bluuebunny/crossref_metadata_2025 huggingface dataset:
Freeze-fracture Transmission Electron Microscopy (FF/TEM) studies of systems containing self-associated structures
Authors: Burns Janet L. , Chestnut Matthew H. , Spontak Richard J.
Published: June 2020

Freeze-fracture transmission electron microscopy (FF/TEM) is a well-established and highly-valued technique, often employed in the study of biological systems which are extremely sensitive to structural alteration (e.g., membranes and tissues). The technique relies on rapid specimen cooling to immobilize detailed microstructure, usually in a hydrated environment, prior to fracture and subsequent surface replication. As Zasadzinski and Bailey point out, though, the principle governing this technique is general and can be applied with equal success to the study of “microstructured” or “complex” fluids, i.e., fluids consisting of self-organized supramolecular structures. In this vein, FF/TEM constitutes a powerful means of characterizing the structural attributes of dispersions, emulsions, gels, and liquid crystalline assemblies at relatively high spatial resolution. Such morphological information can prove valuable in the development of the structure-viscosity relationships needed in processing. Here, we demonstrate the utility of FF/TEM in elucidating the role of self-associated structures in three different systems: a chemical reaction environment, a high-internal-phase emulsion, and a nonaqueous gel.
“Grid-mapped” freeze-fracture: Three-Dimensional confocal laser scanning microscopy for directed fracturing and histological mapping of neurons in spinal cord and brain
Authors: Rash J. E. , Whalen L. R. , Guthrie P. B. , Morita M. , Dillman R. , Fay-Guthrie D.
Published: June 2020

A new correlative microscopic technique, “grid-mapped” freeze fracture, is introduced. This technique allows individual cells in histological slices to be freeze fractured, and their ultrastructural details to be correlated with conventional histological and gross anatomical features. Adult male rats were anesthetized, the sciatic nerve was exposed and crushed, and rhodaminefilled latex microspheres (Lumafluor, Inc) were injected at the crush site to label motor neurons. After 3-7 days, rats were fixed by whole-body perfusion. The brains and spinal cords were removed, embedded in 5% gelatin, and 50 or 100 μm thick slices were cut with a Vibratome. Slices were mapped in three-dimensions (Figs. 1-2 and 3-4) using a Molecular Dynamics Multiport 2001 confocal microscope, and the depths of selected cells were measured (±2μm) from the cut surfaces. After freezing on gold specimen supports, the fracture plane was directed through selected neurons using the precise planar microtome of the JEOL JFD-9000-CR freeze-fracture machine.
Enhanced axial resolution of the cytoskeleton in fluorescence microscopy by standing-wave excitation
Authors: Lanni Frederick , Bailey Brent , Farkas Daniel L. , Lansing Taylor D.
Published: June 2020

When the depth-of-field of a microscope is less than the axial dimension of the specimen, 3d information can be derived from a set of images recorded as the specimen is stepped through the object focal plane of the microscope. This procedure, known as optical sectioning microscopy (OSM), is the same in direct imaging and confocal scanning. For both of these cases in fluorescence microscopy, axial (depth) resolution is more limited than transverse resolution, for fundamental reasons. Our research aim has been to enhance axial resolution in fluorescence OSM (FOSM) while retaining the high-speed information transfer characteristics of direct imaging that are necessary for 3d studies of living cells in culture.

Standing-wave fluorescence microscopy (SWFM) is a direct imaging method in which the object is illuminated by a three-dimensional field of planar interference fringes (standing waves) oriented parallel to the focal plane of the microscope. This field is produced in the specimen by crossing two coherent, collimated, s-polarized beams of equal amplitude directed through the specimen at complementary angles (θ, π -θ) relative to the axis of the microscope.
Three-dimensional structure of dendritic spines, neuronal specializations that impart both stability and flexibility to synaptic function
Authors: Harris Kristen M.
Published: June 2020

Dendritic spines are the tiny protrusions that stud the surface of many neurons and they are the location of over 90% of all excitatory synapses that occur in the central nervous system. Their small size and variable shapes has in large part made detailed study of their structure refractory to conventional light microscopy and single section electron microscopy (EM). Yet their widespread occurrence and likely involvement in learning and memory has motivated extensive efforts to obtain quantitative descriptions of spines in both steady state and dynamic conditions. Since the seminal mathematical analyses of D’Arcy Thompson, the power of establishing quantitatively key parameters of structure has become recognized as a foundation of successful biological inquiry. For dendritic spines highly precise determinations of structure and its variation are proving themselves as the kingpin for establishing a valid concept of function. The recent conjunction of high quality information about the structure, function, and theoretical implications of dendritic spines has produced a flurry of new considerations of their role in synaptic transmission.
In situ cryofixation of rat spinal cord by a new propane jet freezing device
Authors: Greene William B.
Published: June 2020

