bluuebunny/crossref_metadata_2025 huggingface dataset:
Low-energy point-reflection EM
Authors: Spence J. C. H. , Zhang X. , Zuo J. M. , Weierstall U. , Munro E. , Rouse J.
Published: June 2020

The limited penetration of the low-voltage point-projection microscope (PPM) may be avoided by using the reflection geometry to image clean surfaces in ultra-high vacuum. Figure 1 shows the geometry we are using for experimental point-reflection (PRM) imaging. A nanotip field-emitter at about 100 - 1000 volts is placed above a grounded atomically flat crystalline substrate, which acts as a mirror and anode. Since most of the potential is dropped very close to the tip, trajectories are reasonably straight if the sample is in the far-field of the tip. A resolution of 10 nm is sought initially. The specular divergent RHEED beam then defines a virtual source S' below the surface, resulting in an equivalent arrangement to PPM (or defocused CBED). Shadow images of surface asperities are then expected on the distant detector, out of focus by the tip-to-sample distance. These images can be interpreted as in-line electron holograms and so reconstructed (see X. Zhang et al, these proceedings). Optical analog experiments confirm the absence of foreshortening when the detector is parallel to the surface.
Reconstruction of electron holograms recorded in a non-FEG, non-biprism TEM
Authors: Wang Z.L.
Published: June 2020

An experimental technique for performing electron holography using a non-FEG, non-biprism transmission electron microscope (TEM) has been introduced by Ru et al. A double stacked specimens, one being a single crystal foil and the other the specimen, are loaded in the normal specimen position in TEM. The single crystal, which is placed onto the specimen, is responsible to produce two beams that are equivalent to two virtual coherent sources illuminating the specimen beneath, thus, permitting electron holography of the specimen. In this paper, the imaging theory of this technique is described. Procedures are introduced for digitally reconstructing the holograms.
An experimental measurement on the mechanism of charge balance in the environmental SEM
Authors: Phifer D. W. , Joy D. C.
Published: June 2020

Increasingly, electron microscopes are being used to look at non-conducting samples. These samples are prone to buildup of surface charge that must be drained away to prevent distortion of the image (due to the large potential buildup) and damage to the sample surface (due to dielectric breakdown). In conventional high vacuum scanning electron microscopes, the microscopist control beam energy to produce an acceptable yield of electrons while minimizing sample charging on non-conducting samples. This charge balance is achieved by reducing incident beam voltage to produce detectable interactions without excessive charge buildup on the sample surface. Electron yields at the low voltages required are sufficient for imaging when using a field emission gun, but it is not, in general, possible to perform microanalysis because the x ray lines of interest cannot be excited. The environmental scanning electron microscope (ESEM) has demonstrated the ability to overcome this problem by placing the sample in a low vacuum environment.
Size distribution standards measured by digital SEM imaging
Authors: Kelly James F.
Published: June 2020

The National Institute of Standards and Technology (NIST) certifies several size and size distribution Standard Reference Materials (SRMs) using spherical particles ranging in diameter from submicron to several millimeters. Glass beads are used as SRMs for a variety of laboratory applications. Their broad size distribution and spherical shape make them a model material for calibration of instruments for powder measurements.

The glass bead standard (SRM1017b) covering the diameter range from 50μm to 350μm is currently being certified using SEM backscatter electron imaging. The digital images are acquired using an AMRAY SEM connected to a Apple Power Macintosh via a 4pi digital interface. The public domain computer program, NIH Image, is used in the image acquisition and size analysis. Figure 1 shows the spherical nature of the glass beads. Measurement of the circular area of each particle permits the calculation of the particle volume and diameter. The system magnification in terms of pixels per micrometer using an optical stage micrometer calibrated at NIST by laser interferometry.
A security circuit for the image intensifier on the Electron Microscope
Authors: Shih Min-Yi , Hsu Tung
Published: June 2020

Image intensifiers are widely used on the electron microscopes, mostly for observation and recording of weak images. Usually, the image intensifier is not used in diffraction mode, for the strong direct beam may damage the YAG crystal. However, neither the microscope nor the image intensifier is equipped with any protection mechanism, leaving the safety of the intensifier as the responsibility of the operator.

While modifying a JEOL JEM-100C microscope for UHV-REM, we designed a security circuit to blank the electron beam in order not to damage the image intensifier accidentally. The required logic, shown in Fig. 1, indicates the electron beam blanked (1) or unblanked (0) under all possible combinations of three parameters. The requirement is that the beam must be blanked (1) while the view screen is fully raised (1), film not advanced (0), and the SAD button is depressed (1). Blanking is accomplished with a 5V signal applied to a relay and short the deflection coil. The circuit is shown in Fig. 2.
Exploring local properties of biological surfaces with the Atomic Force Microscope
Authors: Hoh Jan H.
Published: June 2020

The atomic force microscope (AFM) is developing into a very general tool for exploring biological surfaces. At present there are four broad areas of applications for the AFM: imaging surfaces, manipulating surfaces, measuring intermolecular forces and determining micromechanical properties. Because the dimensions of the apex of the tip used are typically on the order of 10's of nanometers, local measurements of properties are possible on a much smaller length scale than previously. Several reviews that provide further details on AFM instrumentation and biological applications have been published. These reviews also provide specific references for the range of applications that will be reviewed in this tutorial.

