bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunogold Labeling for Igg, Immunoglobulin Light Chains and P-Component in Fibrillary and Immunotactoid Glomerular Nephritis
Authors: Hearn S , Walton J , Moussa M , Rieckenberg Mark , Hutcheson K
Published: July 2020

Fibrillary and immunotactoid glomerulonephritis (FGN & IGN) are uncommon glomerular diseases, diagnosed by electron microscopic identification of fibril deposits which are negative by the Congo red stain. Micro-fibrils are similar to normal structures in glomerular basement membrane and matrix and they can be missed by EM. Pathogenesis of these glomerulopathies is unclear but they are not believed to be associated with monoclonal gammopathies as in AL amyloidosis. Immunofluorescense (IF) on FGN indicates the fibrils typically are positive for IgG and both immunoglobulin light chains while IGN fibrils are light chain monoclonal. We did immunogold labeling on four cases of FIGN in to improve the diagnosis of these diseases and investigate fibril protein composition.

Renal biopsies from four patients consisted of one IGN and 3 FGN. Tests included frozen section IF (IgG, IgA, IgM, C3), paraffin section histology (Congo Red) and conventional EM (osmium fixation and epoxy embedding).
Predictive Value of P53-Positive Cells for Esophageal Cancer Micrometastasis
Authors: Bailey GW , McKernan S , Price RL , Walck SD , Charest P-M , Gauvin R , Wei C , Mao Y , Krasna M
Published: July 2020

Esophageal cancer is the fastest growing malignancy of any tumor in the United States with an increased incidence of adenocarcinoma across all socioeconomic boundaries. The stage and the residual tumor classification are the most important prognostic factors. However, patients with similar stage of disease show a marked difference in survival. Therefore, there are needs to identify new molecular prognostic markers that may help to better assess the survival probability.

The recently introduced genetic diagnosis of cancer micrometastasis is quite attractive because of its high detection sensitivity. Not infrequently, however, there are marked discrepancies between genetic and conventional histologic diagnoses, especially concerning lymph nodes from carcinoma patients. Alterations in the p53 tumor suppressor gene are the most frequent genetic changes found in breast and lung cancer. The p53 gene functions as a negative regulator of cell growth. Alterations in the gene lead to loss of its usual negative growth regulation and more rapid cell proliferation.
Increase Transforming Growth Factor-Beta and its Receptors in Human Renal Tissue with Rejection
Authors: Song H , Papadimitriou J , Drachenberg C , Weir M R , Wei C
Published: July 2020

Transforming growth factor-bate (TGF-β) is a growth-relating peptide that has been shown to enhance collagen production in vivo and in vitro. TGF-β isoforms include TGF-β 1, β2 and β3. TGF-β receptors subtypes include type I (TGFβRI) and type II (TGFβRII) receptors. Overproduction of TGF-β can lead to real damage.

Renal graft rejection is major problem after kidney transplantation with severe renal damage. We hypothesized that renal tissue level of TGF-β may increase in renal rejection after kidney transplantation. Therefore, the current study was designed to determine the TGF-β 1 isoform and its receptor TGFβRI in human renal tissue with rejection by immunohistochemical staining (IHCS) and compared with normal human renal tissue. The results of IHCS was evaluated by IHCS staining density scores (0, no staining; 1, minimal staining; 2, mild staining; 3, moderate staining; and 4, strong staining). The positive staining area (+%) in entire section was also determined. The sections treated with preabsorbed blocking peptide or nonimmune rabbit serum demonstrated no immunoperoxidase activity.
Increasing Nitric Oxide Synthase in Human Renal Graft Rejection after Kidney Transplantation
Authors: Seta K , Papadimitriou J , Drachenberg C , Weir M R , Wei C
Published: July 2020

Nitric oxide (NO) is a potent endothelial-drived vasorelaxing factor and may involved in salt sensitive hypertension and renal damage. Nitric oxide is produced from L-arginine by activation of nitric oxide synthase (NOS). Three NOS has been identified which are brain NOS (bNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). On the other hand, renal graft rejection is major problem after kidney transplantation with severe renal damage and renal vasoconstriction. While NOS has been reported localized in human and animal kidney, the status of three NOS in human renal tissue with rejection remains poorly defined. Therefore, the present study was designed to determine the expression of bNOS, iNOS and eNOS by immunohistochemical staining (IHCS) in human renal tissue with rejection and compared with that in normal human renal tissue.

Human renal biopsy (n=5) were obtained after kidney transplantation with mild and moderate renal rejection. Normal kidney biopsy was obtained during nephrectomy.
The Effects of Cyclosporin-A on Human Vascular Smooth Muscle Cells Apoptosis
Authors: Kinjo M , McLaughlin J S , Wei. C
Published: July 2020

Cyclosporine-A is the one of the most effective immunosuppressant drug in organ transplantation. However, cyclosporine-A has a potent cytotoxic effect. Cellular apoptosis is being suggested may contribute to the cyclosporine A-mediated cytotoxic action. To date, regarding the effects of cyclosporine-A on apoptosis and apoptosis-related genes expression in vascular smooth muscle cells remain unclear. Therefore, the current study was designed to investigate the actions of cyclosporine-A on apoptosis and p53 expression in human vascular smooth muscle cells.