Spinal cord trauma is a major health problem in terms of morbidity and mortality. It is clear that traumatic spinal cord injury is related to the death of a cord segment which results in the structural and functional disruption of key pathways vital to the maintenance of normal neurological function. The injured area of the cord eventually becomes a zone of segmental necrosis which, in the more severe cases, involves all but a thin rim of sub-pial white matter. The clinical consequences of segmental necrosis can be consistently reproduced in experimental animals by dropping weights onto the surgically exposed cord. At the moment of impact and during the primary injury phase, there are changes in the spinal cord which indicate that neural membranes are altered. The extent and reversibility of these alterations are being investigated for therapeutic treatment. Emphasis has been placed principally on secondary events leading to calcium toxicity. An ultra-rapid freezing technique evolved from the necessity of preserving diffusible ions in situ before and after spinal cord injury.
Cryo-electron microscopy of human erythrocyte membrane skeletons
Authors: Li Li , Josephs Robert
Published: June 2020

The membrane skeleton is a two-dimensional network of structural proteins attached to the cytoplasmic surface of the erythrocyte membrane. Spectrin, actin and band 4.1 are the major components in the skeleton network. It is generally believed that the membrane skeleton plays an important role in the deformability and stability of red cells. In this work we have used cryo-electron microscopy to examine the effect of ionic strength on skeleton size. Since the skeletons were unstained, unfixed and hydrated drying effects and the attendant changes in salt concentrations and pH associated with negative staining did not affect our results. The skeletons were embedded in a thin layer of vitreous ice supported by a fenestrated carbon film, which provided higher contrast than fenestrated carbon grids.

Ghosts derived from normal human red cells were incubated with 4 volumes of 2.5% Triton X-100 in 5 mM sodium phosphate (pH 7.4) for 1 hour at 4°C. The mixture was layered on top of a discontinuous density gradient of 10% and 60% sucrose. After centrifugation, the membrane skeletons were collected and dialyzed against 0.2 mM or 5 mM sodium phosphate buffer (pH 7.4) for 4-6 hours.
Exploration of cell wall architecture with the rapid-freeze deep-etch technique
Authors: Satiat-Jeunemaitre Béatrice , Hawes Chris
Published: June 2020

The comprehension of the molecular architecture of plant cell walls is one of the best examples in cell biology which illustrates how developments in microscopy have extended the frontiers of a topic. Indeed from the first electron microscope observation of cell walls it has become apparent that our understanding of wall structure has advanced hand in hand with improvements in the technology of specimen preparation for electron microscopy. Cell walls are sub-cellular compartments outside the peripheral plasma membrane, the construction of which depends on a complex cellular biosynthetic and secretory activity (1). They are composed of interwoven polymers, synthesised independently, which together perform a number of varied functions. Biochemical studies have provided us with much data on the varied molecular composition of plant cell walls. However, the detailed intermolecular relationships and the three dimensional arrangement of the polymers in situ remains a mystery. The difficulty in establishing a general molecular model for plant cell walls is also complicated by the vast diversity in wall composition among plant species.
Calcium ion imaging in plant cells
Authors: Hepler Peter K. , Callaham Dale A.
Published: June 2020

Calcium ions (Ca) participate in many signal transduction processes, and for that reason it is important to determine where these ions are located within the living cell, and when and to what extent they change their local concentration. Of the different Ca-specific indicators, the fluorescent dyes, developed by Grynkiewicz et al. (1), have proved most efficacious, however, their use on plants has met with several problems (2). First, the dyes as acetoxy-methyl esters are often cleaved by extracellular esterases in the plant cell wall, and thus they do not enter the cell. Second, if the dye crosses the plasma membrane it may continue into non-cytoplasmic membrane compartments. Third, even if cleaved by esterases in the cytoplasm, or introduced as the free acid into the cytoplasmic compartment, the dyes often become quickly sequestered into vacuoles and organelles, or extruded from the cell. Finally, the free acid form of the dye readily complexes with proteins reducing its ability to detect free calcium. All these problems lead to an erroneous measurement of calcium (2).
Correlative light and electron microscopy: Counting centrioles in individual sea urchin zygotes previously followed in vivo
Authors: Sluder Greenfield , Miller Frederick J.
Published: June 2020

Centrosomes are the ensembles of structures that define the poles of the mitotic apparatus. In animal cells, a centrosome typically consists of a pair of orthogonally arranged centrioles associated with osmiophilic pericentriolar material which nucleates the microtubules of the aster and central spindle. The precise doubling, or reproduction, of the centrosome during interphase is an important event in the cell’s preparations for mitosis.

To characterize the mechanisms that control centrosome reproduction we need to follow the behavior of centrosomes within individual cells over several cell cycles. We use sea urchin zygotes as an experimental system because they have a rapid cell cycle, are large enough (100 um diameter) to easily manipulate, and are optically clear. However, the experiments pose two challenges. The first is to follow the behavior of centrosomes within individual zygotes for several hours with the light microscope and then recover just those individuals from a large population for ultrastructural analysis.
New digital image processing technology for FSEM microscopy
Authors: Peters Klaus-Ruediger , Oho Eisaku
Published: June 2020

