block that may be sectioned immediately upon cooling. The thin sections obtained will accept readily any of the staining solutions now available.

Elimination of the anhydride hardener used normally in formulations of this sort does not affect the stability of thin sections under a 50 or 75 KV beam (figures 1 and 3), and sections supported on naked grids will withstand a 100 KV beam (figure 2) if carefully irradiated at low intensity before exposure to cross-over conditions.
Dislocations in Crystals
Authors: Geiss Roy H.
Published: June 2020

Transmission electron microscopy is being utilized today in the study of many important problems in materials science and metallurgy. Such areas of research include annealing and heat treating, strengthening, phase transformations and crystal growth. In many of these studies it is important to assemble quantitative data on the dislocation structures existing in the material. These data might include the determination of Burgers vectors ḇ, glide planes and densities of the various dislocations. The purpose of this paper is to review briefly a few of the fundamentals used in obtaining such data.

The contrast from a dislocation arises from the extra phase factor α=2πg · Ṟ found in the dynamical equations of electron diffraction, where g is the diffraction vector and Ṟ a vector describing the lattice distortion. For a screw dislocation Ṟ is proportional to ḇ and similarly near the glide plane of an edge dislocation.
Stacking Faults in Crystals
Authors: Gallagher P. C. J.
Published: June 2020

Stacking faults are an important substructural feature of many materials, and have been widely studied in layer structures (e.g. talc) and in crystals with hexagonal and face centered cubic structure. Particular emphasis has been placed on the study of faulted defects in f.c.c. alloys, since the width of the band of fault between dissociated partial dislocations has a major influence on mechanical properties.

Under conditions of elastic equilibrium the degree of dissociation reflects the balance of the repulsive force between the partials bounding the fault, and the attractive force associated with the need to minimize the energy arising from the misfits in stacking sequence. Examples of two of the faulted defects which can be used to determine this stacking fault energy, Υ, are shown in Fig. 1. Intrinsically faulted extended nodes (as at A) have been widely used to determine Υ, and examples will be shown in several Cu and Ag base alloys of differing stacking fault energy. The defect at B contains both extrinsic and intrinsic faulting, and readily enables determination of both extrinsic and intrinsic fault energies.
Microstructure of Quasi-Graphitic Carbons
Authors: Ban L. L. , Hess W. M.
Published: June 2020

There are many types of carbon which have the same hexagonal atomic configuration as the layer planes of graphite. These include colloidal carbon blacks and the various cokes, chars and pyrolized fibers that are used commercially. The evaporated carbon films that are utilized in electron microscopy also fit into the same category. Where all these carbons differ from graphite is in their rotational disorder about the c-axis, larger c-spacing (e.g. about 3.5-3.8 Å as compared to 3.35 Å for graphite) and smaller crystallite size.

The recent work of Heidenreich, Hess and Ban has demonstrated phase contrast imaging of the graphite lattice in heat treated carbon blacks using axial illumination. The c-spacing of 3.4 Å and numerous other aspects of microstructure were resolved in an ISAF carbon black heated at 2600-3000°C. This type of carbon has been proposed as a high resolution test specimen and as a means of calibrating magnification at high electron optical magnifications.
The Physics of Track Registration in Solids
Authors: Goland A. N.
Published: June 2020

During the last ten years many observations of charged particle tracks in solids have been reported, and the utilization of this phenomenon in several fields of physics has grown apace. For example, Fleischer, Price, Walker, and their many collaborators have demonstrated the usefulness of track formation as a tool in nuclear physics, radiation dosimetry, geochronology and cosmic-ray physics. All the more reason, then, that one should endeavour to understand the basic mechanisms of track production in different kinds of solids. To do this, one must investigate the relationship between stopping power and track formation, the nature of the damaged region comprising a track, and the annealing behavior of the tracks. The electron microscope can furnish much of the necessary information provided that one is able to interpret the diffraction-contrast image of the tracks.