AFM imaging modes can be divided into two groups based on the detection method, the DC method detects the absolute deflections of a cantilever while the AC method detects the RMS amplitude of an oscillating cantilever. Conventional contact AFM, sometimes called repulsive mode, is based on DC detection of the cantilever movement as the tip moves across a surface, usually in direct contact with the surface. The deflection of the cantilever is used to control the height of the sample, and the force applied to the sample, through a feedback loop.
Image processing and analysis fundamentals for microscopy
Authors: Newell Bruce D.
Published: June 2020

Advances in computers and related digital hardware, coupled with sophisticated software techniques have resulted in microscopy migrating from its historical roots as a subjective, qualitative science towards a more robust position as a truly quantitative technique. Granted, we will probably never totally remove the microscopist from the process of image interpretation (at least those at this conference hope not) but we will certainly continue to progress from describing our image data in qualitative terms (e.g., many/few, large/small, equiaxis/elongated, ordered/random, rough/smooth) to quantitative measurements of number, size, shape, location, texture, and so on.

To move along the path toward quantitative image interpretations requires an understanding of image processing and analysis (IP/A) fundamentals to insure that the data obtained is of the required accuracy and precision. A generalized model of the critical steps in the image processing and analysis chain is given in Figure 1. This tutorial will examine the fundamental issues in each step that impact the quality of the final result and provide a broad overview of techniques that may be applicable.
Correlative microscopy in distrubuted (client/server) computing environments: tools for catalyzing the rapid dissemination of information about the cell biology of the human prostate
Authors: Hakam A. , Gau J.T. , Grove M.L. , Evans B.A. , Shuman M. , Strom S.C. , Bahnson R. , Sethi P.K. , Song K.C. , DiGiorgio C.J. , He F. , Hatton C.S. , Wimberly F.C. , Roskies R.Z. , Deckenbaugh L. , Gross W. , Schubert E. , Becich M.J.
Published: June 2020

Prostate adenocarcinoma is the most common malignant tumor of men in the United States and is the third leading cause of death in men. Despite attempts at early detection, there will be 244,000 new cases and 44,000 deaths from the disease in the United States in 1995. Therapeutic progress against this disease is hindered by an incomplete understanding of prostate epithelial cell biology, the availability of human tissues for in vitro experimentation, slow dissemination of information between prostate cancer research teams and the increasing pressure to “ stretch” research dollars at the same time staff reductions are occurring.

To meet these challenges, we have used the correlative microscopy (CM) and client/server (C/S) computing to increase productivity while decreasing costs. Critical elements of our program are as follows:

1) Establishing the Western Pennsylvania Genitourinary (GU) Tissue Bank which includes >100 prostates from patients with prostate adenocarcinoma as well as >20 normal prostates from transplant organ donors.
Correlative immunolocalization with high-resolution light microscopy and Electron Microscopy using London Resin Gold embedded tissue
Authors: Grove M. L. , Evans B. A. , Misra D. N. , Zhao J. , Alpers D. H. , Becich M. J.
Published: June 2020

Immunoelectron microscopy specimens are often embedded in hydrophilic resins, like London Resin Gold(LRG), which permit antibody staining without etching (or deplasticizing) the sections. This characteristic of hydrophilic resins also allows for immunohistochemistry at the light level on semi thin sections (0.5μm - 1μm). The ability to do immunohistochemistry at both the light and electron microscopic level on the same tissue block allows focused ultrastructural study. Immunohistochemistry on semi-thin sections displays cellular localization of macromolecules, permitting more specificity in the selection of areas for studying intracellular localization ultrastructurally. We have developed a method for immunoperoxidase staining of LRG embedded tissues, utilizing anti-human polyclonal antibodies directed against intrinsic factor (Fig. 1). Intrinsic factor (IF), a cobalamin binding protein, is known to be produced in the stomach, pancreas and salivary glands of most mammals. We are interested in distribution of IF in gastric epithelium, small intestine (ileum) and supporting tissues in both gastrointestinal tract sites. Previously, we have described the cellular localization of IF in human and rat tissues.
Three Dimensional imaging of biological macromolecules
Authors: Koster A.J. , Walz J. , Typke D. , Nitsch M. , Baumeister W.
Published: June 2020

3D imaging of both cellular structures as well as molecular assemblies of biological molecules has become an increasingly useful tool to study structure-function relationships of biological systems. In this paper instrumental and methodological developments are discussed towards automated 3D imaging, which will be illustrated by examples of structures studied in Martinsried. To image individual structures with dimensions in the range of 10-500 nm with a resolution of 1-5 nm, electron tomography is the only technique available. The strategy of choice depends on size and shape of the structure to be reconstructed. Single-tilt axis tomography is suitable for the reconstruction of unique structures (for example, irregularly shaped viruses or cellular structures). Random conical-tilt data collection, as well as angular reconstitution techniques, can be used to reconstruct the average structure of many copies of a particle, such as those present in suspension of one kind of protein. To reconstruct a unique structure with single-tilt axis tomography the tilt range and tilt increments are chosen to meet the resolution desired within the constraint of the allowable electron doses (Table 1).
The functional organization of RNAs in the mammalian cell nucleus
Authors: Huang S. , Spector D.L.
Published: June 2020

