bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphotungstic Acid As An Electron Stain
Authors: Pease Daniel C.
Published: June 2020

Under certain conditions phosphotungstic acid (PTA) can be used as an effective electron stain for demonstrating polysaccharide moieties in tissue sections. There it selectively combines with the glycoproteins of basement membranes, the mucoid layers associated with brush borders, the mucus of goblet cells, the chondroitin sulfate of cartilage matrix, glyco-gen, and “glycocalyces” in general. The initial published investigation indicated that an acid environment was essential for this specificity, but the effect of pH was not systematically explored.

Rib cartilage of newborn rats was chosen for a detailed study of the effects of pH on PTA staining, particularly to provide ancillary information on the formation, secretion and deposition of chondroitin sulfate. Cartilage was prepared without chemical fixation by “freeze-substitution” with 70% glycol, or by “inert dehydration” with glycol at room temperature. Dehydrated material was embedded in prepolymerized hydroxy-propyl methacrylate without an intermediate solvent.
Selective Staining of Acid Mucopolysaccharides By Ruthenium Red
Authors: Luft J. H.
Published: June 2020

Ruthenium red is one of the few completely inorganic dyes used to stain tissues for light microscopy. This novelty is enhanced by ignorance regarding its staining mechanism. However, its continued usefulness in botany for demonstrating pectic substances attests to selectivity of some sort. Whether understood or not, histochemists continue to be grateful for small favors.

Ruthenium red can also be used with the electron microscope. If single cells are exposed to ruthenium red solution, sufficient mass can be bound to produce observable density in the electron microscope. Generally, this effect is not useful with solid tissues because the contrast is wasted on the damaged cells at the block surface, with little dye diffusing more than 25-50 μ into the interior. Although these traces of ruthenium red which penetrate between and around cells are visible in the light microscope, they produce negligible contrast in the electron microscope. However, its presence can be amplified by a reaction with osmium tetroxide, probably catalytically, to be easily visible by EM. Now the density is clearly seen to be extracellular and closely associated with collagen fibers (Fig. 1).
Comparison of Cholesterol Extraction from Tissues During Processing for Electron Microscopic Radioautography
Authors: Hedley-Whyte E. Tessa , Uzman Betty G.
Published: June 2020

Radioautographic studies of lipid metabolism with the electron microscope have been limited by the large losses of lipid which occur during the usual dehydration and infiltration procedures. In this study different methods of processing tissue for electron microscopic radioautography have been compared with respect to the extraction of cholesterol-1, 2-3H from labeled mouse liver and nerve.

In all methods primary fixation had been in 10% formalin for several months followed by washing overnight. Post-fixation for one hour in Dalton's chrome osmium and staining in 1% uranyl acetate in 10% formalin resulted in small but constant losses. Four methods were assessed: 1) graded acetone dehydration with propylene oxide and epon-araldite mixture infiltration, 2) exposure to 0.5% digitonin in 50% ethanol for 1 hour before post-fixing in osmium followed by procedure 1, 3) Durcupan embedding, and 4) limited dehydration followed by embedding in an epon:araldite mixture (modified Idelman).
Freeze-Etching Simplified
Authors: Steere Russell L.
Published: June 2020

Freeze-etching, as a tool in the preparation of biological specimens for electron microscopy, has seen relatively little use since it was introduced in 1957. The involved procedure, originally described, and, until recently, the cost of special equipment, must certainly have played a part in the failure of freeze-etching to become a commonly used technique. Efforts to solve some of the problems have resulted in the development of a modified freeze-etching module that can fit on nearly any vacuum evaporator and will permit the production of good specimens by an unskilled technician in less than one-half hour.

The freeze-etching module (Fig. 1) consists of a collar (A) with two opposing hollow tubes supported by small collars so that the specimen end of the specimen tube (D) can rotate within the rotatable cold shroud (F).
Stellate Cells in the Pituitary Gland of the Salamander
Authors: Cardell Robert R.
Published: June 2020

The fine structure of five cell types in the pars distalis of the salamander [Notophthalmus (Triturus) viridescens viridescens] pituitary gland has been described. Four of these possess characteristic secretory granules (figure 1) and apparently they are involved in the synthesis and secretion of pituitary hormones. The other cell (Cell type #5 or “stellate cell”) does not possess characteristic secretory granules (figures 1 and 2) and they show a morphology which does not suggest a secretory function. The present report extends the observations on the fine structure of the stellate cell and considers, in addition, the distribution of this cell throughout the pars distalis.