Human saphenous vein was obtained from coronary artery bypass surgery (n=5). After saphenous vein removed, the tissue was minced and incubated in the special tissue culture system for 24 hours in the absence or presence of cyclosporine-A (10-6M). To detect the DNA fragmentation, in situ terminal deoxymucleotidyl transferase dUTP nick end labeling (TUNEL) was performed. The p53 level in human vascular smooth muscle cells was determined by immunohistochemical staining (IHCS). An average of 1000 nuclei was analyzed for both TUNEL and p53 EHCS studies.
Renal Manifestations of Hepatitis C Virus Infection: Extremity at a Distance
Authors: Howell D N , Miller S E
Published: July 2020

Hepatitis C virus (HCV) is an RNA virus of the family Flaviviridae. In the decade since its identification,1 it has emerged as a leading cause of chronic hepatitis and cirrhosis. Easily transmissible by parenteral and sexual routes, fastidious in culture, highly limited in host range, and difficult to detect by most microscopic and immunohistochemical methods, HCV qualifies as an extreme pathogen. Another form of extremity exhibited by HCV is the bewildering array of renal disorders that occur in the setting of HCV-induced hepatitis.

Several different mechanisms may underlie the development of renal disease in patients infected with HCV. Immune complexes of anti-HCV antibodies and viral components, frequently bound to anti-immunoglobulin rheumatoid factors, can form in the circulation and be deposited in the kidney. HCV has also been shown to induce immune responses to a variety of autoantigens, some of which are expressed in the kidney. Hepatic injury caused by HCV may produce downstream effects on other organs, including the kidney.
Multiple Microscopic Approaches to the Study of Oral Bacterial Biofilms.
Authors: Palmer Robert J , Kazmerzak Karen , Wu Rosemary , Kolenbrander Paul E
Published: July 2020

Biofilms are collections of microorganisms at interfaces. Because of the high proportion of interfacial regions in the natural environment, biofilms are likely to be the repository for most of the world's microbial biomass. The close proximity of organisms within the biofilm presents the opportunity for intra- and interspecies signaling on a physical level through direct contact of organisms with one another, and on a metabolic level through production/excretion of intermediary molecules of metabolism and of specific cell signaling molecules. One of the best studied biofilms is dental plaque, which has been investigated from a microscopy standpoint since the time of van Loewenhouk and from a classical bacteriological standpoint since the relatively recent (20th century) development of anaerobic culture techniques. Through the even more recent (the last decade) application of molecular methods, it has been shown that perhaps half of all bacteria present in plaque are culturable and, at present, this consists of some 500 species.
Role of Extremophyles in Solar Salt Production
Authors: Davis J S
Published: July 2020

One-third of the worldwide sodium chloride (salt) production (about 200 million tons per year) is manufactured in solar saltworks. A solar saltworks is a series of one or more interconnected, shallow concentrating ponds (40 to 70 cm deep) through which seawater flows, evaporates by wind and sun, and deposits sodium chloride on the floors of crystallizer ponds (30 cm to 50 cm deep). In the downstream flow, salts of low solubility (in respect to sodium chloride) precipitate, with calcium carbonate first dropping out near three times, and calcium sulfate (gypsum) first appearing near four times seawater salinity. Shortly before reaching saturation with sodium chloride (about S.G. 1.214), the brine flows into crystallizer ponds (crystallizers), where evaporation continues until 5 to 20 cm of sodium chloride is deposited on the floors. The supernatant liquid, rich in compounds of calcium, magnesium, chloride, and sulfate, is removed, and the salt is harvested, washed, stockpiled for a time to decrease contaminants, and marketed.
A SEM Study of Epilithic and Endolithic Algae and Cyanophytes in Coquina Beach Rock of North Central Florida
Authors: Love N C , Kelley K L , Davis J S
Published: July 2020

The eastern coast of north central Florida is home to a habitat unique in the southeastern United States. Some areas of the north to north-central Florida beaches consist of outcroppings of coquina, a beach rock formed by shell fragments and sand held together by a calcareous cement. The major constituents of coquina are shell fragments of the surf clam Donax variabilis, while fragments of Area sp. and Dinocardium sp. are also generally present. These same shell fragments are also found loose comprising most of the beach “sand” in this area. The coquina serves as a substrate for a variety of attatched seaweeds and fouling organisms. Microalgae and cyanophytes of interest to this study can be classified as either epilithic or endolithic. Epilithic microorganisms inhabit the surface, using the rock primarily as a site of attatchment. The endolithic microorganisms are those algae and cyanophytes that actively bore into the coquina rock's shell fragments.
Impact of Cryo Techniques on Cytological Studies of Plant Pathogenic Fungi and their Hosts
Authors: Howard R J , Bourett T M , Duncan K E
Published: July 2020

Cryo techniques of specimen preparation have become the standard for cytological studies of biological specimens. From the pioneering work of Feder and Sidman, and Zalokar, the now ubiquitous application of freeze substitution for transmission electron microscope studies of fungi was a direct result of work reported in 1979. Since then, cryopreparative methods have also become the standard for SEM studies and may, for certain purposes, replace conventional methods of fixation for light microscopy as well. Undoubtedly, there are instances where non-cryo methods might be preferred, or where comparisons of results using cryo vs. chemical fixations can provide unique information. On the whole, however, the advantages offered to mycologists and plant pathologists by cryo fixation over any chemical methods are many, and include (a) the opportunity to preserve specimens in a non-aqueous environment, (b) preservation of labile structures such as certain organelles, extracellular matrices, or various cellular content such as ions or that of vacuoles, (c) preservation of cells in a more life-like state,
Ultrastructure of Conidia of the Plant Pathogenic Fungus Entomosporium Mespili
Authors: Richardson Elizabeth A , Mims Charles W
Published: July 2020

