bluuebunny/crossref_metadata_2025 huggingface dataset:
Physical Considerations in Low Energy Biological Analysis
Authors: Joy David C
Published: July 2020

Biological scanning electron microscopy is increasingly performed at low beam energies in order to improve image contrast, reduce charging artifacts, and minimize beam induced damage to the sample. It is natural then to wish to also use these same low accelerating voltage for X-ray microanalysis using an energy dispersive spectrometer (EDS) but a variety of fundamental physical effects affect the performance that can be achieved. An X-ray photon can only be emitted when the incident beam Eo energy exceeds the critical excitation energy Ecrit for that line. As the beam energy is reduced the number of elements that can be excited falls and it is becomes necessary to use L- and M-lines rather than the K-lines accessible at higher energies. At 5keV, the upper limit of ‘low voltage’ microscopy, K-lines can be excited from elements up to calcium, L-lines can be detected up to cesium, and the rest of the periodic table is available using M-lines. The entire periodic table is therefore, in principle, available at an overvoltage U>2, where U = E0/Ecrit But at lower energies the number of accessible excitations falls and some elements cannot, with current technology, then be analyzed and for general purposes an incident energy lower than about 3keV is probably too limiting to be useful.
Microcalorimeter Energy Dispersive Spectrometry for Low Voltage SEM
Authors: Wollman D A , Newbury Dale E , Hilton G C , Irwin K D , Rudman D A , Dulcie L L , Bergren N F , Martinis John M
Published: July 2020

Microanalysis performed at low electron beam energies (≤ 5 keV) is limited by the physics of x-ray generation and the performance of existing semiconductor energy dispersive spectrometry (EDS) and wavelength dispersive spectrometry (WDS). Low beam energy restricts the atomic shells that can be excited for elements of intermediate and high atomic number, forcing the analyst to consider using unconventional M- and N-shells for elements such as Sn and Au. Unfortunately, these shells have very low fluorescent yield, which results in inherently low spectral peak-to-background ratios. The modest energy resolution of semiconductor EDS leads to poor limits of detection for these weakly emitted photons. The situation is further complicated by the inevitable interferences with the much more strongly excited K-shell x-rays of the light elements, particularly carbon and oxygen. WDS has the spectral resolution to overcome the resolution limitations of semiconductor EDS. However, WDS has a low geometric efficiency, and because of its narrow instantaneous spectral transmission, spectral scanning is required to detect and analyze x-ray peaks. Moreover, the high resolution field-emission-gun scanning electron microscope (FEG-SEM) provides only a few nanoamperes of beam current.
On the Contrast of Precipitates Observed with a FE-SEM
Authors: Gauvin Raynald , Hovington Pierre
Published: July 2020

It has been shown recently that precipitates as small as 10 nm can be imaged with a FE-SEM using a Through-The-Lens (TTL) SE detector or the so-called Upper Detector (UD). This is a very significant finding because normally, such small precipitates are observed in the TEM where specimen preparation techniques are generally a timely process. However, from Monte Carlo simulations using CASINO, it has been shown that NbC precipitates embedded in Fe as small than 6 nm can be imaged with BE. The experimental demonstration was difficult because no good BSE detectors were available at that time for low energy work.

Recently, a new BSE detector has been developed for low energy work from the GW corporation, the Centaurus BSE detector. This detector is coupled with an HITACHI S-4700 FESEM In this work, images of Mg2Zn precipitates in Al obtained with the UD and the Centaurus BSE detector are compared.
Quantitative Chemical Mapping with Low-Voltage EDS Spectrum Imaging and Multivariate Statistical Analysis (MSA)
Authors: Anderson IM
Published: July 2020

One of the advantages of performing X-ray microanalysis at low (≤5 kV) operating voltages is that high spatial resolution (≤250 nm) chemical maps of the specimen can be acquired. Spectrum imaging, where a full spectrum is acquired for each pixel in a two-dimensional array, provides the most comprehensive method of characterization, as long as the sampling density (pixel size) is sufficiently smaller than the spatial resolution. Multivariate statistical analysis (MSA) methods are effective in reducing the large (typically ∼10 MByte) raw spectrum images to the modest (typically ∼100 kByte) data files that contain all of the statistically significant information of interest about the specimen. Preliminary analysis of a cross section of a computer chip from a major semiconductor company was previously performed using limited spectrum imaging capabilities available with the 4pi X-ray mapping module, which allowed for simultaneous acquisition of only 48 channels. MSA of images acquired with only a 960 eV portion of the spectrum containing the Al-K, Si-K and W-M lines showed that excellent contrast between the Si- and W-rich regions of the specimen could be achieved in spite of the strong overlap between Si-Kα and W-Mα (∼34 eV separation).
Imaging Self-Assembled Monolayers: a Comparative Study Between AFM and Field-Emission SEM
Authors: Russell PE , Neves BRA , Salmon M E , Troughton EB
Published: July 2020