Digital image acquisition and processing can provide many advantages over conventional analog image information handling, i.e., undisturbed access to the “raw data set”, quantitative analysis of the image information, and reduced costs and increased flexibility of image data handling. However, it may principally change microscopy by providing a new facility for instant exhaustive data presentation in acquired images. Detail imaging is one of the basic microscopic tasks but visual access to detail information is cumbersome and often left to post-session data analysis. A dedicated software/hardware technique is now available for automatic “near-real-time” enhancement of image detail information visually not accessible in the “raw data” image. Pertinent image details include spatial dimensions of only a few pixels in size (spatial details) and intensity variations of only a few intensity steps in height (intensity details). While conventional image enhancement techniques often produce serious image artifacts which exclude a closer inspection of enhanced detail information, the new pixel-accurate processing (PAP) technology allows instant image evaluation at an accuracy-level of the raw data through detail enhancement in full-frame images, digital zoom and noise smoothing.
Atomic-force imaging of biological molecules using an enviromental cell with sample translation capability
Authors: Marchese-Ragona Silvio P. , Christie Briggs , Jobe Renee
Published: June 2020

Atomic force microscope images of well-characterized biological macromolecules always appear wider and flatter than expected. These dimensional artifacts can be explained in part by tip convolution and tip compression as well as the relative strengths of the tip-sample and tipsubstrate interactions. The effects of tip convolution and tip compression can be manipulated by knowing the tip geometry and applied force, however the tip-sample interaction strength is highly dependent on the environment in which the specimen is scanned. In order to manipulate the environmental conditions under which images are obtained we have constructed an enviromental cell (Fig. 1.) that allows us to simultaneously image molecules in the topographic and lateral force modes in almost any gas or liquid at ambient temperatures and pressures. The design of the enviromental cell allows the sample to be translated in the x and y directions, and also allows the sample to be viewed optically at magnifications up to 400 times.
A rapid silver-impregnation technique on ultra-thin sections for Electron Microscopy
Authors: Chien K. , Heusser R.C. , Jones M.L. , Van de Velde R.L.
Published: June 2020

Silver impregnation techniques have been used for the demonstration of the complex carbohydrates in electron microscopy. However, the silver stains were believed to be technically sensitive and time consumming to perform. Currently, due to the need to more specifically evaluate immune complex for localization in certain renal diseases, a simplified procedure in conjunction with the use of the microwave has been developed and applied to renal and other biopsies. The procedure is as follows:

Preparation of silver methenamine solution:

1. 15ml graduated, clear polystyrene centrifuge tube (Falcon, No. 2099) was rinsed once with distilled water.

2. 3% hexamethylene tetramine (methenamine) was added into the centrifuge tube to the 6ml mark.

3. 3% silver nitrate was added slowly to the methenamine to the 7ml mark while agitating. (Solution will instantly turn milky in color and then clear rapidly by mixing. No precipitate should be formed).

4. 2% sodium borate was added to the solution to the 8ml mark, mixed and centrifuged before use.
High-performance EM-002B optical column construction for future expansion
Authors: Shirota K. , Moriyama K. , Mikami S. , Ando A. , Nakamura O. , Yanaka T.
Published: June 2020

Since modern analytical transmission electron microscopes must have a wide range of illumination conditions (from “mm” to “nm” probe size), an additional lens (one of the condenser lenses, usually called the “mini-lens”) is arranged immediately above the objective lens pole pieces. As a result, it has become very difficult to install an exchange mechanism for the objective pole pieces, which used to be done routinely.

To overcome this, TOPCON Electron Microscope EM-OO2B incorporated a new mechanism which can be exchanged quite easily and reliably by the user. This mechanism makes a space to exchange pole pieces, without column disassembly, by precisely driven external mechanisms (Fig. 1). The time required for a typical user to carry out such exchange is usually 15 to 20 minutes, and it will take not more than two hours for high resolution image or analysis after exchange. This time is also shortened by the fact that an anti-contamination cold trap is not generally required in the case of EM-OO2B.
The new CM-Series TEMs: integration of a five-axis motorized, fully computer-controlled goniometer
Authors: de Jong Marc J.C. , Asselbergs P. Emile S.J. , Otten Max T.
Published: June 2020

A new step forward in Transmission Electron Microscopy has been made with the introduction of the CompuStage on the CM-series TEMs: CM120, CM200, CM200 FEG and CM300. This new goniometer has motorization on five axes (X, Y, Z, α, β), all under full computer control by a dedicated microprocessor that is in communication with the main CM processor. Positions on all five axes are read out directly - not via a system counting motor revolutions - thereby providing a high degree of accuracy. The CompuStage enters the octagonal block around the specimen through a single port, allowing the specimen stage to float freely in the vacuum between the objective-lens pole pieces, thereby improving vibration stability and freeing up one access port. Improvements in the mechanical design ensure higher stability with regard to vibration and drift. During stage movement the holder O-ring no longer slides, providing higher drift stability and positioning accuracy as well as better vacuum.
Development of an automated analyzer for TEM diffraction patterns
Authors: Jackson A. G. , Park J. , Wood D. , LeClair S.
Published: June 2020

An application of self-adaptive algorithms to TEM is presented. Analysis of diffraction patterns is an integral part of an experiment because of the need to control contrast conditions. Obtaining two-beam conditions or locating a zone axis can be time consuming or not possible, because of restrictions in the tilt ranges of the holder being used. The present approach is to manually search reciprocal space in the instrument to find zones of interest and that are accessible, visually identify the zone axis, or, analyze the patterns and plot on a stereogram the locations of major zones. Once the experimenter has plotted the orientation, then locating desired zone axes is accomplished by manually tilting. This process can be relatively easy, as in the case of a cubic crystal, or very difficult as in the the cases of hexagonal, trigonal, triclinic, monoclinic or orthorhombic crystals, particularly when the crystalline region is a few microns or less in size.
Three-dimensional light microscopy, optimized staining, and automated image analysis of cell nuclei in thick tissue slices
Authors: Lin W. , Holmes T. , Ancin H. , Roysam B. , Szarowski D.H. , Turner J.N.
Published: June 2020