Entomosporium mespili has emerged as a significant pathogen of red tip (Photinia × fraseri), a popular and widely grown ornamental in the southeastern United States. This fungal pathogen produces its distinctive multi-celled, insect-like asexual spores or conidia (Fig. 1) in structures known as acervuli (Fig. 2) that rupture the surfaces of infected leaves. This study examines the fine structure of these conidia using a combination of scanning and transmission electron microscopy. In the case of transmission electron microscopy, conidia were prepared for study using either plunge freezing or high pressure freezing followed by freeze substitution.

Each mature conidium of E. mespili consists of four to six cells (Fig. 1). These include one apical cell and one basal cell and two to four small lateral cells attached to the basal cell. The apical and lateral cells each possess a long, slender appendage. Excluding these appendages, the length of a mature conidium is usually 20-24μm while the diameters of the apical and basal cells are 8-11μm and 6-9μm respectively.
Ultrastructure of the Infection of Poinsettia by Oidium SP. using High Pressure Freezing and Freeze Substitution
Authors: Celio G J , Richardson E A , Mims C W
Published: July 2020

Cryofixation is becoming more widely used to study host-pathogen relationships in fungal diseases of plants. This presentation describes results we have obtained using high pressure freezing and freeze substitution to study powdery mildew disease of poinsettia ﹛Euphorbia pulcherrima) caused by Oidium sp.

Approximately 0.5 mm leaf disks bearing sporulating colonies of Oidium sp. were excised and placed in a 15% dextran solution contained in brass planchets. Samples were frozen using a Balzer's HPM 010 High Pressure Freezing Machine and substituted according to the procedures of Hoch.6 Thin sections of embedded leaves were cut using a diamond knife, collected on gold slot grids, and placed on formvar-coated racks. Sections were poststained with uranyl acetate and lead citrate and examined using a Zeiss EM 902A transmission electron microscope.

Outstanding preservation of haustoria, the specialized nutrient-absorbing structures produced in host epidermal cells by Oidium, was obtained. Both young, unlobed (Fig. 1) as well as mature, highly lobed (Fig. 2) haustoria were observed.
Semiconductor Defect Studies Using Scanning Probes
Authors: Hsu Julia W P
Published: July 2020

Understanding how defects alter physical properties of materials has lead to improvements in materials growth as well as device performance. Transmission electron microscopy (TEM) provides an invaluable tool for structural characterization of defects. Our current knowledge of crystallographic defects, such as dislocations, would not have been possible without TEM. Recently, scanning tunneling microscopy and scanning force microscopy (SFM) have shown the capability of imaging surface defects with atomic or near-atomic resolution in topographic images. What is more important is to gain knowledge on how the presence of a certain type of defects changes the physical properties of materials. For example, how is the carrier lifetime altered near electrically active defects? How does photoresponse vary near grain boundaries? Where are defect levels in the forbidden bandgap? This talk will discuss several examples of how scanning probe microscopies (SPMs) can contribute to this aspect of defect studies in semiconductors.
Polarization Screening and Image Formation in SSPM, EFM and Piezoresponse Imaging of Ferroelectric BaTiO3 (100) Surface
Authors: Kalinin Sergei V , Bonnell Dawn A
Published: July 2020

Possible applications of ferroelectric materials in non-volatile memories, MEMS, microwave ceramics, PTCR devices and sensors draw significant interest to these materials. Operation of most of these devices relies heavily on the surface (FRAM and other thin-film devices) and interface (PTCR, varistors) properties of ferroeiectrics, particularly on the polarization and charge distribution in the surface or interface region. Electrostatic scanning probe techniques such as electrostatic force microscopy (EFM), scanning surface potential microscopy (SSPM) and piezoresponse imaging (PRI) can be successfully employed for the characterization of ferroelectric surfaces on the micron and submicron level. The former technique is based on the detection of the resonant frequency shift of mechanically driven cantilever, which is proportional to gradient of electrostatic force acting on the tip. The latter two techniques are based on the voltage modulation approach, i.e. during imaging the piezoelectric actuator driving the cantilever is disengaged and the AC bias is applied directly to conductive tip.
Quantitative Study Of MgO (110) Surface Faceting Angles by AFM and SEM
Authors: Giese D R , Lamelas F J , Owen H A , Gajdardziska-Josifovska M
Published: July 2020