The stellate cell displays many slender, cytoplasmic extensions which penetrate between the secretory cells (figure 1). These extensions contact processes from other stellate cells and desmosomes are found at the points of contact. Moreover, desmosomes are frequently located between the stellate cells and the secretory cells (figure 2). Thus, the stellate cells are not only linked to each other throughout the pars distalis, but to each secretory cell as well. Stellate cell processes contact the outer basement lamina of the pericapillary space and often such processes follow the contour of the basement lamina for considerable distances. They do not penetrate, however, into the pericapillary space.
The Morphology of the Pecten Oculi and the Conus Papillaris
Authors: Dunn R. F.
Published: June 2020

In this study, the pecten of the house finch, and the conus of the gecko, Eublipharis, were utilized. The structure of the house finch pecten resembled that of the pigeon. The capillary endothelium is characterized by an extensive luminal and abluminal system of plasma membrane plications (Fig. 1, LP and AP). The luminal ridges vary in length from 1.3 - 2.9μ, and are 40-90 mμ in width. The luminal and abluminal plications are separated by the narrow, rather dense endothelial cytoplasm which contains free ribosomes and dense mitochondria in addition to granular cytomembranes. The abluminal ridges are generally shorter, and rest on a basement membrane. Many of the pecten surface capillaries are separated from the vitreous by only a short distance of about 1μ. In other regions the capillaries are surrounded by pigmented cells, which contain smooth surfaced, very osmiophilic granules up to 1.6μ in diameter, a few smaller, irregularly shaped granules and the usual compliment of cytoplasmic organelles (Fig. 2).

In sharp contrast to the pecten, the endothelium of the conus is relatively smooth and lacks any surface foldings as well as any fenestrations (Fig. 3). The endothelial basement membrane is separated from that of the pigment cell by a variable extracellular connective tissue space containing many collagen fibrils. The pigment cells contain melanin granules similar to those of the pecten (Fig.4).
Turbomolecular Pumped Electron Microscopy of Rubella Virus (In BHK Celis)
Authors: Taylor A. B. , Cole G. C. , Holcomb M. A. , Baechler C. A.
Published: June 2020

An aliquot from a continuous fermenter culture of baby hamster kidney cells (BHK-21 Clone PD-4) (Wistar) maintained in Ca free Eagle's Basal Medium containing 2% Kaolin adsorbed fetal calf serum was planted in spinner flasks at 300,000 cells per ml, total volume 600 ml. After equilibration for one day at 35°C to insure that cells were in log phase, the culture was infected with the M-33-AGMK25 BHK-219 strain of rubella at an input multiplicity of about 6 TCID50 per cell. The virus was identified with specific rubella antiserum.

Preliminary experiments had shown that such cultures would reach a peak or plateau HA titer of approximately 1:64, 24 hrs after inoculation and would continue to yield virus for 6 to 12 days. One hundred ml aliquot harvests were withdrawn daily and the culture was returned to volume with growth medium and incubation continued. The harvested cells were spun down rapidly at 2500 rpm per 15 mins., fixed in 3.7% gluteraldehyde in Ca free phosphate buffer saline, and post fixed in osmium tetraoxide. After dehydration, the cells were embedded in Epon 812 and cured approximately 20 hrs at 60°C.
On The Mechanism of Formation of Stacking Faults in Aged Nickel-Base Alloys
Authors: Rowe G. M. , Rand W. H. , Kear B. H.
Published: June 2020

The formation of stacking faults upon aging a solution treated and quenched nickel-base alloy has been reported previously. More recent work has revealed a similar behavior in several γ’ precipitation hardened nickel-base alloys aged in the temperature range l400°-1650°F. The faults intersect both γ (nickel solid solution) and γ’ (Ni3Al solid solution) phases, and are of extrinsic type. Fault formation is attributed to a chemical segregation effect involving the climb of Frank partials, similar to that reported for austenitic steels.

Fig. 1 shows extrinsic faults associated with a slip band in Udimet 700 lightly deformed at 70°F prior to aging. For the specified [011] normal to the foil, the slip trace is (11), and the faults lie in (111) and (11). Using the g · b = 0 or ± 1/3 (s = 0) criterion for dislocation invisibility, Figs, 1 (a-c) show that the outer partial for a (111) fault must be 1/6 [11] Shockley, or 1/3 [111] Frank.
Microstructure and Lattice Defects in Polycrystalline Nuclear Ceramics
Authors: Daniel J. L. , Mayhan S. J.
Published: June 2020

Transmission electron microscopy of several nuclear ceramics has been extended to thin sections of as - fabricated poly-crystalline materials, by use of a thinning technique utilizing only common metallographic practices. The method is based on work by Doherty and Leombruno. However, while mechanical thinning (polishing) produces large, evenly thinned specimens, the surface of ceramic materials retains many shallow scratches and defects introduced by the polishing medium. On the other hand, the chemical thinning methods commonly applied produce only very small areas which are thin enough for examination by transmission electron microscopy, since preferential attack occurs on grain boundaries, inclusions, second phases, etc. By combining the chemical polish with the mechanical thinning procedures, large, relatively clean areas of ceramic materials can be produced. Another significant advantage is that in the course of thinning, the same specimens can be examined frequently and in detail by light microscopy, some physical measurements can be made along the way (e.g., microhardness, spectral transmission, autoradiography), and all observations can be closely correlated finally with the high resolution electron microscopy.
Residual Gas Reactions in the Electron Microscope: I. Qualitative Observations on the Water Gas Reaction
Authors: Hartman Richard E. , Hartman Roberta S. , Ramos Peter L.
Published: June 2020

The action of water and the electron beam on organic specimens in the electron microscope results in the removal of oxidizable material (primarily hydrogen and carbon) by reactions similar to the water gas reaction .which has the form:

The energy required to force the reaction to the right is supplied by the interaction of the electron beam with the specimen.