Self-assembled monolayers (SAM) and multilayers of organic materials have been intensely studied in the past years, due to their numerous potential applications as, for example, lubricants, corrosioninhibitors and/or adhesion-promoters [1,2]. Due to the reduced height of SAM, typically a couple of nanometers, the various Scanning Probe Microscopy (SPM) techniques, especially Scanning Tunneling Microscopy (STM) and Atomic Force Microscopy (AFM), have been the tools of choice in the morphological and structural study of those systems [1,2]. However, these SPM techniques have some limitations. One of them is the reduced scanning area, preventing the assessment of SAM coverage in large substrate areas, which is one of the key issues in studies of the potential applicability of SAM. Furthermore, due to their imaging principle, the SPM techniques are not suited to analyze SAM on substrates where the roughness is of the order of hundreds of nanometers or higher, impeding their application on most non-ideal (atomically flat) substrates.
Scanning Electron Microscopic Analysis of Fossil Bone (Mosasaurs) from Marine Deposits of the Upper Cretaceous of North America
Authors: Amy Sheldon M
Published: July 2020

Research into bone histology of fossil material is a growing area of inquiry in vertebrate paleontology. Incorporating histological study into ontogenetic investigation presents new opportunities for gaining data to better understand ontogeny, ecology, and evolution of mosasaurs. Adding scanning electron microscopy to the growing technology being applied to fossil research can only further the understanding of long extinct animals.

Mosasaurs were totally marine lizards which inhabited both open ocean and epicontinental seas during the late Cretaceous (Turonian through Maestrichtian), approximately 90-65. million years ago. Mosasaurs have been collected from many areas of North America. The specimens used in this study were collected from Alabama, Kansas, South Dakota, and Texas. I have restricted the fossil material for destructive analysis to ribs and vertebrae. Ribs have not been shown to have importance in morphological analysis relating to systematics or evolution, therefore their destruction does not imped the research of others. Vertebrae were isolated on outcrop but could be identified to genus and growth stage.
Displacement Mapping During In-Situ Straining in the SEM
Authors: Biery Nicholas E , Pollock Tresa M , Nuhfer NT , Graef Marc De
Published: July 2020

We have developed a technique for measuring small strains during in-situ tensile tests in a Scanning Electron Microscope (SEM). The technique uses pattern recognition algorithms to locate markers on a gridded sample before and after deformation. The distortions in the grid are used to calculate the strains at each of the markers. The strains are then displayed in contour plots and correlated with the microstructure of the sample to help understand the local progression of strain as a function of applied load and microstructure.

Tensile experiments were performed on gridded notched and unnotched samples of a Ti-48Al-2Cr- 2Nb alloy (at%) in a Philips XL-30 FEGSEM using a 10,000 N Kamrath-Weiss in-situ straining stage. Gold was evaporated onto the samples through nickel grids with a mesh size of 17 μm. Electron backscatter images of the samples taken during testing were analyzed using a set of, interactive routines written in the Interactive Data Language (IDL).
Omega Filter design for a 200kv TEM
Authors: Tsuno K , Kaneyama T , Honda T , Ishida Y , Munro E , Rouse J
Published: July 2020

A new omega filter has successfully been installed in 200kV TEMs(see FIG. 1). Energy filtering TEM (EFTEM) is extremely useful to enhance contrast of biological specimens, to observe defects in thick semiconductor tips, to obtain a good compositional map in grain boundaries, to give structure image in the thick specimen regions and to obtain quantitative data by removing plasmon loss signals. In-column filters such as omega filter has advantages over postcolumn filters from points of cancellation of aberrations, to obtain iso-chromatic images and a freedom of selecting variety of magnifications.

Here, we discuss conditions of designing an omega filter. In usually, curved axis systems, which are common to energy analysis equipment, has large second order aberrations. Those aberrations are compensated in analyzers with single sector magnet with multi stage sextupole correctors. However, the adjustment of those correctors is difficult and residual aberrations destroy quality of the filtered image.
Modification of an Energy Filtering Transmission Electron Microscope for Low Energy Loss Imaging and Chromophore Mapping
Authors: Davis J A , Heng Y M , Barfels M M G , Ottensmeyerf F P
Published: July 2020

We have recently replaced the electron energy filter of the Zeiss EM 902 with an improved PMP filter. The design, incorporating concave curved entrance and exit faces and convex curves on the side facing the electrostatic mirror was based on the theoretical calculations of Jiang and Ottensmeyer and Barfels et al. Care was taken in the design of the filter to allow its introduction into the existing geometry of the microscope and its use of the existing electronics. As a result, dismantling the existing prism housing and support (which were used as the base for the new prism), reinstallation, and adjustment required only three working days. The resolution in tjie energy dispersive plane of the new filter was measured using the 15.0 eV plasmon peak of aluminum as an internal standard. Such measurements yielded a FWHM of 1.1 eV over the full image plane (fig. 1) with an optimum resolution of approximately 0.8 eV possible with corrections to the stability of the voltage supply and further small alignment adjustments.