Several models have been proposed for the functional organization of RNA within the eukaryotic nucleus and for the relationship of this organization to the distribution of pre-mRNA splicing factors. One model suggests that RNAs which must be spliced are capable of recruiting splicing factors to the sites of transcription from storage and/or reassembly sites. In order to further evaluate this model we have transiently transfected HeLa cells with constructs which express RNA transcripts containing introns, lacking introns, or containing an intron with a deletion at the 3' splice site. The expression of RNAs was detected by in situ hybridization and their association with splicing factors was evaluated by immunostaining using specific antibodies (Y12, SC35) in the same cells. We have found that the majority of the RNA transcripts produced from constructs which express intron-containing genes such as β-globin, tropomyosin, and HIV tat are associated with splicing factors. In contrast, RNAs lacking introns, such as βgalactosidase, and adenovirus VAI, are not associated with splicing factors in the nucleus.
High-resolution nucleic acid sequence mapping via in situ hybridization at the Electron Microscope level
Authors: Hamkalo B.A. , Narayanswami S. , Kausch A.P.
Published: June 2020

The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the relative positions of sequences on very small chromosomes may not be feasible.
Domain organization of allele-specific DNA replication within the GABAAreceptor gene cluster
Authors: LaSalle Janine M. , Lalande Marc
Published: June 2020

Parental imprinting is a gamete-specific modification that distinguishes the paternal and maternal chromosomes in higher eukaryotes, resulting in allele-specific changes in chromatin organization, transcription and replication. One example of parental imprinting in humans is revealed by two distinct genetic diseases, Prader-Willi syndrome (PWS) and Angelman syndrome (AS) which both map to chromosome 15q11-13. PWS is caused by the absence of a paternal contribution to 15q11-13, while AS results from the lack of a maternal copy of the region. Within this chromosomal subregion lies a cluster of GABAAreceptor β3 and α5 subunit genes (GABRB3andGABRA5) which are separated by about 100 kb and arranged in opposite transcritional orientations (Figure 1). Allele-specific asynchronous DNA replication has previously been found to be associated with imprinted chromosomal regions.In order to further study the association between DNA replication and imprinting, allele-specific replication was assayed by fluorescence in situ hybridization (FISH). Biotin-labeled phage probes detected by FITC hybridized to each chromosome as either a singlet (unreplicated state) or a doublet (replicated state). Cells demonstrating asynchronous replication (one singlet and one doublet) for each probe are shown in Figure 2.
Organization of transcription and pre-mRNA splicing within the mammalian cell nucleus
Authors: Spector D.L. , Huang S. , Kaurin S.
Published: June 2020

We have been interested in the organization of RNA polymerase II transcription and pre-mRNA splicing within the cell nucleus. Several models have been proposed for the functional organization of RNA within the eukaryotic nucleus and for the relationship of this organization to the distribution of pre-mRNA splicing factors. One model suggests that RNAs which must be spliced are capable of recruiting splicing factors to the sites of transcription from storage and/or reassembly sites. When one examines the organization of splicing factors in the nucleus in comparison to the sites of chromatin it is clear that splicing factors are not localized in coincidence with heterochromatin (Fig. 1). Instead, they are distributed in a speckled pattern which is composed of both perichromatin fibrils and interchromatin granule clusters. The perichromatin fibrils are distributed on the periphery of heterochromatin and on the periphery of interchromatin granule clusters as well as being diffusely distributed throughout the nucleoplasm. These nuclear regions have been previously shown to represent initial sites of incorporation of 3H-uridine.
Nucleoskeltal proteins with bifunctional roles in the nucleus and mitotic apparatus: a paradigm for protein dynamics in the cell cycle
Authors: Brinkley B. R. , He D. , Zeng C. , Scott B. , Turner D.
Published: June 2020

The eukaryotic cell nucleus, once believed to contain structureless nucleoplasm surrounding chromatin and the nucleolus is now thought to contain an extensive nucleoskeletal matrix on which chromatin, RNP and a complex array of transcription factors, hormone receptors and other regulatory factors are spatially arranged. A distinct nucleoskeleton has been difficult to identify in the intact nucleus, due in part to masking by a dense array of chromatin fibers. However, if chromatin is extracted by nuclease digestion and high salt, an underlying anastomosing network of 9-13 nm core filaments can be demonstrated. The nucleoskeleton resembles the cytoplasmic intermediate filament complex but is confined entirely within the nucleus where it connects the nuclear lamina with various nuclear organelles, forming an integral lattice of fibers collectively called the nuclear matrix. Although the molecular composition of the nucleoskeleton remains elusive, its integrity apparently requires RNP and a growing list of nuclear matrix proteins.