The mass of water striking the specimen is given by:

where u = gH2O/cm2 sec, PH2O = partial pressure of water in Torr, & T = absolute temperature of the gas phase. If it is assumed that mass is removed from the specimen by a reaction approximated by (1) and that the specimen is uniformly thinned by the reaction, then the thinning rate in A/ min is

where x = thickness of the specimen in A, t = time in minutes, & E = efficiency (the fraction of the water striking the specimen which reacts with it).
Development of Electron Mirror Microscope
Authors: Someya T. , Watanabe M.
Published: June 2020

Our electron mirror microscope has been improved by adopting magnetic lenses, which enable a wide range of magnification to be obtained. This improved version, known as the JEM-M1 (35kV acceleration) uses the straight design and shadow projection method.

The construction of the newly designed instrument is illustrated in Fig. 1 and Fig. 2 shows a sectional view of its electron optical column. The electron gun assembly is located under the console table. Incident beam alignment is carried out by means of the deflection coil knob, the electron gun horizontal movement knob and the condenser lens knob located on the table. The zircaloy crystal located above the screen and on the optical axis enables the alignment procedure to be carried out easily and simply because it radiates light when impinged by the electron beam. The projector lens, having a short focal length, combined with the condenser lens, forms a small source for the shadow projection image on its back focal plane. The intermediate lens, acting on the reflected beam, produces image magnification by changing the excitation current. Magnification in the range x200 – x1500 is possible without any appreciable distortion.
Some Aspects of Contrast in High Voltage Electron Microscopy
Authors: Metherell A. J. F. , Fisher R. M. , Duff W. R. , Buchanan R.
Published: June 2020

In view of greater penetration at high accelerating voltages, the column approximation method of calculating cartographic plots of image intensity bear examination. Results of theoretical considerations of several aspects of contrast in high voltage microscope will be presented and illustrated with relevant electron micrographs.

(1) Column Approximation - In the column approximation for a crystal with a crystal with a small distortion due to a dislocation, stacking fault, etc. two assumptions are made; (a) that the direct and diffracted waves propagating through the crystal do not diverge and (b) that the same value of the Bragg deviation parameter x can be used for the waves excited on the different branches of the dispersion surface, regardless of the angle the local crystal distortion makes with the Brillouin zone boundary. Takagi has obtained differential equations which take into account the divergence of the beams as they pass through the crystal.
Feasibility of Molecular Structure Determination With a High Resolution Electron Microscope
Authors: Welton T. A.
Published: June 2020

An ultimate design goal for an improved electron microscope, aimed at biological applications, is the determination of the structure of complex bio-molecules. As a prototype of this class of problems, we propose to examine the possibility of reading DNA sequence by an imaginable instrument design. This problem ideally combines absolute importance andrelativesimplicity, in as much as the problem of enzyme structure seems to be amuchmore difficult one.

The proposed technique involves the deposition on a thin graphite lamina of intact double helical DNA rods. If the structure can be maintained under vacuum conditions, we can then make use of the high degree of order to greatly reduce the work involved in discriminating between the four possible purine-pyrimidine arrangements in each base plane. The phosphorus atoms of the back bone form in projection (the helical axis being necessarily parallel to the substrate surface) two intertwined sinusoids. If these phosphorus atoms have been located up to a certain point on the molecule, we have available excellent information on the orientation of the base plane at that point, and can then locate in projection the key atoms for discrimination of the four alternatives.
A High Resolution Scanning Microscope
Authors: Crewe A. V. , Wall J. , Welter L. M.
Published: June 2020

A scanning microscope using a field emission source has been described elsewhere. This microscope has now been improved by replacing the single magnetic lens with a high quality lens of the type described by Ruska. This lens has a focal length of 1 mm and a spherical aberration coefficient of 0.5 mm. The final spot size, and therefore the microscope resolution, is limited by the aberration of this lens to about 6 Å.