The number, size, shape and three-dimensional (3D) distribution of cell nuclei in thick tissue samples is often critical to our understanding of tissue level organization and function. Examples include the analysis of neurons in brain tissue as a function of development, electrical activity, and exposure to toxins as well as the development of whole embryos. The sample is stained with a fluorescent dye selective for nucleic acid, and observed with a confocal light microscope. The classic Feulgen stain, although primarily an absorption stain, is often used for this purpose. However, it is unreliable for fluorescence due to the mixture of compounds usually present in Fuchsin. We have optimized a procedure using acriflavine as the Schiff reagent for staining thick (100 - 500 μm) brain slices. In addition, Ancin and Roysam have developed software for automatically analyzing 3D objects, which are classified by volume, shape, intensity and relative 3D spatial locations.
Cytochemical localization of glycogen phosphorylase activity in rat liver
Authors: Michaels John E. , Cardell Robert R.
Published: June 2020

Glycogen phosphorylase (GP) is a key enzyme in liver glycogen breakdown. GP activity is altered with its state of phosphorylation. In the current study, the intralobular distribution of GP activity was observed histochemically in frozen sections of rat liver during fasting and after stimulation of glycogen synthesis.

Normal and adrenalectomized (ADX) rats were fasted overnight to reduce liver glycogen to minimal levels. Fasted ADX rats received 2 mg dexamethasone (DEX) 0-8 h prior to sacrifice to stimulate glycogen synthesis. Liver was removed, rapidly frozen in isopentane cooled in liquid nitrogen, then cryostat sectioned. In order to determine sites of GP activity, sections were incubated in medium that contained glucose 1-phosphate as substrate. Under the incubation conditions used, GP synthesized glycogen as the reaction product. Glycogen was identified by two staining methods: 1) iodine staining has been shown to be rather specific for newly synthesized glycogen produced during the histochemical procedure (Figs.1-6), whereas 2) periodic acid-Schiff (PAS) stained both native and nascent glycogen.
Immunogold electron microscopic localization of phosphoenolpyruvate carboxykinase in rat liver
Authors: Gao Kuixiong , Morris Randal E. , Giffin Bruce F. , Cardell Emma Lou , Cardel Robert R.
Published: June 2020

Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-limiting gluconeogenic enzyme which catalyzes the conversion of oxaloacetate to phosphoenolpyruvate. A gradient of PEPCK from periportal hepatocytes to pericentral hepatocytes has been demonstrated in normal rat liver; however, the subcellular distribution of PEPCK molecules within hepatocytes could not be determined from these light microscope studies.

We have used two immunogold histochemical approaches to study the subcellar distribution of PEPCK in normal rat hepatocytes. In the first procedure 10μm cyrosections of 4% paraformaldehyde perfusion-fixed rat liver were collected on silanated slides and immunostained with goat anti-rat PEPCK (normal goat serum served as a control), and incubated in a secondary antibody (5nm gold-labeled rabbit anti-goat IgG) and a tertiary antibody (5nm gold-labeled goat anti-rabbit IgG). The immunogold stained samples, with or without silver enhancement (IGSS), were counterstained with pyronine Y, re-embedded in Visio- Bond and removed from the slides and semithin sectioned for epipolarized illumination combined with transmitted light microscopy (Figs. 1 and 2), or re-embedded in epoxy resin and sectioned for electron microscopy (EM, Fig. 4) In the second procedure 25, 50 and 100 μm free-floating vibratome sections of well fixed rat liver (4% paraformaldehyde plus 0.5% glutaraldehydeperfusion-fixed, immersion fixed overnight at 4°C) were immunogold stained using a 5nm gold labeled secondary antibody, with and without IGSS, osmicated, embedded in epoxy resin and sectioned for EM (Figure 3).
The catalytic activity of adenosine monophosphate deaminase in murine duodenum as revealed by transmission electron microscopy
Authors: Bielat K. , Tritsch G.
Published: June 2020

AMP deaminase was visualized in a manner analogous to our previous approach with adenosine deaminase. The chloro-analog of AMP, i.e., 6 Chloropurine riboside 5' monophosphate (CPRMP) (from Sigma) was shown to be a substrate of this enzyme which liberates Cl- which is precipitated with added Ag+, and, after exposure to light electron dense Ag° grains are deposited at loci of enzyme activity. The substrate at a concentration of 1.1 mM in 50 mM HEPES buffer, pH 7.2, in the presence of 150 mM K+ (as the acetate), 3mM ATP and 10¼M pentostathi (deoxycoformycin) was incubated with freshly excised tissue from a female C-57 BL/6 mouse. The substrate concentration is 1.4 times Km for rabbit muscle enzyme (Sigma), and K+ and ATP are allosteric activators of this enzyme. Because many cells have ecto 5'-nucleotidase activity, pentostathi was added to prevent manifestation of adenosine deaminase activity of dechlorination of any nucleoside that might be formed.
Pathology of the spermatic middle piece and infertility
Authors: Gallegos de L. G. , Ramírez B. E. , Gómez G. L. , Diaz G. M.
Published: June 2020

On the pathology of spermatozoa associated to infertility, it has been described a variety of alterations that include subtle axonemal defects such as lack of dineyn arms, and altered microtubular pattern (9+0). Also it has been reported acrosomal agenesy in round head sperms, and altered acrosomal modelation. All these alterations become responsible of sperm motility disarrangement that impair procreation. In these cases it is convenient the ultrasructural study with the purpose of a more correct and precise diagnosis and prognosis.