Although relatively insoluble within the nuclear compartment, the entire nucleoskeletal framework and associated chromatin is efficiently dismantled, packaged, partitioned and reassembled into daughter nuclei during mitosis.
Growth cone dynamics of embryonic grasshopper pioneer neurons in situ
Authors: O'Connor Tim P.
Published: June 2020

During development of the nervous system, neurons extend axons over relatively long distances to contact their targets. A variety of molecules in the extracellular environment are instrumental in guiding a neuronal process. The motile tip of the process, the growth cone, senses and transduces this guidance information, resulting in a local reorganization and consolidation of the cytoskeleton. Although much work has been dedicated to isolating the molecules that guide a neuronal growth cone, relatively little is known about the dynamic processes that occur when a growth cone turns in response to guidance information. Recently, a number of biological systems have been developed that enable time lapse imaging of growth cones as they extend axons in situ. One of these systems is the embryonic grasshopper limb fillet.

In the grasshopper embryo, a pair of sibling neurons, named the Til pioneers, are the first neurons to extend axons toward the central nervous system (CNS).
Optical imaging of brain slice preparations using voltage sensitive dyes
Authors: Nelson D.A. , Katz L.C.
Published: June 2020

Fast responding, biological membrane-soluble voltage sensitive dyes are probes which make it possible to directly monitor the electrical activity of neuronal tissues with both high temporal and high spatial resolution. High temporal resolution is desirable because of the millisecond time scale on which neuronal events occur. High spatial resolution is desirable to better reveal the complex interactions between different regions of neural circuits.

Taking full advantage of the promise of voltage sensitive dyes can be both difficult and expensive. Tissues stained with these dyes undergo very small (often less than one part in a thousand) relative changes in fluorescence in response to changes in transmembrane potential. The signal-to-noise ratio is similarly small for these responses. Finally, the responses are very fast. Most major components of the signal rise to their maximum and then disappear within 15 milliseconds of stimulation (figure 1).

Because standard video cameras are inadequate for recording the very small and very fast optical signals transduced by voltage sensitive dyes, specialized detection equipment is required. This entails a high cost per pixel, often limiting the practial number of detector elements to a few hundred or even less.
Difference fourier maps reveal structural changes for intermediates in the bacteriorhodopsin photocycle
Authors: Glaeser R. M. , Han B.-G. , Hendrickson F. M. , Vonck J.
Published: June 2020

Bacteriorhodopsin (bR) is a light-driven proton pump that exists in the cell membrane of halobacteria. Proton pumping across the membrane is achieved through a photochemical cycle that involves several discrete structural intermediates. These intermediates are characterized by discrete changes in the visible and the resonance Raman spectra of the chromophore (a retinal group linked to the protein by a Schiff base) and in the infrared spectrum of the protein itself. Protein structural changes are thought to change access to the protonated Schiff base via inward and outward facing aqueous channels, respectively, during different stages of the photocycle, thus allowing for vectorial proton movement across the cell membrane. A schematic model of the functional changes in structure that occur throughout the photocycle is given by the cartoon shown in Figure 1.

We have recently begun electron diffraction studies on the intermediates in the bR photocycle, with the goal of using difference Fourier maps to see directly the structural changes that correspond to the previously observed spectroscopic changes.
Assembly of VP26 in herpes simplex virus-1 capsid implicated by 3-D structure of recombinant capsid
Authors: Zhou Z. Hong , He Jing , Jakana Joanita , Tatman J. D. , Rixon Frazer J. , Chiu Wah
Published: June 2020

Herpes simplex virus-1 (HSV-1) is a ubiquitous virus which is implicated in diseases ranging from self-curing cold sores to life-threatening infections. The 2500 Å diameter herpes virion is composed of a glycoprotein spike containing, lipid envelope, enclosing a protein layer (the tegument) in which is embedded the capsid (which contains the dsDNA genome). The B-, and A- and C-capsids, representing different morphogenetic stages in HSV-1 infected cells, are composed of 7, and 5 structural proteins respectively. The three capsid types are organized in similar T=16 icosahedral shells with 12 pentons, 150 hexons, and 320 connecting triplexes. Our previous 3D structure study at 26 Å revealed domain features of all these structural components and suggested probable locations for the outer shell proteins, VP5, VP26, VP19c and VP23. VP5 makes up most of both pentons and hexons. VP26 appeared to bind to the VP5 subunit in hexon but not to that in penton.
Multiple isomorphous replacement for phase determination in protein crystals
Authors: Burmester Christoph , Holmes Kenneth C. , Schröder Rasmus R.
Published: June 2020

Electron crystallography of 2D protein crystals can yield models with atomic resolution by taking Fourier amplitudes from electron diffraction and phase information from processed images. Imaging at atomic resolution is more difficult than the recording of corresponding high resolution electron diffraction patterns. Therefore attempts have been made to retrieve phase information from diffraction from heavy atom labelled protein crystals. The expected differences between native and labelled crystals are small, therefore a high experimental accuracy is necessary. This is achieved by the use of energy filter TEM and image plates, as dicussed in. Here we present electron diffraction data obtained from frozen hydrated 3D protein crystals with an energy filter microspcope and a specially designed image plate scanner. Data were recorded for the native crystal as well as for two different heavy atom derivatives. Differences between the native and the derivate forms can be detected and are significant.