The lens has been constructed very carefully, maintaining a tolerance of + 1 μ on all critical surfaces. The gun is prealigned on the lens to form a compact unit. The only mechanical adjustments are those which control the specimen and the tip positions. The microscope can be used in two modes. With the lens off and the gun focused on the specimen, the resolution is 250 Å over an undistorted field of view of 2 mm. With the lens on,the resolution is 20 Å or better over a field of view of 40 microns. The magnification can be accurately varied by attenuating the raster current.
The Possibility of Improving Scanning Microscope Images by After-Treatment
Authors: Thomson M. G. R. , Crewe A. V.
Published: June 2020

The scanning electron microscope is particularly suited to the after-treatment of the image because the information is obtained in serial form, and may be processed using electronic analogue or digital computers. The techniques employed at present include logarithmic amplification, differentiation, and the subtraction of a constant intensity; all of these being intended to render low contrast detail more visible, and so making staining of the specimen less necessary.

One further technique is the removal from the final picture of some of the effects of the finite size of the scanning electron beam. Essentially this involves construction of the Fourier transform of the picture, division by the Fourier transform of the scanning spot, and Fourier transformation of this result. It is possible to perform these operations in the case either of a scanning or conventional microscope by digitising the picture, doing the calculations numerically, and reconstructing the improved picture.
Lorentz Microscopy: Effect of Tilting on Magnetic Domains in Single Crystal Iron Films
Authors: Allard L. F. , Rowe A. P. , Fan P. L.
Published: June 2020

In order to observe magnetic domain walls by Lorentz microscopy techniques it is often necessary either to operate the microscope with the objective lens off, thus severely limiting the magnification, or to move the specimen from its usual position or make some other modification so that the field to which it is subjected is not so strong that it saturates the specimen. However, conditions in the JEM-6A have proved favorable for observation of domains in single crystal iron films by the out-of-focus method without any modifications, using either the regular specimen stage with the small bore pole piece or the tilting stage with the large bore pole piece. The tilting stage is particularly useful for these studies because the domains are very sensitive to small differences in inclination in the field.
Investigation of Interfaces Between Steel and Vapor Phase Deposited Tungsten by Optical and Electron Microscopy and by Electron Probe Microanalysis
Authors: Meyer Karl H. , Stulpe Klemens , Lehner John P.
Published: June 2020

Specific materials requirements such as high temperature strength in combination with corrosion and erosion resistance and limitations in cost and weight often can only be satisfied when a composite material is utilized. A typical example is shown in the form of a thin tungsten layer deposited by the vapor phase process onto the bore of gun barrels to increase their operational life. Initial attempts to plate 4140 steel resulted in flaking-off of the tungsten coating from the substrate but after development of proper pre-treatment procedures of the substrate and of specified deposition temperature, flow ratios and flow rates, firm mechanical bonds between tungsten and substrate were obtained. Requirements for firm bonds are the absence of surface oxide films, small differences in thermal expansion coefficients, interdiffusion of the two metals into each other and other factors.
Observations in Polycrystalline Ceramics by Electron Transmission Microscopy
Authors: Tighe N. J.
Published: June 2020

Polycrystalline ceramic compacts can be thinned for transmission electron microscopy by ionic bombardment. The present paper describes this technique and presents some results of studies on ceramic bodies of different composition, grain size and porosity. Thinning was done in an apparatus which was designed by Paulus and Reverchon who used it to thin single crystal specimens. The apparatus uses two ion sources so that specimens placed between them can be thinned from both sides.

The polycrystalline specimens were thinned using argon ions at an accelerating voltage of 5 - 8 kV and a beam current of ∼ 100 μA/cm2. The thinning rate depended on the voltage and beam current and varied with the specimens and was 0.1 to 1 μm/hr. The thinning progress was monitored with a microscope mounted on the vacuum chamber and by removing the specimen from the chamber and measuring the thickness with an optical microscope. The specimens were prepared for the ion thinning by mechanical grinding and polishing.
Electron Microfractography Applied to Large Pipe Bending Fatigue Tests
Authors: Wolff Ursula E.
Published: June 2020

Among the different causes of pipe failures, fatigue cracking has to be considered in any design program. Since the integrity of nuclear reactor piping is of particular importance, an AEC pipe study program includes a task to assess quantitatively the crack propagation rate through pipes for cracks originating at surface flaws. This problem has been attacked theoretically by the use of fracture mechanics, and practically by striation measurements on fracture surfaces produced by full-scale bending fatigue tests. This paper deals with the practical aspect of applying electron microfractography to such fractures as a means to determine crack propagation rates in pipes of different dimensions and materials, and with different flaw locations.