Linear damage regions have been found both in thin films and in bulk samples. While tracks appear in metallic and nonmetallic thin films they have only been found in nonmetallic bulk solids. The latter include a wide variety of plastics, glasses and minerals.
Radiation Damage in Vanadium
Authors: Rau Robert C. , Ladd Robert L. , Moteff John
Published: June 2020

Transmission electron microscopy has been used to study the microstructure of vanadium irradiated at reactor ambient temperature (∼ 70°C) to a fast (E > 1 MeV) neutron fluence of 5 x 1019 n/cm2. Observations were made of the as-irradiated material, and after one-hour vacuum annealing at various temperatures ranging from 330°C to 1175°C.

In the as-irradiated condition, shown in Fig. 1, a very high density of small defect clusters was present. These clusters appeared as black dots averaging approximately 25-50 Å in diameter, and were estimated to be present in quantities of 1016 to 1017 per cm3. Post-irradiation annealing caused the clusters to increase in average size and decrease in number, as shown in Figs. 2 and 3, until after the highest temperature anneal, 1175°C, the cluster density was legs than 1014 per cm3 and the average cluster size was approximately 500Å. After annealing at temperatures of 510°C or above, many of the clusters were seen to be resolvable as dislocation loops. Tilting experiments indicated that these loops were probably interstitial in nature.
Optical Image-Reconstruction From Fraunhofer Electron-Hologram
Authors: Tonomura A. , Fukuhara A. , Watanabe H. , Komoda T.
Published: June 2020

Since the principle of holography was discussed by Gabor in the intention of improving the quality of electron-microscopic images, several authors have tried electron beam holography of in-line Fresnel type. However, the reconstructed images are seriously disturbed by a virtual image, i.e. accompanied with Fresnel fringes. Meanwhile, in the field of light optics various types of the holography technique and their applications have been proposed and developed under the decisive influence of growing laser technology.

This paper deals with electron-beam version of Thompson's in-line Fraunhofer holography, which was already mentioned in Gabor's work but has not yet been tried in electron-optics. The principle of the method is as follows. A beam of quasimonochromatic electrons is collimated by two lenses and illuminates a small object on a plane S as shown in Fig. 1(a). Diffraction fringes produced on a plane A in a far-field region from S are magnified and recorded on a photographic film at H. This film, which is just a Fraunhofer hologram, is inserted in a collimated light beam from a laser as in Fig. 1(b) and an image of the object is reconstructed onto a plane B.
Applications of Scanning Microscopy in Biomedical Research
Authors: Hayes T. L.
Published: June 2020

Biomedical applications of the scanning electron microscope (SEM) have increased in number quite rapidly over the last several years. Studies have been made of cells, whole mount tissue, sectioned tissue, particles, human chromosomes, microorganisms, dental enamel and skeletal material. Many of the advantages of using this instrument for such investigations come from its ability to produce images that are high in information content. Information about the chemical make-up of the specimen, its electrical properties and its three dimensional architecture all may be represented in such images. Since the biological system is distinctive in its chemistry and often spatially scaled to the resolving power of the SEM, these images are particularly useful in biomedical research.

In any form of microscopy there are two parameters that together determine the usefulness of the image. One parameter is the size of the volume being studied or resolving power of the instrument and the other is the amount of information about this volume that is displayed in the image. Both parameters are important in describing the performance of a microscope. The light microscope image, for example, is rich in information content (chemical, spatial, living specimen, etc.) but is very limited in resolving power.
A Combined Scanning Electron Microscope/Electron Microprobe Analyzer
Authors: Macres V.G. , Preston O. , Yew N.C. , Buchanan R.
Published: June 2020

The instrument described here is the Materials Analysis Company Model 400S combined scanning electron microscope/electron micro-probe analyzer. It was designed specifically to incorporate the most advanced features of a high performance electron microprobe analyzer with those of a medium resolution (1000A°) scanning electron microscope. The high effective x-ray take-off angle of the instrument (38.5°) offers low x-ray absorption, and thus allows the analysis of fairly rough specimens. The large depth of focus of the scanned electron images further enhances the capability of examining rough specimens.

The electron-optical column comprises a triode electron gun, double condenser lens and objective lens. The electron gun uses a conventional hairpin filament, autobiased Wehnelt cylinder and anode. An externally controlled filament/Wehnelt cylinder height adjustment is provided for optimizing gun performance at all operating potentials. The double condenser lens is unitized and has two lens regions and a common energizing coil.
Annealing Effects in Ten Films of Thoria
Authors: Bronisz S. E. , Douglass Dana L.
Published: June 2020

Thin films of thoria, either cleaved from air-fired material or vacuum deposited on polished copper substrates, were examined by transmission electron microscopy. As prepared, the two types of samples were considerably different, but after being heated in the electron microscope they were closely similar.