Electron diffraction patterns from frozen hydrated catalase crystals were recorded on an EFTEM Zeiss 912 Ω (120kV, zero loss mode, energy width ΔE=10eV, electron dose 5 e-/A2) using image plates and a quasi confocal scanner readout.
Aldehyde gold clusters for molecular labeling
Authors: Hainfeld James F. , Furuya Frederic R.
Published: June 2020

Glutaraldehyde is a useful tissue and molecular fixing reagents. The aldehyde moiety reacts mainly with primary amino groups to form a Schiff's base, which is reversible but reasonably stable at pH 7; a stable covalent bond may be formed by reduction with, e.g., sodium cyanoborohydride (Fig. 1). The bifunctional glutaraldehyde, (CHO-(CH2)3-CHO), successfully stabilizes protein molecules due to generally plentiful amines on their surface; bovine serum albumin has 60; 59 lysines + 1 α-amino. With some enzymes, catalytic activity after fixing is preserved; with respect to antigens, glutaraldehyde treatment can compromise their recognition by antibodies in some cases. Complicating the chemistry somewhat are the reported side reactions, where glutaraldehyde reacts with other amino acid side chains, cysteine, histidine, and tyrosine. It has also been reported that glutaraldehyde can polymerize in aqueous solution. Newer crosslinkers have been found that are more specific for the amino group, such as the N-hydroxysuccinimide esters, and are commonly preferred for forming conjugates. However, most of these linkers hydrolyze in solution, so that the activity is lost over several hours, whereas the aldehyde group is stable in solution, and may have an advantage of overall efficiency.
The effect of dates on the ultrastructure of hepatocytes of mice inoculated with Cerastes Cerastes venom
Authors: Al-Sallal A. , Amr Z. , Al-Bagdadi F.
Published: June 2020

Upon fractionation of Cerastes Cerastes venom it was found to contain L-aminoacidoxidase, phosphodiesterase, hyaluronidase and phospholipase A. The pathological effects of snake venom on the kidney, muscle tissues, liver, spleen and lymph nodes5 were investigated. In this study we report the effects of dates on the ultrastructural morphology of hepatocytes of mice envenomized intraperitoneally with Cerastes Cerastes venom.

The venom used in this study was collected from Cerastes Cerastes snake, which were usually found in Wadi Araba, south of Jordan. The extracted venom was lyophilized and kept at -20 degrees centigrade. Three white albino mice were starved for 60 hours and then fed dates. Then after twelve hours the mice were injected intraperitoneally with a lethal dose of Cerastes Cerastes. After 42 hours the mice were still alive. Control mice were not starved nor fed with dates,they were also envenomized intraperitoneally with lethal dose of Cerastes Cerastes venom, and the liver of all mice was dissected immediately after death.
Ultrastructural analysis of the physical interactions that occur between Baccillus SP. and polyurethane during biodegradation
Authors: Norton W.N. , Howard G. , Blake R.
Published: June 2020

The scientific community is confronted with a number of significant environmental issues, including the dilemma of how to maintain, safely and effectively, the enormous quantity of organic and inorganic hazardous wastes that are produced in the U.S. alone. Substantial interest in the concept of bioremediation, the use of microorganisms to accelerate the degradation of environmental contaminants, has been generated during the past decade. Bacillus sp., has demonstrated an ability to degrade water dispersible polyurethane, a molecule normally exceedingly difficult for microbial organisms to metabolize. The primary objective of this investigation is to obtain basic ultrastructural information on the physical nature of polyurethane biodegradation conducted by Bacillus.

A bacterial suspension of l×l09 cells was placed in one liter of a stock solution of polyurethane (3mg polyurethane/L distilled water). A comparable number of cells was maintained in 1 L of growth media and served as a control. At 2, 8, and 24 h subsequent to the initial exposure, samples were obtained from the experimental and control flasks and processed for phase-contrast microscopy, TEM and SEM.
Ultra-spatially resolved synchrotron powered FT-IR microspectroscopy of individual cells of biological material
Authors: Wetzel David L. , Reffner John A. , Williams Gwyn P.
Published: June 2020

Synchrotron radiation is 100 to 1000 times brighter than a thermal source such as a globar. It is not accompanied with thermal noise and it is highly directional and nondivergent. For these reasons, it is well suited for ultra-spatially resolved FT-IR microspectroscopy. In efforts to attain good spatial resolution in FT-IR microspectroscopy with a thermal source, a considerable fraction of the infrared beam focused onto the specimen is lost when projected remote apertures are used to achieve a small spot size. This is the case because of divergence in the beam from that source. Also the brightness is limited and it is necessary to compromise on the signal-to-noise or to expect a long acquisition time from coadding many scans. A synchrotron powered FT-IR Microspectrometer does not suffer from this effect. Since most of the unaperatured beam’s energy makes it through even a 12 × 12 μm aperture, that is a starting place for aperture dimension reduction.
High-resolution chemical mapping using tapping mode AFM with phase contrast
Authors: Chernoff Donald A.
Published: June 2020