Thus, we studied the subcellular spermatic pathology in semen of infertile men obteined by masturbation. The specimens were proccesed for electron microscopy by the usual method, after washing in phosphate buffer. Thin sections were observed in Carl Zeiss EM 109 electron microscope.

In this series of 21 patients studied, 43% have showed alterations of the flagellar middle piece. At the optical microscope, the sperms showed short tail and were immotile or presented a big cytoplasmic drop and movement grade I or II (according to WHO classification).
Sulfhydryl bond formation is a prerequisite for proper cycling of centrosomes and chromosomes in mammalian tissue culture cells
Authors: Schatten Heide , Paweletz Neidhard , Balczon Ron
Published: June 2020

To study the role of sulfhydryl group formation during cell cycle progression, mammalian tissue culture cells (PTK2) were exposed to 100¼M 2-mercaptoethanol for 2 to 6 h during their exponential phase of growth. The effects of 2-mercaptoethanol on centrosomes, chromosomes, microtubules, membranes and intermediate filaments were analyzed by transmission electron microscopy (TEM) and by immunofluorescence microscopy (IFM) methods using a human autoimmune antibody directed against centrosomes (SPJ), and a mouse monoclonal antibody directed against tubulin (E7). Chromosomes were affected most by this treatment: premature chromosome condensation was detected in interphase nuclei, and the structure in mitotic chromosomes was altered compared to control cells. This would support previous findings in dividing sea urchin cells in which chromosomes are arrested at metaphase while the centrosome splitting cycle continues. It might also support findings that certairt-sulfhydryl-blocking agents block cyclin destruction. The organization of the microtubule network was scattered probably due to a looser organization of centrosomal material at the interphase centers and at the mitotic poles.
Light-microscopic identification of golgi staining patterns in embedded blocks of muscle tissue prior to sectioning for electron microscopy
Authors: Fujimaki Noboru , Peachey Lee D. , Ishikawa Harunori
Published: June 2020

The Golgi stain is very useful for selective staining of membrane systems in muscle cells, especially for HVEM and IVEM where thick sections can be examined and three-dimensional information obtained. However, the staining intensity and pattern are highly variable from preparation to preparation, among fibers in the same preparation, and within individual fibers. Staining can be absent altogether, or can involve the T-system (T) or the sarcoplasmic reticulum (SR) alone, or both T and SR. Considerable time can be spent sectioning blocks and scanning the sections in the electron microscope, searching for the desired staining pattern, sometimes without success. We have found that examination of the trimmed face of an embedded block under a dissecting microscope with oblique reflected light reveals a pattern of colors in muscle fibers and parts of muscle fibers. The origin of the colors and the reason that the different staining patterns show different colors are not clear. Nevertheless, the colors can be related to the staining pattern that will be seen in the electron microscope in sections cut from the same blocks, thus facilitating trimming of the block to areas of interest, both saving time and reducing the likelihood of missing rare staining patterns or paticularly interesting parts of the embedding.
Soybean chloroplast responses to enhanced ultraviolet irradiation
Authors: Crang Richard F.E. , Vassilyev Audrey E. , Miroslavov Yevgeney A.
Published: June 2020

Environmental concerns over the degradation of the earth’s stratospheric ozone layer have been expressed for the past decade in recognition that with ozone depletion, enhanced ultraviolet irradiation will be received at the earth's surface. Such increase in ultraviolet irradiation can be hypothetically determined by making appropriate computer calculations based on proposed cloud cover, season, latitude, elevation, and percent of stratospheric ozone depletion. We have proposed a 40% reduction in the ozone layer corresponding to a daily increase of 19.1 kJ in the limits of ultraviolet-B (UV-B) spectral irradiation (280-320 nm). This is within the range of realistic possibilities based on current estimated ozone depletion rates for the next 40-50 years. We wish to determine the extent to which chloroplasts are ultrastructurally altered compared with those from plants raised under ambient conditions lacking an UV-B irradiation component.

Uninoculated seeds of soybean (Glycine max), cv. “Forrest” were sown in standardized greenhouse soil in 4" clay pots, watered daily, and fertilized once per week.
Structure of seeds and developing seedlings of Hydrilla verticillata
Authors: Holmberg D. L. , Ryan F. J.
Published: June 2020

Until recently, it was believed that Hydrilla verticillata, a widespread aquatic weed, did not generally produce seeds. There were reports that the monoecious biotype from the U. S. was sometimes capable of producing viable seeds, although these were not described in any detail. Recent work at the USDA ARS Aquatic Weed Research Laboratory, Ft. Lauderdale, FL, identified a monoecious biotype of H. verticillata from Penang Island (Malaysia) that is self-fertile and reliably produces viable seed. This biotype may also be crossed with other biotypes including the female dioecious plant from Florida. This work was undertaken to describe the anatomical characteristics of seeds and developing seedlings, using light and electron microscopy.