The cleaved samples were obtained by means of extraction replication of fracture surfaces of polycrystalline thoria. The thin flakes ranged from about 0.1 to 20 μm in diameter. Most of them were single crystals exhibiting the diffraction effects expected of crystalline materials and containing many long dislocations. Upon heating with the unapertured electron beam the dislocations disappeared, the crystals became more electron transparent, and the striated microstructure shown in Fig. 1 developed. The orientations of most of the cleaved crystals were equally divided among ﹛110﹜, ﹛111﹜, and ﹛112﹜. The striae were usually parallel to <110> or <135>.
A Method For Determining The Anomalous Absorption Parameter of a Crystal
Authors: Bell W. L. , Thomas G.
Published: June 2020

The two-beam dynamical theory gives the intensity diffracted by a perfect wedge-shaped, absorbing crystal at the Bragg angle for diffraction as:

where and are all dimensionless parameters of absorption and thickness. In common notation and are the mean and anomalous absorption distances, to is the extinction distance and z is the thickness of the foil; all are generally expressed in Angstroms.

Values for the absorption parameters are obtained using the extrema values from an intensity profile and determining the envelope for the intensity variations.
Electron Microscopy of Diamond Synthesized by Explosive Shock
Authors: Trueb Lucien F.
Published: June 2020

The microstructure of early shock-synthesized diamond was discussed in a previous report; this paper is concerned with the characterization of improved material which is now available from E. I. du Pont de Nemours & Company in various size ranges suitable for lapping and polishing.

This new type of synthetic industrial diamond is produced by shocking dense graphite-metal mixtures at high explosive pressures. It consists of two types of particles which are shown in Figure 1; polycrystalline aggregates which are rounded and have a mottled surface, and single crystals or fragments thereof which have straight edges and flat surfaces. Figure 2 shows the two types of particles in transmission; they both partly consist of the hexagonal modification of diamond. Single crystals have either a cubic or a hexagonal habit and they often contain large numbers of stacking faults. Polycrystalline particles consist of densely aggregated bundles of acicular crystallites having an average width of 150 A and a length varying between 0.05 and 0.3 p.; a strong preferred orientation is invariably present (Figure 3).
High-resolution scanning electron microscopy (HRSEM) of mitochondria from various rat tissues
Authors: Lea P.J. , Hollenberg M.J.
Published: June 2020

Our current understanding of mitochondrial ultrastructure has been derived primarily from thin sections using transmission electron microscopy (TEM). This information has been extrapolated into three dimensions by artist's impressions (1) or serial sectioning techniques in combination with computer processing (2). The resolution of serial reconstruction methods is limited by section thickness whereas artist's impressions have obvious disadvantages.

In contrast, the new techniques of HRSEM used in this study (3) offer the opportunity to view simultaneously both the internal and external structure of mitochondria directly in three dimensions and in detail.

The tridimensional ultrastructure of mitochondria from rat hepatocytes, retinal (retinal pigment epithelium), renal (proximal convoluted tubule) and adrenal cortex cells were studied by HRSEM. The specimens were prepared by aldehyde-osmium fixation in combination with freeze cleavage followed by partial extraction of cytosol with a weak solution of osmium tetroxide (4). The specimens were examined with a Hitachi S-570 scanning electron microscope, resolution better than 30 nm, where the secondary electron detector is located in the column directly above the specimen inserted within the objective lens.
Determination of connections between cellular structures using 3-d reconstruction from multiple-view stereopair images of thick sections
Authors: Marko M. , Leith A. , Parsons D.
Published: June 2020

An important application for serial section reconstruction is determining whether a structure is composed of separate parts, or is continuous. Structures which are apparently separate on random sections may actually be continuous within the 3-D volume. The presence of connections between different types of organelle compartments within a cell should give important information about metabolic control of the cell. The determination of whether an organelle type is composed of multiple compartments, or actually exists as one or a few complexes, should also be of metabolic significance. The metabolic importance of some connections, such as gap junctions, between adjacent cells has already been well established.