Six-inch diameter pipes with a wall thickness of 7.5, 10.8, or 18 mm were notched inside or outside and failed in reverse bending fatigue by growing a crack through the wall thickness. The crack front was marked at intervals by reducing the strain amplitude for a number of cycles.
Observations of the Thermal Decomposition of Brucite
Authors: Burnett P. L. , Mitchell W. R. , Houck C. L.
Published: June 2020

Natural Brucite (Mg(OH)2) decomposes on heating to form magnesium oxide (MgO) having its cubic ﹛110﹜ and ﹛111﹜ planes respectively parallel to the prism and basal planes of the hexagonal brucite lattice. Although the crystal-lographic relation between the parent brucite crystal and the resulting mag-nesium oxide crystallites is well known, the exact mechanism by which the reaction proceeds is still a matter of controversy. Goodman described the decomposition as an initial shrinkage in the brucite basal plane allowing magnesium ions to shift their original sites to the required magnesium oxide positions followed by a collapse of the planes along the original <0001> direction of the brucite crystal. He noted that the (110) diffraction spots of brucite immediately shifted to the positions required for the (220) reflections of magnesium oxide. Gordon observed separate diffraction spots for the (110) brucite and (220) magnesium oxide planes. The positions of the (110) and (100) brucite never changed but only diminished in intensity while the (220) planes of magnesium shifted from a value larger than the listed ASTM d spacing to the predicted value as the decomposition progressed.
Identification of Nodules Present on Reduced Iron Ore Particles
Authors: Bailey G. W.
Published: June 2020

In the fluidized reduction of iron ore, certain operating conditions result in the formation of nodular growths on the ore particles. These growths begin to appear even at very low metallization. The size and number of these nodules have varied with the reducing conditions and have been found to be particularly dependent on reduction time. The nodules are usually only a few microns in size. The structure and composition of these nodules have been investigated using a variety of physical techniques including:

∘ Optical microscopy

∘ Electron microscopy

∘ Electron probe analysis

∘ X-ray diffraction

Nodular growths were detected initially on reduced iron ore particles by optical microscope examination of mounted and polished cross-sections of the partially reduced particles (Figure 1). In an effort to determine the composition of the nodules, electron probe analysis was conducted on the mounted specimens.
Laboratory and in-situ analysis of comet dust
Authors: Brownlee D.E. , Albee A.L.
Published: June 2020

Comets are primitive, kilometer-sized bodies that formed in the outer regions of the solar system. Composed of ice and dust, comets are generally believed to be relic building blocks of the outer solar system that have been preserved at cryogenic temperatures since the formation of the Sun and planets. The analysis of cometary material is particularly important because the properties of cometary material provide direct information on the processes and environments that formed and influenced solid matter both in the early solar system and in the interstellar environments that preceded it.

The first direct analyses of proven comet dust were made during the Soviet and European spacecraft encounters with Comet Halley in 1986. These missions carried time-of-flight mass spectrometers that measured mass spectra of individual micron and smaller particles. The Halley measurements were semi-quantitative but they showed that comet dust is a complex fine-grained mixture of silicates and organic material. A full understanding of comet dust will require detailed morphological, mineralogical, elemental and isotopic analysis at the finest possible scale. Electron microscopy and related microbeam techniques will play key roles in the analysis. The present and future of electron microscopy of comet samples involves laboratory study of micrometeorites collected in the stratosphere, in-situ SEM analysis of particles collected at a comet and laboratory study of samples collected from a comet and returned to the Earth for detailed study.
Development of accurate linewidth measurement techniques for in-process wafers
Authors: Van Asselt Robert. L. , Becker Heinrich
Published: June 2020

Measurements of the device geometries on “in-process” wafers present some interesting challenges. Sample coating is not possible and the measurement probe must not damage the devices. The Scanning Electron Microscope (SEM) is the standard tool for submicrometer measurements. However, operating at the low electron beam accelerating voltage required to avoid damage to integrated circuits introduces problems in resolution. Also, the measurement accuracy may be limited by the effects of surface charging and topography. Further, SEM linewidth standards do not exist at the present time. Optical measurements are attractive because, in general, they display greater precision, are typically less expensive to implement and have a higher throughput. However, diffraction effects associated with the complex three-dimensional geometries of integrated circuit structures make accurate measurements very difficult. The "blur" regions that occur in the optical image at each edge must be interpreted to predict the actual location of the structure edges.
Nanometer-scale motion analysis of microtubule-based motor enzymes
Authors: Gelles J. , Schnapp B.J. , Steuer E. , Sheetz M.P.
Published: June 2020

Kinesin and cytoplasmic dynein are mechanochemical ATPases thought to transport vesicular organelles within cells by moving them along microtubules. Molecules from purified enzyme preparations will bind to plastic microspheres (beads) and move them along microtubules in vitro at rates of ∽0.5 μm s-1. Bead movement can be directly observed by light microscopy in an assay that provides an opportunity to study motions driven by small numbers of enzyme molecules. Measurement of such motions with nanometer-scale precision yields information about the molecular events which drive bead movement.