The stability of polar and neutral MgO surfaces has been addressed by theory and experiment as an ideal example of ionic oxides with rock-salt structure, but with unresolved controversial results. For example, energy-minimization theory predicted that higher energy polar MgO (111) and neutral MgO (110) surfaces could thermally facet into the lowest energy MgO (100) planes. Early low-energy electron diffraction (LEED) experiments reported diffraction spot splitting, interpreted as evidence for thermal faceting to ﹛100﹜ planes. However, a recent study found MgO (111) faceting was caused by chemical etching with nitric or phosphoric acid, and not promoted by annealing. Quantitative atomic force microscopy (AFM) and scanning electron microscopy (SEM) measurement of the facet angles revealed them to be vicinal planes rather than the predicted ﹛100﹜ planes. A recent AFM study reported thermal faceting of MgO (110) into vicinal planes.
Imaging Ultrathin Organic Films on the Nanometer Level Using Surface Plasmons
Authors: Bailey GW , McKernan S , Price RL , Walck SD , Charest P-M , Gauvin R , Fried G , Bohn W
Published: July 2020

Surface plasmons have been used to detect single molecular layers for many years. The most common examples are biosensor systems and the detection of self-assembled monolayers (SAMs). A surface plasmon is a two-dimensional electromagnetic wave that travels on the surface of a noble metal, most commonly gold or silver. This electromagnetic wave is extremely sensitive to changes in the dielectric constant 100 nm above the metal surface. When the dielectric constant above the metal changes due to the addition of a molecular layer, the intensity of the surface plasmon will decrease. In this way monolayers may be indirectly imaged on a surface by mapping the surface plasmon intensity.

Although there are many reasons for imaging ultrathin films or molecular layers over large distances (10-100 μm), there are several difficulties involved. Typically the substrate has a roughness greater than the height of a single monolayer. This can mask the presence of the monolayer on large area scans.
Determining Grain Boundary Potential from Electrostatic Force Based Scanning Probe Imaging
Authors: Bonnell Dawn A , Kalinin Sergei V
Published: July 2020

A combination of electrostatic force microscopy (EFM) and scanning surface potential microscopy (SSPM) is used to quantify nanometer scale field variations for the general case of electronically inhomogeneous surfaces. The specific example illustrated here is the intersection of a Σ5 grain boundary in donor doped SrTiO3 intersecting the (100) surface. ‘The topographic structure is compared to the surface potential, the positive and the negative electrostatic force images in figure 1. The EFM contrast changes sign with the polarity of the applied bias, as expected for an electrostatic attractive/repulsive interaction. There is a significant localization of surface potential at the grain boundary that is manifest as a protrusion in the SPPM image.

To quantify the properties at the surface, rather than above the surface where the measurements are acquired, a relationship connecting the sample-tip interaction is required. It has been shown that models based on simple geometric assumptions do not represent the behavior well.
Direct Electron Exposed Silicon Detectors in EELS
Authors: Orsholm Claes , Csillag Stefan
Published: July 2020

Samples to be studied using EELS techniques are more or less sensitive to the effects of the radiation exposed to them by the beam. Quite often the sample is damaged due to the high radiation doses in the areas where the beam has been focused. Secondly, the build up of carbon contamination on the surface of the sample caused by the beam affects the results of the analysis if the sample itself contains carbon. The solution to these problems is to minimize the exposure times needed to record the spectrum, which will lower the radiation-doses on the sample. In addition, problems related to beam instabilities would be reduced due to the shortened acquisition times. However, in order still to achieve an acceptable signal-to-noise ratio the need for a detector with high detective quantum efficiency is essential. We have investigated the possibilities using silicon detectors such as photodiode arrays (PDA) and charge coupled devices (CCD) in direct electron detection mode using 100 keV incident electrons.
New Multifunctional Detector Unit for SEM
Authors: Dolukhanyan T , Galstyan V , Nielsen C , Sung C
Published: July 2020

In order to increase analytical capabilities of SEM we have developed a multifunctional detector unit, which allows the simultaneous and highly efficient registration of the information from the chosen area of the specimen using backscattered electrons (BSE) and cathodoluminescent (CL) radiation. At the same time secondary electrons (SE) could be collected by the regular Everhart - Thornley detector. Thus, the unit allows to perform microstructural investigations in three modes of SEM operation simultaneously - BSE, CL and SE. A schematic diagram of the unit is presented in Fig. 1.

In the plate made of scintillating material, and installed between the pole piece of SEM and the specimen, the bottom side is a part of spherical surface. The surface covers completely the whole top surface of the specimen and collects BSE and CL radiation in the solid angle ∼2 π sr.
Electrostatic Aberration Correction in LV-SEM
Authors: Maas DJ , Henstra A , Krijn MPCM , Mentink SAM
Published: July 2020

The resolution of a low-voltage electron microscope is limited by the chromatic and spherical aberration of the objective lens, see Fig. 1. The design of state-of-the-art objective lenses is optimised for minimal aberrations. Any significant improvement of the resolution requires an aberration corrector. Recently, correction of both Cc and Cs has been demonstrated in SEM, using a combination of magnetic and electrostatic quadrupoles and octupoles (Zach and Haider, 1995). The present paper presents an alternative design, which is based on a purely electrostatic concept, potentially simplifying the ease-of-use of an aberration corrected microscope.