One method to prove that two membrane-bound structures connect is to trace the membrane continuously from one structure to the other. This is difficult when using thick sections because the membranes may be nearly invisible if they are at an oblique angle to the direction of view.
Measurement of [Ca] in single cells and pH in single organelles by fluorescence microscopy
Authors: Maxfield F.R. , Shelanski M.L.
Published: June 2020

Microscope spectrofluorometry and digital image processing provide the ability to study changes in ion concentrations in living cells with high temporal and spatial resolution. We have used fluorescein labeled macromolecules to measure the pH of specific endosomal compartments (1-3). The ratio of fluorescence intensities at 450 nm and 490 nm excitation provides a measure of the pH (4). The acidification of endosomes detected by this technique provide an explanation for endosome functions including intracellular sorting of ligands and receptors, release of iron from transferrin, and penetration of viruses and toxins into the cytosol (3).

Using the tetracarboxylate calcium indicator dyes synthesized by R. Tsien and his colleagues (5), the same instruments can be used for measuring intracellular free calcium, [Ca2+]i, in single cells. We have used the system to measure [Ca2+]i changes associated with cell motility (6-9).

Cells are examined using a Leitz Diavert microscope with a computer-controlled illumination and photometry system.
Submicrometer optical metrology
Authors: Larrabee Robert D.
Published: June 2020

The National Bureau of Standards (NBS) has had a continuing program for over 10 years [1-5] to develop optical feature-size standards for the integrated circuit industry. As the dimensions of interest to this industry have evolved into the submicrometer region, feature-size measurements have become more difficult because the dimensions of interest have become comparable to (or smaller than) the wavelength of light used for their measurement. In this domain of feature sizes, diffraction produces large effects in the optical image and makes that image difficult to interpret. The effects of diffraction mask the location of the feature edges that define the dimensions of interest.
Enhancement of outer cell layers of a methanotrophic bacterium using uranyl acetate en bloc
Authors: Putzer K. , Fassel T.A. , Remsen C.C.
Published: June 2020

Obligate methanotrophic bacteria are autotrophic microorganisms capable of oxidizing methane to supply both their carbon and energy requirements. Methanotrophs are placed in one of two groups based on certain biochemical features and the arrangement of intracytoplasmic membranes (ICM). Type I methanotrophs contain ICM arranged as bundles of disc-like vessicles in the interior of the cell, while Type II methanotrophs display paired peripheral ICM. The organism used in this study, Methylosinus trichosporium OB3b, is a Type II methanotroph and has a capsular structure surrounding the cell as well as several complex cell wall structures described as cup-like, filamentous, and spike-shaped. This study examines the external surface of M. trichosporium OB3b following en bloc staining with uranyl acetate.

Cells were harvested in mid-log phase, fixed in 0.5% glutaraldehyde and postfixed in 1% OsO4. Cells were enrobed in 4% agar prior to dehydration in a graded ethanol series.
Quantitative x-ray elemental imaging in stimulated single intact skeletal muscle fibers: The fate of calcium in the presence of ryanodine
Authors: Sommer J. , Ingram P. , LeFurgey A. , Nassar R. , High T.
Published: June 2020

We are involved in a continuing series of experiments aimed at a complete description,in terms of morphology and quantitative topochemistry, of the time course of spatial distributions of physiologically important elements during excitation-contraction coupling (ECC) at different time intervals (fractions of msec) following electrical stimulation of single, intact frog skeletal muscle fibers. In this present study wg report such distributions for Ca after 1,2 and 3 min of electrical stimulation in the presence of 2x10-4 M ryanodine, an alkaloid that, in time, causes irreversible muscle contractures.

Single, intact frog skeletal muscle fibers were quick-frozen, cryosectioned, freeze-substituted and in one case freeze-fractured. The freeze-dried cryosections were subjected to electron probe X-ray microanalysis (EPXMA) in a JEOL 1200EX analytical electron microscope equipped with a Tracor Northern X-ray detector and a fully quantitative imaging system. Both, 64/64 pixel images (ambient temp.), and small raster probes (cold stage,-115 °C) for better statistics, were obtained, each from the same section.