We devised a method to measure the relative position o£ an enzyme-driven 150nm bead with a precision better than 1 nm at 30 Hz2. This method is based on digital processing of bead images in differential interference contrast microscopy using a cross-correlation/centroid algorithm.
Techniques for analyzing conventional histologic sections
Authors: Shelburne J.D. , Ingram P. , Tucker J.A.
Published: June 2020

Despite the many known artifacts attendant to conventional processing and staining, one of the most common types of medical microprobe analysis involves the use of routine histologic sections. Formalin-fixed, paraffin embedded tissue sections are particularly useful for cases involving inorganic particulates or insoluble, tightly bound exogenous metals. These sections are technically easy to prepare and usually offer much greater surface area than is available with plastic embedding for electron microscopy. Figure 1 reviews our approach to the analysis of this type of specimen.

After identifying a feature of interest such as unknown particles, the microscopist then must decide if the feature is prevalent and homogeneous or rare and unique. In the former case, serial sections are cut from the paraffin block at 5-10 μm and picked up onto spectroscopically pure, ultrasmooth carbon planchets. Paraffin tissue sections readily adhere to the carbon substrate after heating at 60°C for 30 minutes, followed by deparaffination in xylene and air drying.
3-D imaging of cells using a confocal laser scanning microscope and digital image processing
Authors: Jovin Thomas M. , Robert-Nicoud Michel , Arndt-Jovin Donna J. , Schormann Thorsten
Published: June 2020

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.

We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.
Ceramic sol-gel transformations studied using cryo-microscopy
Authors: Bailey J. K. , Mecartney M. L.
Published: June 2020

The preparation of ceramics from chemical precursors yields high quality materials. Sol-gel processing, one of these preparation methods, offers controlled purity, low temperature processing, and production of difficult shapes, such as fibers and coatings. Beginning as either a colloidal dispersion or a solution of inorganic monomers, the sol undergoes a gelation or polymerization reaction producing a solid body that incorporates the initial solvent. This body is then dried to remove the solvent and densified to produce either a porous or dense material as desired. In sol-gel processing, control over and understanding of the developing gel microstructure and how this microstructure influences the sintered properties is of vital importance. Furthermore, it has been difficult to determine the structure unambiguously.

Using cryo-microscopy, direct observation of the microstructure of colloidal sols and the formation of the gel has been described in previous work.
Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics
Authors: Jacobson K. , Ishihara A. , Holifield B. , Zhang F.
Published: June 2020

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.
Ultrastructural Studies on Initial Stages of Human Coronary Artery Disease
Authors: Gabrovsek J. , Robertson A. L. , Dianovsky J.
Published: June 2020

Electron microscopy evaluation of pathological changes in human tissues are often marred by inadequate specimen preservation due to unavoidable postmortem changes and/or fixation artifacts. In the present study, preclinical coronary athero-arteriosclerosis was systematically studied in specimens obtained from 35 infants and children of both sexes. They were selected on the basis of having no clinical history of cardiovascular disease and have died of sudden infant death syndrome or acute trauma less than 48 hours after hospital admission. All cases were autopsied within 8-24 hours postmortem following one of the two protocols found most effective in the preservation of ultrastructural details: 1) If direct perfusion-fixation was not possible due to heart trauma, samples of the right coronary artery (RCA), left anterior descending (LAD) and left circumflex (LCX) were first fixed by immersion in 2.5% glutaraldehyde-2% paraformaldehyde-0.1 M cacodylate buffer at pH 7.4 (modified Karnovsky's solution) for 3 hours at 4°C. Matching samples were processed for light microscopy-histochemistry, while those for ultrastructural evaluation were routinely obtained from ring sections 10 mm from the origin of each coronary artery.
Image Processing of Thin Three-Dimensional Crystals of the Light-Harvesting Chlorophyll a/b-Protein Complex from Chloroplast Membranes
Authors: Kühlbrandt Werner
Published: June 2020

Thin three-dimensional, hexagonal crystals of the light—harvesting chlorophyll a/b—protein complex (LHC II) from pea chloroplast membranes diffract electrons to 3.7 Å resolution when preserved in glucose or tannin. The symmetry of the diffraction pattern (6mm), the dimensions of the unit cell in projection (a = 127 Å) and micrographs of negatively stained specimens suggested that the hexagonal crystals were stacks of two-dimensional crystals of p321 symmetry. Low—dose (1 —2 electrons/Å2) electron micrographs of thin three-dimensional crystals preserved in tannin were recorded in an attempt to determine the structure of this integral membrane protein complex at high resolution, initially in projection. The best images showed sharp diffraction spots at 12 — 14 Å resolution when examined by optical diffraction. Image areas measuring up to 40 x 40 mm2 were densitometered at a step size corresponding to 3 Å or less at the specimen and computer processed to correct for lattice distortions, using programmes by R.Henderson and J.M.Baldwin. Fourier transforms of the distortion—corrected images showed reflections above background level to 6 Å resolution.
Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography
Authors: Winkelmann Donald A.
Published: June 2020

The primary role of the interaction of actin and myosin is the generation of force and motion as a direct consequence of the cyclic interaction of myosin crossbridges with actin filaments. Myosin is composed of six polypeptides: two heavy chains of molecular weight 220,000 daltons and two pairs of light chains of molecular weight 17,000-23,000. The C-terminal portions of the myosin heavy chains associate to form an α-helical coiled-coil rod which is responsible for myosin filament formation. The N-terminal portion of each heavy chain associates with two different light chains to form a globular head that binds actin and hydrolyses ATP. Myosin can be fragmented by limited proteolysis into several structural and functional domains. It has recently been demonstrated using an in vitro movement assay that the globular head domain, subfragment-1, is sufficient to cause sliding movement of actin filaments.