In 1936 Scherzer showed that the fundamental lens aberrations of round lenses are positive definite, in absence of time-varying fields and/or space charge. Negative lens aberrations, required for the correction of Cc and Cs, can only be obtained using non-round lenses, e.g. quadrupoles and octupoles (Scherzer, 1947).
The Energy Spectra of Secondary Electrons
Authors: Joy David C , Braski David
Published: July 2020

It has been estimated that more than 90% of all scanning electron microscope (SEM) images ever published have been obtained using secondary electrons (SE) which are defined as being those electrons emitted with energies between 0 and 50eV. The properties of these secondary electron are therefore of considerable interest and importance. However, although secondary electrons have been intensively studied since their discovery by Starke in 1901 the majority of the work has been aimed at determining the SE yield coefficient and its variation with energy for elements and compounds. The energy spectrum of secondary electrons has received far less attention although it is evident that the form of the spectrum must have an effect on the image contrast observed in the SEM because SE detectors are energy selective devices. The few studies that have been made have mostly concentrated on spectra obtained from clean samples observed under ultra-high vacuum conditions. This is understandable, because it is certain that the presence of a surface layer of contamination will change the SE spectrum to some degree or other, but it is unfortunate because all specimens in real SEMs are dirty and it is information about this situation that is required.
Imaging of Insulators in LVSEM and Related Physical Mechanisms
Authors: Cazaux Jacques
Published: July 2020

From the best fit of two adjustable parameters, B (escape probability) and ƛ (empirical escape depth of the secondary electrons, s.e.), the constant loss theory accounts fairly well for the s. e. emission from materials: δ (E0) = B [E0/E(e.h)] [ƛ/R] [1 - exp. -(R/ ƛ)] (1) (with E(e.h): energy required for generating a s.e. inside the specimen; R: range of the incident electron related to the primary beam energy, E0, via a power law of the form: R=C E0n). The influence of the angle of incidence, i, may also be estimated by changing R into R cos i (see fig la for the example of diamond with B/ E(e.h) = 22 keV-1; ƛ ≈ 10 nm). For insulators, δ is obtained from short pulse experiments (to prevent charging) and, in the 1-5 keV range, it is often one order of magnitude (or more) larger than that of metals via a larger ƛ value (also more sensitive to the temperature, to the crystalline state and resulting from a random walk involving many elastic and inelastic-phonon- scattering events).
Imaging and Microanalysis of Non-Conducting Materials in the Lowvoltage FE-SEM: Challenges and Strategies
Authors: Liu Jingyue
Published: July 2020

Under high-energy electron bombardment, electrical charge can rapidly build up in non-conducting materials. The electron-induced charging process is very complex and is still not well understood. Injection of energetic electrons into insulators generates electron-hole pairs which can be localized at pre-existing or newly created charge-trap centers. Charging of insulators results from a balance between the emission of secondary electrons and the diffusion and trapping of incident electrons. In the SEM, charging is usually a dynamic process: the degree of specimen charging depends on the energy and the dose of the incident electrons, the image magnification, the pixel dwell time (PDT), and the history of the electron irradiation of the specimen. Although the degree of charging can be reduced by varying the energy of the incident electrons, charging of non-conducting specimens cannot be completely eliminated due to variations in the local SE yield across the specimen surface and the presence of charge-traps inside the specimen.
Applications of High Resolution Microcalorimeter Type X-Ray Spectrometers in Material Analysis
Authors: Höhne Jens , Bühler Matthias , Hertrich Theo , Hess Uwe
Published: July 2020

As the semiconductor industry further shrinks feature sizes, new analytical solutions are required for elemental composition analysis. Conventional EDX analysis is limited by the obtainable energy resolution. In order to analyze extremely small impurity particles, material doping, interdiffusion effects or small chip structures, the diameter of the area hit by an electron beam and particularly the volume of electron-surface interaction has to be reduced accordingly. This can be achieved by reducing the electron beam energy.

X-ray spectroscopy is most frequently used for damage free material testing. Nowadays, semiconductor based energy dispersive X-ray spectrometers (EDS) are employed on a routine basis and are frequently used in combination with scanning electron microscopes (SEMs). But performance is limited by their rather poor energy resolution, which is typically in the 130eV range for a 6keV X-ray line, and their limited capability of resolving X-ray lines within short spectral ranges. Further, overlaps of spectral lines are most critical in the low energy range.
Low Voltage Topographic Imaging of Biological Samples in the SEM
Authors: Walther Paul
Published: July 2020

Joy and Pawley (1992) defined the limitations of spatial resolution in the SEM as follows. “The spatial resolution of the scanning electron microscope is limited by at least three factors: the diameter of the electron probe, the size and shape of the beam/specimen interaction volume with the solid for the mode of imaging employed and the Poisson statistics of the detected signal.” The interaction volume of the primary beam in the sample increases with increasing accelerating voltage (Vo), because electrons with higher energy penetrate deeper into the sample. The interaction volume affects the spatial resolution, because the electrons that finally form the image do not only originate from the impact point of the primary beam, but from the much larger interaction volume. It is not simple to understand how this affects spatial resolution, since resolution is also heavily influenced by the properties of the sample surface (the surface coating) and it is the issue of ongoing debates.
Field Emission SEM with a Newly Developed FEGUN and Conical Strongly Excited Objective Lens
Authors: Bailey GW , McKernan S , Price RL , Walck SD , Charest P-M , Gauvin R , Kazumori H , Yamada A , Mita M , Nokuo T , Saito M
Published: July 2020

A newly developed cold FE-GUN which enables to us to obtain large probe current and low emission noise, and conical strongly excited objective lens has been installed on the JSM-6700F Scanning Electron Microscope (SEM). In the range of accelerating voltages from 0.5 to 15kV, this instrument has got much better resolution as compared with in-lens type SEM (Ohyama et al 1986)(Fig. 1). We can obtain high-resolution secondary electron images with large samples (ex. 150mm ϕ×10mmH).