Introduction. A challenging problem in materials analysis is imaging the microstructure of composite surfaces. Some examples of composite surfaces are: contamination deposits, discontinuous (i.e. defective) thin films, devices built of composite materials, and cross-sectional specimens of composite materials. Techniques such as XPS and SIMS can provide detailed information about the chemical species present on the surface, but only average information is obtained for areas smaller than a few (μm wide. Auger can have sub-micron spatial resolution, but it provides little useful information about organic materials and it is difficult to use on insulating surfaces. In contrast, we have developed a new method for imaging chemical domains with spatial resolution of 10-20 nm. In principle, this method may be used on any surface.

The TappingMode™ AFM1 uses an oscillating cantilever whose motion can be characterized by its amplitude and phase relative to its driver. In its most common mode of operation, this AFM uses the amplitude signal to sense the specimen surface, control the Z piezo and create a height image.
High-resolution SEM of clostridial attachment structures
Authors: Panessa-Warren B. , Tortora G.T. , Warren J.B.
Published: June 2020

Clostridium sporogenes forms small (1 μm), dehydrated endospores that are resistant to the effects of solvents, acids, alkali, pressure, UV radiation and most sporocides. In the past it was felt that the exosporial membrane served no function in the infection and colonization process, however our recent work on intact spores has demonstrated that the exosporial membrane seems to play a significant role in the initial attachment of the spore to a nutritive substrate. With the improved resolution of the SEM, it has been possible to examine intact spores attached to various substrates and characterize each stage of attachment. This paper examines the exosporial membranes of activated C.sporogenes spores using conventional specimen preparation with imaging using a lanthanum hexaboride gun, and specimens imaged with a field emission SEM. Spores were grown on Trypticase Soy agar with 0.5% glucose (TSA) added and blood CDC (BCDC) agar and attachments and viability monitored by plate counts and light microscopy of unfixed spores (both phase and malachite green/safranin staining).
Modulated polarization microscopy displays the dynamics of cytoskeletal structures in living, motile cells
Authors: Kuhn J. R. , Poenie M.
Published: June 2020

Cell shape and movement are controlled by elements of the cytoskeleton including actin filaments an microtubules. Unfortunately, it is difficult to visualize the cytoskeleton in living cells and hence follow it dynamics. Immunofluorescence and ultrastructural studies of fixed cells while providing clear images of the cytoskeleton, give only a static picture of this dynamic structure. Microinjection of fluorescently Is beled cytoskeletal proteins has proved useful as a way to follow some cytoskeletal events, but long terry studies are generally limited by the bleaching of fluorophores and presence of unassembled monomers.

Polarization microscopy has the potential for visualizing the cytoskeleton. Although at present, it ha mainly been used for visualizing the mitotic spindle. Polarization microscopy is attractive in that it pro vides a way to selectively image structures such as cytoskeletal filaments that are birefringent. By combing ing standard polarization microscopy with video enhancement techniques it has been possible to image single filaments. In this case, however, filament intensity depends on the orientation of the polarizer and analyzer with respect to the specimen.
Ultrastructure of the basal cells of the uterine glands of mares infused with povidone iodine solution
Authors: Al-Bagdadi F. , Richardson G. , Eilts B. , McCoy D. , Braun W. , Archbald L. , Olsen L. , Thompson D. , Titkemeyer C.
Published: June 2020

Gentamicin and povidone iodine infusion solutions have been used separately, to treat endometritis in mares. The effect of povidone iodine on the histology of the endometrium has been reported. This study describes the morphology of the fine structure of the endometrial epithelium and its response to povidone iodine infusion with the emphasis on the basal cell of the uterine gland.

Ten light horse mares of breeding age were selected and divided into two groups. Five mares were used as controls and five mares were infused with 1 % povidone iodine solution. Estrous of the mares was controlled with two doses of prostaglandin, 11 days apart, so that all the mares were in the first 3 days of estrus at the beginning of the experimental period (day 0). Uterine biopsies were obtained on days 0, 3, 5, 7, 10, 15, 20, and 30 of the experimental period. The biopsies were obtained from the endometrium at the junction of the uterine body and the uterine horn using alternate sides for subsequent biopsies.
Calcitonin effects on myoblasts: an ultrastructural study
Authors: González Santander R. , Toledo Lobo M.V. , Martínez Alonso F.J. , Martínez Cuadrado G. , González-Santander Martínez M. , Monteagudo M.
Published: June 2020

Several studies have provided detailed descriptions about the effects of calcitonin (CT) on different cell types such as osteoclasts, multinucleate giant cells and trophoblastic cells. CT main role is associated with the maintenance of peripheral Ca2+homeostasis and it may also modulate free intracellular Ca2+. Myoblasts are particularly sensitive to changes in extra and intracellular Ca2+concentration, so we have studied CT effects on myotome myoblasts differentiation.