The discovery of conditions for crystallization of the myosin subfragment-1 (S1) has led to a systematic analysis of S1 structure by x-ray crystallography and electron microscopy. Image analysis of electron micrographs of thin sections of small S1 crystals has been used to determine the structure of S1 in the crystal lattice.
Electron Spectroscopic Imaging of DNA and Protein-DNA Complexes
Authors: Bazett-Jones D.P. , Brown M.L.
Published: June 2020

Elemental distributions in cells and molecular spreads can now be produced at the spatial resolution attainable in the electron microscope by the collection of X-rays or by the collection of and imaging with inellastically scattered electrons. With the latter method, known as Electron Spectroscopic Imaging (ESI), an image is produced with electrons that have lost characteristic amounts of energy from ionizing or exciting specific elements in the specimen. ESI can generate an elemental map of a specimen at a resolution of about 0.5 nm. It can be carried out in a fixed beam microscope equipped with a parallel energy filter inserted into the column of the microscope below the specimen (1,2). An instrument equipped with a prism-mirror-prism electron spectrometer was used in this study to image purified DNA molecules and a complex of the transcription factor TFIIIA with DNA.

Transcription of most genes is activated by the binding of transcription factors to promoter elements.
Current State of Biological High-Resolution Scanning Electron Microscopy
Authors: Peters Klaus-Ruediger
Published: June 2020

A new generation of high performance field emission scanning electron microscopes (FSEM) is now commercially available (JEOL 890, Hitachi S 900, ISI OS 130-F) characterized by an "in lens" position of the specimen where probe diameters are reduced and signal collection improved. Additionally, low voltage operation is extended to 1 kV. Compared to the first generation of FSEM (JE0L JSM 30, Hitachi S 800), which utilized a specimen position below the final lens, specimen size had to be reduced but useful magnification could be impressively increased in both low (1-4 kV) and high (5-40 kV) voltage operation, i.e. from 50,000 to 200,000 and 250,000 to 1,000,000 x respectively.

At high accelerating voltage and magnification, contrasts on biological specimens are well characterized1 and are produced by the entering probe electrons in the outmost surface layer within -vl nm depth. Backscattered electrons produce only a background signal. Under these conditions (FIG. 1) image quality is similar to conventional TEM (FIG. 2) and only limited at magnifications >1,000,000 x by probe size (0.5 nm) or non-localization effects (%0.5 nm).
High-Resolution Topographic Imaging of Enamel Crystal Surfaces
Authors: Apkarian R.P. , Gutekunst M.D.
Published: June 2020

Previous interpretations of enamel crystal morphology have been handicapped by preparation techniques and the understanding of enamel crystals was derived from TEM and conventional SEM studies looking into lesion formation in enamel. Early studies focused on the demineralization and effects of mineralization on enamel structure. Enamel crystals were previously described as flattened hexagonal structures 100 μm long and 35-40 nm in diameter using conventional SEM and 47.3 nm in diameter using TEM. A methodology has been developed and employed in order to accurately image hydroxyapatite crystals in normal human teeth. The combination of an ultrathin coating of sputtered chromium (Cr) film and SE-I imaging herein provide topographic contrasts of crystal surfaces. Since the majority of the SE-I information should be produced close to the surface, the resultant image is specimen surface specific. Optimal metal coating thicknesses of low atomic number were utilized so that the generation of the SE-I signal contrast which occurs on the enamel crystal surface was used to form the image, thus avoiding image contrast contribution by electrons scattered in the metal coat.
Submicron linewidth measurement for VLSI
Authors: Bennett Marylyn Hoy , Lilley H. Shannon
Published: June 2020

For several years, SEM-based inspection and measurement has been a necessary tool for in-process IC characterization. SEM inspection phenomena are now well understood due to sample-beam interaction modeling, instrument calibration using a NBS standard, and years of experience in low voltage applications. However, the practice of submicron very large scale integration (VLSI) linewidth measurement has outdistanced understanding of the measurement phenomena. As the automated SEM-based instrument is the only available tool, we must improve its calibration techniques. The approach commonly taken in the semiconductor industry is to calibrate instrumentation using a homemade metal pitch "standard", then characterize the SEM and linewidth measurement data on various sample substrates in relation to the "standard". Accepted methods of statistical analyses can then be applied to the data to at least insure precision and repeatability. Accuracy, however, remains unknown. There are currently significant efforts underway in the areas of linewidth measurement modeling, and fabrication on a measurement standard. Until the completion of these projects, intermediate improvements may be available.
Distribution of c-protein isoforms in sarcomeres of developing chick skeletal muscle
Authors: Fischman D. A. , Dennis J. E. , Obinata T. , Takano-Ohmuro H.
Published: June 2020