Our old type objective lens (Kazumori et al 1994) has the limitation of working distance (WD), but the new lens enables us to work at very short WD at accelerating voltage of 15kV. As a result the spherical (Cs) and chromatic (Cc) aberration constants are 1.9mm and 1.7mm respectively at a WD of 3mm.

In order to get large probe current, we increased emission current and got near the distance between the t ip of emi tter and the pr inciple plane of condenser lens.
Fractal Characterization of the Fractured Surface of a Duplex Stainless Steel and Their Relation with the Strength and Ductility
Authors: Hilders O A , Sáenz L , Peña N , Ramos M , Quintero A , Caballero R , Berrío L
Published: July 2020

Due to the very good combination of the most outstanding properties of ferrite and austenite, the microstructure of duplex stainless steels allows them to obtain high strength and toughness levels even at low temperatures. As a result of these combined effects, duplex stainless steels have become very popular for many applications. In practice, the prolonged use of these materials at temperatures below approximately 500°C may cause an embrittlement of the ferrite phase, which has been called 475°C embrittlement. Thus, the isothermal aging at 475°C can be exploited to produce a variety of strength values associated with the corresponding decreases in ductility and variations of the fractal dimension of the fracture surfaces. No experimental measurements of the fractal dimension - tensile properties relationships are available for many commercial metallic alloys, then, the present experiments on a duplex stainless steel were conducted to show that the fractal dimension, D, many be used as a characterization parameter in fracture morphology - mechanical properties studies.
The Construction of an EBSD Stage for the Electroscan E3 SEM
Authors: Yurek Peter , McKernan Stuart , Lee Kyung-Ho
Published: July 2020

With recent advances in computing power and application specific software it has become practical for smaller microcopy labs to either purchase or build a electron backscatter diffraction (EBSD) systems. This system can be used to probe the crystallography and microstructure of many different materials. One set of such materials is geological samples from deep within the earth's crust. In collaboration with the Geology and Geophysics department an EBSD system has been built to function with the Philips Electroscan E3 Environmental Scanning Electron Microscope (ESEM). Using an existing low light level SIT camera, the system was completed with the addition of a phosphor screen and appropriate software and acquisition cards.

One main advantage of the ESEM for EBSD is that since it is an environmental SEM samples can be imaged without coating. For insulating materials this is means that the sample can be imaged without coating.
Improved Signal Processing in Multi-Photon Imaging
Authors: Fisher Wait G , Wachter Eric A
Published: July 2020

Multi-photon excitation has been used in microscopy for nearly a decade, providing a number of demonstrated advantages over other methods for fluorescence imaging. Because excitation is achieved using longer, less energetic light, photodamage and photobleaching of the sample are reduced. Furthermore, since excitation occurs only at the focal point, this approach allows the practical collection of three-dimensionally resolved fluorescence images of live cells. However, due to the small two-photon cross-section of most fluorophores, pulsed lasers are required to generate detectable signal levels. This is due to the quadratic dependence of twophoton absorption on the instantaneous power of the laser. Typically, these lasers are pulsed at very high repetition frequencies, on the order of 106 pulses per second with pulse durations of a few hundreds of femtoseconds. For example, a titanium:sapphire (Ti:S) laser mode-locked at 76 Mhz can provide up to 100,000 watts of instantaneous power and is ideal for exciting two-photon events.
Two-Photon Microscopy of Single Molecules
Authors: Fourkas J T , Previte M J R , Farrer R A , Olson C , Peyser L A
Published: July 2020

The ability to observe the fluorescence arising from single molecules has revolutionized our ability to study the structure and dynamics of materials on a microscopic level and to probe the properties of individual members of a heterogeneous ensemble. A variety of near-field and far-field excitation techniques have been employed to study single molecules. Multiphoton excitation (MPE) techniques have a number of advantages that make them particularly attractive for singlemolecule detection. First, because the excitation and fluorescence wavelengths are significantly different from one another, Rayleigh and Raman scattering can easily be filtered out, leading to a low number of background counts. Second, because the probability for MPE depends on the excitation intensity to the second or higher power, the excitation is localized to the point in space where the excitation beam is most tightly focussed, thus providing three-dimensional resolution.
Second Harmonic Imaging Microscopy: A New Non-Linear Optical Modality for Cell Membrane Physiology
Authors: Campagnola Paul , Lewis Aaron , Loew Leslie M
Published: July 2020

Confocal microscopy is an excellent high resolution method to image fluorescently labeled cells. However, the use of confocal microscopy to monitor physiological events, such as membrane potential changes, in living cells is hampered by photobleaching and phototoxicity. To reduce the collateral damage from excitation of fluorescent probes outside the optical slice, Webb and co-workers introduced the use of two-photon excited (TPE) fluorescence in laser scanning microscopy.1 Two-photon absorption depends on the square of the incident light intensity; this has the effect of confining excitation to the plane of focus where the photon flux density is greatest. The wavelength of the exciting light is in the near infrared facilitating penetration of thick tissues. Due to these significant advantages this methodology is rapidly gaining popularity as a tool for live cell and tissue imaging.