Seeds and seedlings of the Penang Island biotype and Florida X Penang Island cross were processed for light and scanning electron microscopy (SEM). Fixed and fresh material was used. Seeds and seedlings were fixed in 2% paraformaldehyde and 0.1% gluteraldehyde in 0.1M phosphate buffer, pH 7.0. and dehydrated to 100% ethanol.
Use of low-voltage SEM (LVSEM) to evaluate cellular morphology and extracellular polysaccharide production by Pseudomonas fragi (ATCC 4973), Listeria monocytogenes (scott A), and Salmonella typhimurium
Authors: Schwach Tina , Zottola E. A.
Published: June 2020

LVSEM offers the microbiologist a unique view of the microbial cell surface. Colony morphology is used by microbiologists to distinguish between organisms. Some bacteria exhibit different colony morphologies when grown on solid media. P. fragi ATCC 4973 exhibits both rough (R) and smooth (S) colonies. It is not known why different morphologies form, but there is some speculation that their expression may be affected by nutrient conditions,other physical parameters, such as growth in a nonstatic environment, and may involve extracellular polysaccharide (EPS) formation. EPS has also been implicated in bacterial attachment to surfaces.

In this study, three organisms of importance to the food industry were grown in Trypticase Soy Broth (TSB) at 21C. Growth rates, EPS production, and cell surface morphologies were evaluated in both 3% TSB and 0.3% TSB. The lower concentration was used to simulate decreased availability of nutrients. Two glass chips (3mm × 6mm) were added to small petri dishes containing the culture medium and the organism.
Role of Kupffer cells in CCl4-induced hepatocytic toxicity: A preliminary study
Authors: Yamashiro S. , Bast T. , Towner R.A. , Janzen E.G. , Reinke L.A.
Published: June 2020

Carbon tetrachloride (CCl4) hepatotoxicity is one of the most frequently used experimental models to study the mechanism of cell damage by metabolites of xenobiotics (1,2). Cytochrome P-450 enzymes are considered to be involved in the metabolic activation of CCl4 to generate the trichloromethyl (.CCl3), which in turn may be converted to the peroxy radical (.00-CCl3) (3,4). In our previous studies, we have demonstrated CCl4-induced edematous response by using proton magnetic resonance imaging and electron microscopy (2,5). However, these studies have not given any consideration to Kupffer cells, which could act as an interphase between the sinusoid and hepatocytes.

Three male Wistar rats (200-250 g) were given two doses of CCL4 one hour apart (160 μl/kg bodyweight, with 5% Emulphor in 0.85% saline) intraperitoneally (i.p.). The same number of rats received phenyl tert-butyl nitrone (PBN), 25 mg/200 g in a 0.07 M pH 7.4 phosphate buffer and corn oil i.p. 30 min prior to the administration of CC14 as above.
Human, bovine, and pig teeth surface characterization of enamel and dentin: An SEM study
Authors: Fortin Daniel
Published: June 2020

Many researchers use extracted human teeth to evaluate the adhesive strength of restorative materials. Attachment to tooth hard tissues in the oral cavity is a complex and difficult problem that has challenged investigators. An important step in dealing with this problem is a thorough characterization of the substrate. Extracted human teeth are, however, becoming increasingly difficult to obtain due to recent progress in conservative dental treatment. Other mammalian teeth, such as bovine teeth have been tested. Although slightly different in composition and structure, bovine teeth have been used as substitute for human teeth and their use has been documented in different papers. While using such teeth does not lead to results that are identical with those obtained when the enamel and dentin of human teeth is used, they do produce results that are comparable and certainly useful in evaluating the influence of various methods of enamel and dentin treatment.

In order to find a substitute for human teeth in laboratory studies, surface characterization of enamel and dentin from bovine and pig teeth were compared using the SEM to human teeth.
Rapidly developing tissue undergoing dramatic decrease in size
Authors: Lindley V. , Pfeiler E.
Published: June 2020

The purpose of this study was to look at rapidly developing tissues in a system that is undergoing a dramatic decrease in size. Metamorphosing bonefish (Albula sp.) larvae (leptocephali) undergo major changes in morphology and biochemical composition in a period of less than two weeks. Total body length decreases by 60-65%, losing approximately 80% of the total body mass, an average loss of 3.5 um/min. Biochemical changes include about a 50% decrease in total lipid and about an 80% decrease in total carbohydrate. Metamorphosing larvae do not begin feeding until late in the process; nutritional requirements are probably provided by breakdown and metabolism of endogenous lipid and carbohydrate. Light microscopy (LM) and transmission electron microscopy (TEM) were performed on the fore-, mid- and hindgut (and adjacent tissues) of early, intermediate and advanced metamorphosing larvae. Changes in ultrastructure correlate well with the biochemical and physiological data. The most dramatic changes are seen in the gut.
Electron Microscopic study of inner ear of guinea pig with special reference to morphological changes in vestibular macula in hypercholesterolemia
Authors: Qiao Yan , Asano Goro , Kashiwado Izumi , Hattori Yasuo
Published: June 2020

Vertigo may be induced by various factors such as vascular changes including arteriosclerosis and hypercholesterolemia in the inner ear. In orter to clarify the mechanism of vertigo, the inner ear of guinea pig in hypercholesterolemia condition was investigated by electron microscopic techniques.