C-protein is a 150 kDa protein found within the A bands of all vertebrate cross-striated muscles. By immunoelectron microscopy, it has been demonstrated that C-protein is distributed along a series of 7-9 transverse stripes in the medial, cross-bridge bearing zone of each A band. This zone is now termed the C-zone of the sarcomere. Interest in this protein has been sparked by its striking distribution in the sarcomere: the transverse repeat between C-protein stripes is 43 nm, almost exactly 3 times the 14.3 nm axial repeat of myosin cross-bridges along the thick filaments. The precise packing of C-protein in the thick filament is still unknown. It is the only sarcomeric protein which binds to both myosin and actin, and the actin-binding is Ca-sensitive. In cardiac and slow, but not fast, skeletal muscles C-protein is phosphorylated. Amino acid composition suggests a protein of little or no αhelical content. Variant forms (isoforms) of C-protein have been identified in cardiac, slow and embryonic muscles.
The Ultrastructure of Fibrin Prepared from Fibrinogen Haifa (γ275 Arg→His)
Authors: Siebenlist K.R. , Mosesson M.W. , Diorio J.P. , Tavori S. , Tatarsky I. , Rimon A.
Published: June 2020

Fibrinogen is composed of six polypeptide chains, 2 Aα, 2 Bβ and 2 γ chains, covalently linked at their amino termini by disulfide bonds. Fibrinogen is activated to α,β-fibrin by thrombin which cleaves 2 A (FPA) and 2B (FPB) fibrinopeptides from the N-termini. Release of FPA and FPB exposes'A'and'B' polymerization sites, respectively. The resulting fibrin self-associates into a branched 3-dimensional matrix. Two other types of fibrin can be formed in vitro: ct-fibrin [FPA release]and β-fibrin [FPB reelease). Fibrinogen Haifa is a congenital abnormal fibrinogen variant characterized by normal fibrinopeptide release, abnormal fibrin polymerization and replacement of γ275 Arg by His. The propositus has experienced thrombotic episodes. In this study the ultrastructure of fibrin formed from fibrinogen Haifa was compared to that of normal fibrin in order to better define the effect of the γ275 Arg → His substitution on its molecular function.
Ultrastructural Study of Peroxisome Proliferation in Hepatocytes by Quick-Freezing and Deep-Etching Method
Authors: Ohno S.
Published: June 2020

It had been emphasized that luminar continuities between sER and peroxisomes were detected by conventional electron microscopy. However, recent studies ruled out the luminar continuities between sER and peroxisomes. Lazarow et al. reported that peroxisomal proteins were synthesized on free ribosomes and postulated the existence of “peroxisomal reticulum” distinct from the ER. The object of this study is to clarify the proliteration mechanism of peroxisomes after administration of a peroxisome proliferator, DEHP (di-2- ethylhexyl phthalate).

Mice treated with 2% DEHP for 1, 3, 5 and 7 days and normal mice were perfused with 2% paraformaldehyde in 0.1M phosphate buffered solution, pH 7.4, (PB) for 5 min. The livers were cut into small pieces, washed in PB to remove cytoplasmic soluble proteins and were fixed again with 2% paraformaldehyde-0.25% glutaraldehyde for 30 min. They were quickly frozen in isopentane-propane mixture (around -190 C) and fractured in liquid nitrogen to remove the damaged surface tissues. They were deeply etched in Eiko FD-3S machine (-95°C, 2-6xl0-7 Torr) and rotary shadowed with platinum.
Freeze-Fracture Observations of Hamster Sperm-Egg Fusion
Authors: Clark Judy , Koehler James K.
Published: June 2020

A number of TEM and SEM studies have made use of the hamster in vitro fertilization system to observe the process of sperm-egg fusion. We have extended these studies to include observations of freeze fracture replicas at the site of the sperm-egg interactions prior to, during and immediately following their fusion.

Zona-free eggs were collected following superovulation of female hamsters according to the methods of Yanagimachi et al. Sperm were capacitated and prepared for incubation with eggs as in Clark et al. Eggs and sperm were co-incubated 5-15 minutes, washed and immediately fixed and prepared for freeze fracture replicas and thin sections as previously reported.

In order to investigate the role of lipid components during the fusion process, some preparations were fixed in the presence of filipin, which binds to β-OH sterols in membranes and forms complexes that are visible in freeze fracture replicas. Thin sections were also prepared and viewed to help confirm and support observations of the replicas.