To further exploit non-linear optical processes in laser scanning microscopy, we have developed surface second harmonic generation (SHG) as a powerful new imaging modality.
Third Harmonic Microscopy: Imaging Applications in the Biological and Material Sciences
Authors: Squier Jeff , Müller Michiel
Published: July 2020

Tightly focused beams of ultrashort optical pulses such as produced with a high numerical aperture optic as found in a microscope exhibit an interesting property: a strong third harmonic signal is produced when the axial focal symmetry is broken through a change in material properties. This change can be manifested as either a change in refractive index and/or a change in the nonlinear susceptibility of the specimen. Conversely, third harmonic production is inhibited in those cases when the axial focal symmetry is unbroken. This property can be effectively exploited to produce volumetric images of biological and non-biological specimens. The third harmonic is localized to interfaces within the specimen making it possible to produce optical sections in much the same manner as in confocal, and two-photon fluorescence microscopy. Unlike these techniques however, no exogenous fluorophore is necessary for labeling - the signal is produced by endogenous interfaces.
Confocal Fluorescence Microscopy with CCD Sensors Speed by Parallelization, Low Cost by Using More Electronics
Authors: Großkopf Rudolf E
Published: July 2020

CCD sensors are able to image a few megapixels within one frame. The image is scanned electronically within the semiconductor chip (instead of mechanical-optical scanning with moving mirrors, Nipkow discs or acousto-optical devices). Through parallelization. the novel concept yields speed at an unprecedented degree for confocal imaging Routine applications requiring high speed and low costs will profit from this principle. Thus, confocal imaging technology will take the same path television technology has taken—from mechanical scanning to the broadest possible application of electronics

In order to go this way, a pinhole matrix is used on the illumination side. It has as many pinholes and the same pitch as the pitch and number of pixels of the CCD (Figure 1). In front of the receiver, a second pinhole matrix with the same pitch and number of pinholes is used All pinholes of both matrices and the pixels of the CCD are in confocal position.
A Robust Two-View Method for Increasing the Imaging Depth and Correcting for Signal Attenuation in Confocal Microscope Images
Authors: Can Ali , Lasek Shade , Szarowski Donald H , Turner James N , Roysam Badrinath
Published: July 2020

Confocal Microscopy is now an essential, and widely used tool for biological research. Particularly valuable is its ability to image thick sections of intact tissue. A compelling need in this field is to improve the imaging depth, as well as quantitative accuracy, and minimize the possibility of missing important structures/phenomena due to attenuation of the imaging signal. Previous methods work by amplifying the signal from deeper parts of the specimen. In these methods, amplification of noise is an unavoidable artifact. We describe a novel method that not only corrects confocal stacks for attenuation without noise amplification, but also enhances the achievable imaging depth. Interestingly, it does not require explicit estimation of the extinction factor, It relies on a synergistic combination of specimen preparation and image reconstruction algorithms.

The key idea is to image the specimen from two angles (Fig. 1), separated by 180° and then to reconstruct the complete thick image using computational methods.
Multi-Photon Fluorescence Spectroum of Common Nucleic Acid Probes
Authors: Bailey GW , McKernan S , Price RL , Walck SD , Charest P-M , Gauvin R , Cheng P C , Lin B L , Kao F J , Sun C K , Johnson I
Published: July 2020

Fluorescent probes are commonly used in biological fluorescence microscopy for tracking specific structures and sub-cellular compartments, and for indicating cellular ionic conditions. Recent development in multi-photon fluorescence microscopy has greatly expanded the usage of fluorescent probes in biomedical research. Considering its non-linear nature, two-photon excitation may generate very different fluorescence spectral response in the sample when compared with single photon excitation. It is thus necessary to measure the two-photon spectra of various fluorescent probes, so that two-photon fluorescence microscopy may be operated effectively and the images properly interpreted. This report represents the first installment of a continued effort in characterizing the multi-photon fluorescence spectra of commonly used bio-probes.

Two-photon fluorescence spectra excited with near infrared at 780nm were obtained with a SpectraPro-500 spectrophotometer (Acton Research) equipped with a TE-cooled PMT and coupled to a Spectra-Physics Tsunami Ti-sapphire laser pumped by a Coherent Verdi solid-state laser operated at 85MHz, l00fs pulse.
FRET: A Spectral Ruler for Interacting Molecules Involved in Apoptosis
Authors: Centonze Victoria E , Liang Xiao H , Casanova Elizabeth A , Levine Beth , Herman Brian
Published: July 2020

Fluorescence Resonance Energy Transfer (FRET) is a process by which a fluorophore (the donor) in the excited state transfers its energy to a neighboring fluorophore (the acceptor) non-radiatively through dipole-dipole interactions. Since the efficiency of energy transfer varies as the inverse of the sixth power of the distance separating the donor and acceptor chromophores, for FRET to occur the distance between the two molecules cannot exceed 10 to 100 angstroms (1 to l0nm). The combination of FRET and optical microscopy allows examination and quantitation of dynamic molecular interactions between cellular constituents at resolutions beyond the Abbe diffraction limit of light microscopy. Through the microscope one may detect FRET by an overall decrease in fluorescence emission of the donor with a concomitant increase in fluorescence emission of the acceptor.
Studying Subnuclear Dynamics in Living Cells
Authors: Spector David L
Published: July 2020

Green fluorescent protein (GFP) and its variants have become powerful tools with which to study the dynamics of many fundamental processes in living cells. I will first discuss the advantages of GFP and important parameters that one must consider in performing live-cell microscopy experiments including how to keep cells happy on the microscope stage. I will then go on to discuss work from our laboratory using multiple microscopic approaches to address questions related to gene expression.