The guinea pigs have served as experimental materials. They were divided into two groups: one received non-administration of cholesterol and the other received the administration of cholesterol. The vestibular macula in inner ear were observed at 1, 2 and 3 months after the administration of cholesterol and also vascular function was investigated by using horseradish peroxidase as tracer.

Ultrastructural changes such as the depletion of microvilli on the cell surface and increased microvesicles were detected in the cytoplasm of sensory cell after cholesterol administration. After two months, lipid droplets were demonstrated in the cytoplasm, associated with intercellular edema. The cytoplasmic projection with vacuolization was demonstrated in the surface of sensory cells in the vestibular macula after three months of cholesterol administration.
Ultrastructural changes in the cochlea of the inner ear in guinea pigs with hypercholesterolemia
Authors: Kashiwado Izumi , Yuge Kurata , Hattori Yasuo , Qiao Yan , Asano Goro
Published: June 2020

Although sensory neural hearing loss stemming from hypercholesterolemia has received attention in recent year, nothing has been made known about inner ear injuries resulting from this condition.

In this study, we prepared guinea pig models with hypercholesterolemia and observed inner ear with a transmission electron microscope, and also investigated the effect of vitamin E therapy on injuries.

Materials and Methods: Ninetyth white female Hartley guinea pigs weighing 250-300g each were divided into three groups. Group A consisted of the control animals, which were fed by a stock diet. Group B was fed by a hyperlipid diet (2.5% cholesterol, 0.25% bile acid, and 7.5% cattle fat). Group C was given a vitamin Eadded hyperlipid diet. All animals were sacrificed at either one, two or three months after treatment, and cochlear duct vasculature was observed with a transmission electron microscope. Cholesterol, triglyceride, and TBARS values were concurrently measured in serum.

Results: In group B and C, cholesterol and triglyceride levels remainedon control levels. The TBARS level was elevated in group B but remained unchanged in groups A and C.
Thickness measurements by Electron Energy Loss Spectroscopy (EELS)
Authors: Ho Ruoya , Zhao Lijie , Wang Yun-Yu , Shao Zhifeng , Somlyo Andrew P.
Published: June 2020

An estimate of specimen mass-thickness is an essential requirement for evaluate with EELS the absolute elemental concentration in biological specimens. The conventional method used for measuring specimen thickness by EELS is:

where t is the specimen thickness, λi is the total inelastic mean free path, It is the total count in an EELS spectrum and I0 is the count in the zero loss peak. This equation is rigorously correct, only if the electrons are collected over all scattering angles and the spectrum covers all energy losses. But in most experiments with a finite energy loss region, because of the limited collection semi-angle, we can only collect a fraction of scattered electrons. Omitting the high loss electrons will result in a cut-off error that is usually less than 5%, if we use an energy window from 0 eV to 150 eV or above. But the effect of the limited semi-angle is more serious. Fig. 1 shows the ln(It/I0) measured on the same specimen in both TEM and STEM mode at 80 keV with a magnetic sector spectrometer equipped with a parallel detector on Philips 400 FEG.
TEM and AEM study of polycrystalline CdS/CdTe heterostructures for photovoltaic applications
Authors: Jones K.M. , Al-Jassim M.M. , Birkmire R.W. , McCandless B.E.
Published: June 2020

CdTe-based solar cells are attracting increasing attention due to their potential for lowcost, large-area high-efficiency photovoltaic applications. Recently, conversion efficiencies exceeding 15% have been reproducibly demonstrated for small area devices. However, the morphology, microstructure and interface properties have not been studied in a systematic manner. Additionally, these structures are normally subjected to at least one heat treatment step prior to device fabrication. The effects of such heat treatment on the microstructure and the CdS/CdTe interface are not well understood. In this work TEM, AEM and secondary ion mass spectrometry (SIMS) were used to study the morphology, microstructure and the CdS/CdTe interface as a function of deposition parameters and post deposition heat treatment. The solar cells used have the following structure:

The CdS and CdTe films were grown by physical vapor deposition. The thickness of these films is 0.5μm for CdS and 2-3μm for CdTe. In some cases, the CdS was heat treated at 400°C for 20-30 min prior to the deposition of CdTe. After the deposition of the latter, the samples were heat treated in the presence of CdCl2 at 400°C for 20-30 min in air.
Computer-aided analytic and visualization tools for atructural studies
Authors: Carragher Bridget
Published: June 2020