Chick embryos from 56-59 h. of incubation (Hamburger and Hamilton stages 16-17) were injected with 0,25 IU of CT dissolved in 125 ul of 0,9 % sterile saline solution (CALSYNAR-50, from Rorer Labs.- synthetic lyophilized salmon CT made by Armour Pharmaceutical Co., Kankakee, Illinois, USA). CT was injected into the ammniotic cavity through a window in the egg shell. Afterwards, the window was closed and the eggs returned into the incubator, until embryos had incubated for 103 h., to reach H.H. stage 24. Some embryos were only injected with saline solution (controls).
DNA levels in somatic and germ line cells of a freshwater crustacean, Mesocyclops edax
Authors: Rasch E. M. , Wyngaard G.A.
Published: June 2020

Chromatin diminution--the fragmentation and elimination of chromosome Regions--provides an unusual opportunity to study genomic reorganization during development. Some species of copepods regularly excise and discard large amounts of nuclear DNA from presumptive somatic cell lines during early cleavage stages . To study this phenomenon in M. edax we determined DNA-Feulgen levels for more than 5,600 individual nuclei from squash preparations of 30 female and 25 male adults collected from lakes in Nova Scotia, Virginia and Florida. Fixation in 3:1 methanol/acetic acid was followed by squashing individual specimens in 45% acetic acid, freezing each slide in liquid N2 and thawing in absolute ethanol before air drying. Each series of slides was stained with the feulgen reaction for DNA and measured with a Vickers M86 scanning and integrating microdensitometer at 560 nm, using chicken RBC nuclei as an internal reference standard of 2.5 pg DNA per cell. This allowed us to ask several questions: are there differences in genome size (1) among specimens from different collecting localities, (2) between females and males at any single locality, and (3) between cells of germ line and somatic cell lineages?
The geometry of the EJSR Z-rete in avian cardiac muscle
Authors: Sommer J.R. , High T. , Taylor I.
Published: June 2020

The junctional SRs (JSR) in skeletal and cardiac muscle in general, including extended junctional SR (EJSR) in avian hearts, are morphologic and functional homologies as shown by biochemical and morphometric evidence, including the recent demonstration that EJSR contains ryanodine receptors and binds [3H]-Ryanodine. Bird hearts have two aberrant membrane features: 1. absence of transverse tu-bules (TT) and, 2. extension of JSRs (thus: E-JSR) from peripheral couplings into the interior of the myocytes forming EJSR Z-retes that both surround and pervade Z-discs. Absence of TT calls for an alternative mechanism for global muscle activation in bird hearts for which EJSR may provide the anatomical substratum, a possibility supported by both experimental evidence, and recent theoretical considerations. Propagated calcium-induced calcium release (CICR) has been difficult to reconcile with the graded response of cardiac muscle to stimulation.2,cf.5Propagation of an action potential along the Z-retes can be excluded. In the absence of TT, saltatory CICR as an alternative to propagated cardiac activation, is made plausible by the existence of EJSR which, (a) is demonstrably calcium-sensitive, (b) is well within < than 0.5μm of the regulatory proteins of cardiac myocytes and, (c) carries rows of proven, vicinal calcium release channels.
An SEM study of raphide crystal initials in the leaves of vitis (grape)
Authors: Arnott H. J. , Webb M. A. , Lopez L. E.
Published: June 2020

Many papers have been published on the structure of calcium oxalate crystals in plants, however, few deal with the early development of crystals. Large numbers of idioblastic calcium oxalate crystal cells are found in the leaves of Vitis mustangensis, V. labrusca and V. vulpina. A crystal idioblast, or raphide cell, will produce 150-300 needle-like calcium oxalate crystals within a central vacuole. Each raphide crystal is autonomous, having been produced in a separate membrane-defined crystal chamber; the idioblast''s crystal complement is collectively embedded in a water soluble glycoprotein matrix which fills the vacuole. The crystals are twins, each having a pointed and a bidentate end (Fig 1); when mature they are about 0.5-1.2 μn in diameter and 30-70 μm in length. Crystal bundles, i.e., crystals and their matrix, can be isolated from leaves using 100% ETOH. If the bundles are treated with H2O the matrix surrounding the crystals rapidly disperses.
Wheat necrosis induced by Pyrenophora tritici-repentis toxin
Authors: Freeman Thomas , Rasmussen Jack , Francl Leonard , Meinhardt Steve
Published: June 2020

Tan spot is a serious foliar disease of wheat caused by Pyrenophora tritici-repentis. This fungal pathogen produces a proteinaceous phytotoxin that induces necrosis in susceptible but not resistant cultivars. Larez et al. reported generalized membrane breakdown and cell death in advance of fungal spread, an effect presumably due to excreted fungal products, including the necrosis toxin. The direct ultrastructural effects of necrosis toxin isolated from P. tritici-repentis have not been previously examined.