Many of the basic processes that are essential for normal cell growth and division are regulated in the nucleus, ie. DNA replication, transcription, RNA processing and transport. The organization of these functions in the nucleus must be highly controlled in order to enable cells and/or the entire organism to function properly. However, relatively little has been known about the relationship of nuclear organization to gene expression.
Field Emission Scanning Electron Microscopy Of Mouse Cerebellar Synaptic Contacts
Authors: Castejón OJ , Apkarian R P , Castejón H V
Published: July 2020

Samples of albino mice cerebellar cortex were processed by the cryofracture method for scanning electron microscopy and examined with the field emission scanning electron microscope (FESEM). Albino mouse cerebellar cortex was excised, cut into 1-2 mm slices and inmersed in 4% glutaraldehyde in O. l M phosphate buffer solution, pH 7.4, for 24h at 4°C; and postfixed for 1 h in a similarly buffered 1% osmium tetroxide solution. Specimens were dehydrated in a graded serie of ethanol (30, 50, 70, 80, 90 2x100%) prior to wrapping individual tissue pieces in preformed absolute ethanol filled parafilm cryofracture packets. Rapid freezing of packets was performed by plunging into LN2. First, the packet was transferred from the LN2 storage vessel with LNT chilled forceps in order to avoid themial damage. Secondly, the cooled fracture blade was removed from the LN2, the packet was orientated under the blade, and immediately struck with a heavy tool.
Ordered Intracellular Reca-Dna Assemblies
Authors: Wolf S G , Levin-Zaidman S , Frenkiel-Krispin D , Shimoni E , Sabanay I , Minsky A
Published: July 2020

The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role. We find that induction of the SOS system in wild-type E. coli bacteria results in fast and massive intracellular coaggregation of RecA and DNA into lateral assemblies, which comprise substantial portions of both the cellular RecA and the DNA complement. The structural features of the coaggregates and their relation to in-vitro RecA-DNA are consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur.

Bacterial chromatin is demarcated in electron micrographs of metabolically active cells as amorphous ribosome-free spaces that are irregularly spread over the cytoplasm (Fig. A). Wild-type E. coli cells exposed to DNA-damaging agents that induce the SOS response reveal a strikingly different morphology (Fig. B).
Three-Dimensional Reconstruction of a Node of Ranvier Obtained by Serial Section Electron Tomography
Authors: Sosinsky G , Deerinck T , Greco R , Ellisman M
Published: July 2020

Nodes of Ranvier are sites on myelinated axons where the insulating layers of myelin are interrupted. They represent an excellent example of a complex cellular structure that contains highly differentiated sub-regions. At these sites of cell-cell specialization, ion fluxes occur which are required for propagation of action potentials. Myelinated axons and their nodes of Ranvier represent an important evolutionary advance for vertebrates that permit very rapid propagation of action potentials without large increases in axonal diameter. This physiological achievement is based on adaptations at a molecular level resulting in an elegant cooperation between glial cells and the axons of neurons.

The organization and sub-specialization of the molecular components of the node of Ranvier are complex but often inter-related. The nodal region of axons are enriched in several channel and pump proteins including the voltage-gated Na+ channel, Na+-K+ ATPase and a number of different K+ channel isotypes.
Localization of Misprocessed Pulmonary Surfactant Protein C in Tubular Myelin Enriched Fractions By Cryo Immunogold Labeling
Authors: Na C-L , Evans E A , Akinbi H T , Weaver T E
Published: July 2020

Pulmonary surfactant is secreted by alveolar type II cells and reduces the surface tension at the air-liquid interface of alveoli. After pulmonary surfactant is secreted into the alveolar space, it transforms into tubular myelin, a highly ordered 3-dimensional lattice-like structure. Pulmonary surfactant protein C (SP-C), one of four pulmonary surfactant associated proteins, is synthesized as a proprotein which is processed to biologically active 35 amino acid mature peptide by proteolytic cleavage of N- and C-terminal peptides from the SP-C propeptide (Weaver, 1998). Processing of SP-C is linked to the expression of pulmonary surfactant protein B (SP-B): In SP-B deficient mice, SP-C is misprocessed and present in the bronchoalveolar lavage (BAL; Vorbroker et. al., 1995a). Although the intracellular localization of SP-C is well established (Vorbroker et. al., 1995b), there is no ultrastructure study available regarding the localization of misprocessed SP-C in the airway. In this study, we used transgenic mice expressing a truncated human SP-B propeptide (hSP-BΔC+/+) bred into the murine granulocyte macrophage colony stimulating factor (GMCSF) and SP-B double knockout background (hSP-BΔC+/+: GMCSF-/-: mSP-B-/-) as a model to localize the misprocessed SP-C by cryoimmunogold labeling.