Structural biologists typically acquire data in the form of a two-dimensional image (or set of images) from which the three-dimensional structure of the object of interest must be inferred. Examples can be found over a range of sizes spanning many orders of magnitude, and covering structures from the macroscopic to the atomic scale. A correspondingly wide range of different instruments is used in the collection of this data, from CT/MRI scanners, through light and electron microscopes, and recently, atomic force instruments. The images which are collected from these instruments may be in the form of a series of 2D slices through the 3D data set (and these may be either physical sections or optical sections) or a series of to mographic 2D projections of the 3D dataset. In either case it is highly likely that computer software tools will be used on the data set eitheras an aid in the qualitative interpretation of the structure or as a means of extracting quantitative morphological measurements.
Scanned tip measurement of deep vertical facets on a flat surface: Measuring roughness on gaas laser waveguide mirrors
Authors: Shen Chang , Fraundorf Phil , Harrick Robert W.
Published: June 2020

Monolithic integration of optoelectronic integrated circuits (OEIC) requires high quantity etched laser facets which prevent the developing of more-highly-integrated OEIC's. The causes of facet roughness are not well understood, and improvement of facet quality is hampered by the difficulty in measuring the surface roughness. There are several approaches to examining facet roughness qualitatively, such as scanning force microscopy (SFM), scanning tunneling microscopy (STM) and scanning electron microscopy (SEM). The challenge here is to allow more straightforward monitoring of deep vertical etched facets, without the need to cleave out test samples. In this presentation, we show air based STM and SFM images of vertical dry-etched laser facets, and discuss the image acquisition and roughness measurement processes. Our technique does not require precision cleaving. We use a traditional tip instead of the T shape tip used elsewhere to preventing “shower curtain” profiling of the sidewall. We tilt the sample about 30 to 50 degrees to avoid the curtain effect.
Architecture/environments for image processing: Hardware and software for the microscopist
Authors: Bright David S.
Published: June 2020

Digital images are large arrays of data with array elements all treated in a similar way. “Architecures for image processing” immediately suggests parallel processors because of the enormous increase in speed. These processors enhance images in real time - TV rates - but this is less of a consideration where image collection takes more than a few seconds. Further, these machines are expensive and programming is difficult. For example, when the image is divided among processors as in Fig. 1 and 2, problems arise at the edges of the pieces. Issues such as cost, flexibility, ease of use and data integrity are often of more interest to the microscopist.

Desktop computers present an ideal environment for off line processing of images with a few thousand pixels on an edge. These computers are adequately fast, they have sufficient storage and communication capability, and the software is less expensive than equivalent software for workstations or larger computers.
Three dimensional deblurring of transmitted-light brightfield micrographs
Authors: Bhattacharyya Santosh
Published: June 2020

Three dimensional microscopic structures play an important role in the understanding of various biological and physiological phenomena. Structural details of neurons, such as the density, caliber and volumes of dendrites, are important in understanding physiological and pathological functioning of nervous systems. Even so, many of the widely used stains in biology and neurophysiology are absorbing stains, such as horseradish peroxidase (HRP), and yet most of the iterative, constrained 3D optical image reconstruction research has concentrated on fluorescence microscopy. It is clear that iterative, constrained 3D image reconstruction methodologies are needed for transmitted light brightfield (TLB) imaging as well. One of the difficulties in doing so, in the past, has been in determining the point spread function of the system.

We have been developing several variations of iterative, constrained image reconstruction algorithms for TLB imaging. Some of our early testing with one of them was reported previously. These algorithms are based on a linearized model of TLB imaging.
Some problems and solutions in electron energy loss spectroscopy of cryosections
Authors: Shao Zhifeng , Ho Ruoya , Somlyo Andrew P.
Published: June 2020

Electron energy loss spectroscopy (EELS) has been a powerful tool for high resolution studies of elemental distribution, as well as electronic structure, in thin samples. Its foundation for biological research has been laid out nearly two decades ago, and in the subsequent years it has been subjected to rigorous, but by no means extensive research. In particular, some problems unique to EELS of biological samples, have not been fully resolved. In this article we present a brief summary of recent methodological developments, related to biological applications of EELS, in our laboratory. The main purpose of this work was to maximize the signal to noise ratio (S/N) for trace elemental analysis at a minimum dose, in order to reduce the electron dose and/or time required for the acquisition of high resolution elemental maps of radiation sensitive biological materials.

Based on the simple assumption of Poisson distribution of independently scattered electrons, it had been generally assumed that the optimum specimen thickness, at which the S/N is a maximum, must be the total inelastic mean free path of the beam electron in the sample.
Determination of nucleic acid content of viruses by electron spectroscopic imaging and correlation averaging
Authors: Misra M. , Conway J. F. , Trus B. L. , Steven A. C.
Published: June 2020

Energy filtering offers the potential to form element-specific images, and thus to quantitate and map various elements in biological specimens. To assess the sensitivity and reproducibility with which we can detect phosphorus using current technology, we have performed ESI experiments in a model system comprising three well-characterized viruses - tobacco mosaic virus (TMV), LA virus of yeast, and bacteriophage T4 - that differ widely in their contents and spatial distributions of nucleic acid. Here we make a preliminary report of experiments in which the data were recorded in a Zeiss EM902 electron microscope equipped with a Henry-Castaing energy filter.

An aliquot of the viral mixture was adsorbed to a thin, grid-mounted, carbon film, blotted to a thin film, rapidly frozen in liquid ethane, and transferred into the microscope in the frozen-hydrated state, where it was freeze-dried at 100°K. A SIT camera was used to record images at energy losses of 105, 120 and 150 eV respectively, with an energy window of 10eV.