Crude extract from a culture of isolate 86-124 (1) was thawed and centrifuged for 20 min at 35O00g. The supernatant was filtered through a 0.45 μm Whatman nylon filter and placed in dialysis tubing with a 3,500 molecular weight cut off. The crude extract was dialyzed overnight against 10 volumes of 20 mM sodium acetate buffer, pH 4.8. The dialyzed extract was loaded onto a fresh 10 ml S-sepharose Fast Flow column (Pharmacia) previously equilibrated with 20 mM sodium acetate.
Amelanotic malignant melanoma: recognizing aberrant premelanosomes
Authors: Bovard Karen K. , Bewtra Chhanda
Published: June 2020

The key to diagnosing cases of melanoma, a tumor of melanocytes, is to recognize cytoplasmic pigment called melanin. In most cases this can be done at the light microscopy level; however, in the rarer amelanotic (non-pigmented) melanomas, melanin is not present in detectable amounts. Thus it is necessary to do an ultxastructural investigation to detect melanosomes, the specific organelles of the melanocyte that synthesize melanin. Other non-melanoma cells can also possess melanin by internalization of melanin granules by phagocytosis or passive transfer (e.g. macrophages, keratinocytes) but they lack the premelanosome organelle, the precursor to the fully developed melanosome. In premelanosomes, the internal structures are not obscured by melanin. Thus it is the diagnostic feature of choice for the pathologist. In malignant melanoma, aberrant forms of premelanosomes and melanosomes are more typical than in normal melanocytes., These aberrant forms may not be well recognized. Amelanotic melanomas give us a good opportunity to study these structures. In studying premelanosomes,the investigator must be aware of variations of size, shape, appearance in different planes of sections, and knowledge of the four stages of normal melanosome development (melanogenesis).
The removal and morphology of intact biofilms from implanted venous access devices
Authors: Gregory M.A. , Hadley G.P.
Published: June 2020

The insertion of implanted venous access systems for children undergoing prolonged courses of chemotherapy has become a common procedure in pediatric surgical oncology. While not permanently implanted, the devices are expected to remain functional until cure of the primary disease is assured. Despite careful patient selection and standardised insertion and access techniques, some devices fail. The most commonly encountered problems are colonisation of the device with bacteria and catheter occlusion. Both of these difficulties relate to the development of a biofilm within the port and catheter. The morphology and evolution of biofilms in indwelling vascular catheters is the subject of ongoing investigation. To date, however, such investigations have been confined to the examination of fragments of biofilm scraped or sonicated from sections of catheter. This report describes a novel method for the extraction of intact biofilms from indwelling catheters.

15 children with Wilm’s tumour and who had received venous implants were studied. Catheters were removed because of infection (n=6) or electively at the end of chemotherapy.
Immunohistochemical localization of prolactin receptor in human digestive tissues
Authors: García-Caballero T. , Gallego R. , Fraga M. , Pintos E. , Beiras A.
Published: June 2020

Prolactin (PRL) is primarily recognized for its lactogenic effect. However, it is known that this polypeptide hormone exerts a great variety of biological functions acting on reproduction, osmoregulation, growth, metabolism, immunomodulation and even on behavior. The actions of PRL initiate with hormone binding to a specific cell surface receptor that belongs to the cytokine/GH/PRL receptor family or the hematopoietin receptor family The PRLR is widely distributed in numerous tissues. The aim of the present work was to investigate by immunohistochemistry the cellular distribution of PRLR in the human gastrointestinal tract and associated glands (liver and pancreas).

Samples of normal esophagus, stomach, small and large intestine, liver and pancreas were obtained from surgical pieces or recent autopsies. These samples were immersion-fixed in 10% buffered formalin for 24 hr, dehydrated and embedded in paraffin routinely. B6.2 anti-PRLR and avidin-biotin-peroxidase complex (ABC) procedure were employed. The B6.2 mouse monoclonal antibody (prepared against a membrane enriched fraction of human metastatic breast cancer), was generously provided by Dr. Barbara K. Vonderhaar and used at a dilution of 1:250, for 1 hr.
Sequential staining and de-staining of a single section by a panel of fluorescently-tagged lectins
Authors: Michael Stanley C. , Phillips Thomas E.
Published: June 2020

Numerous previous studies have used fluorescently tagged lectins to demonstrate the mucin glycoprotein secretory product of intestinal goblet cells are heterogeneous in respect to their lectin binding sites . Lectins stain the mucin secretory product in partially overlapping subpopulations of goblet cells. There are several difficulties in using a panel of fluorescently tagged lectins to determine the overlapping sites of binding. First, steric hindrances resulting from the binding of the first lectin compete with the subsequent binding of other lectins to the same or adjacent sites. Secondly, the number of fluorescent tags that can be practically differentiated by routinely configured microscopes is generally limited to two or three. As an alternative approach, we investigated whether 8 M urea could remove previous bound lectins without damaging the carbohydrate binding sites. Microwave treatment with 8 M urea has previously been used to recover antibody binding sites masked by fixation and embedding.

Small pieces of distal colon were removed from anesthetized rats and fixed for 2 hours at room temperature in 2% freshly-depolymerized paraformaldehyde in 70 mM NaCl, 30 mM HEPES, 2 mM CaCl2, pH 7.4.