This improved energy resolution permits the detection and imaging of signals in the low loss region previously inaccessible to us in the EM902.
Improving the Positional Accuracy of the Goniometer on the Philips CM200 TEM.
Authors: Pulokas J , Green C , Kisseberth N , Potter CS , Carragher B
Published: July 2020

We have developed a system (1) for automatically acquiring large numbers of high quality transmission electron micrographs under low dose conditions. This system has been implemented on a Philips CM200 Transmission Electron Microscope equipped with a Gatan MSC CCD camera. Implementing the automated data acquisition system requires that target locations be identified in a low magnification image and then accurately located at the center of the viewing area. The magnification is subsequently increased, the image is focused on an area adjacent to the target area and the final image is acquired. Centering an identified target location for subsequent high magnification imaging typically requires moving the specimen by many thousands of nm and accurately locating the target to within a few hundred nm. This movement is too large to be achieved using the image shift coils, which would be very accurate, and instead must be achieved using the goniometer.

We have measured the accuracy of the goniometer on the Philips CM200 and the results are shown in fig 1. Data were obtained by selecting a target area from a low magnification image [660x], moving to this target and then measuring the accuracy of the requested movement by cross correlation.
Evaluation of Low Noise Objective Lenses for Detection of Weak Fluorescence
Authors: Nakano Yoshitaro , Sase Ichiro
Published: July 2020

The optimal method for evaluating fluorescence microscope has not been proposed. In this study we developed two quantitative methods using biological and non-biological materials as standard specimens. Based on these methods, a new water immersion lens was tested and its high sensitivity to fluorescence was demonstrated.

First, particles of a fluorescent glass (Luminass-1; Sumita Optical Glass) were used for the standard specimen. Particles with a few micrometer diameter were embedded in silicon oil on a quartz slide glass covered with a quartz glass slip and then observed. Under the fluorescence microscope, the spectra of emission light from the excited area with or without the particle were measured by a monochromator and a photomultiplier tube (PMT). The signal-to-background ratio (S/B) was calculated from these spectra (Fig. 1, Table 1). This specimen showed a significant advantage due to less photobleaching that permits a stable and long observation time.
Parallel Auger Spectroscopy with the Hyperbolic Field Analyser
Authors: Jacka M , Kirk M , El Gomati M , Prutton M
Published: July 2020

The analysis of Auger and other excited electrons in the energy range from about 50eV to 2500eV provides a nearly non-destructive means to determine the surface composition of materials. It is both highly surface sensitive (a few atomic monolayers) and capable of high spatial resolution. This makes it an attractive alternative to energy dispersive x-ray analysis for the examination of ever smaller structures such as in semiconductor devices. For many applications however one important drawback has been the relatively long time required to make useful measurements - usually many seconds or minutes. Spectrum-imaging, in which an entire spectrum is acquired for each pixel in a digital scanned image, is one example for which Auger, spectroscopy becomes a prohibitively lengthy process. Similarly, the study of electron beam sensitive materials is difficult because the measurement time is often longer than the damage time. One method of reducing the measurement time is by acquiring the whole energy spectrum of interest in parallel. This is achieved with the Hyperbolic Field Analyser. (In electron energy loss spectroscopy this approach has lead to the highly successful Gatan energy filter.)
Real-Time Full Motion Stereo Microscopy Over Internet
Authors: Case Gary N , Vouk Mladen A , Mackenzie John M
Published: July 2020

One of the problems with remote imaging which purports to represent the real world (Tele-presence) is that the images are two-dimensional and the “real” world is three dimensional. When viewing many objects, the lack of depth perceptiorican be a serious deficiency. For example, one would want a micro-surgeon to have a good idea how deep to cut into tissue during a tele-presence operation. There is a wealth of three dimensional information in light, SEM, AFM, confocal, and computer reconstructed or simulated objects. To transfer that information faithfully over the networks using broadcast quality high definition video transmission technologies such as MPEG2 may require as much as 20 Mbps per channel. Furthermore, stereo imaging is particularly sensitive to any phase delays and jitter between the two eye channels. Control of these parameters requires advance quality of service features that current internet does not provide, but that may be available on high-performance intranets. All this may present a problem for routine use of the stereo facility.

The next generation of Internet, however, will be able to routinely provision much higher bandwidth and other quality of service, in general through protocols that will allow for not only full screen full resolution television but also the use of two channels (left and right) that can stay in fairly close synchrony.
A Stacked CMOS Active Pixel Image Sensor for Charge Particle Detection and the Application to SIMS
Authors: Yurimoto H , Nagashima K , Kunihiro T , Takayanagi I , Nakamura J , Kosaka K
Published: July 2020

A stacked CMOS active pixel image-sensor (APS) has been developed for detecting various kinds of charged particles and its noise performance has been measured and analyzed. The sensitivity for ions and electrons with keV energy level utilizes for ion microscopy such-as SIMS and electron microscopy, respectively.

Charge particles such as ions and electrons with kinetic energy of keV order are useful probes for surface analysis of material. A measurement system which yields two-dimensional image of charge particles is highly demanded. The conventional two-dimensional detection system is composed of a micro channel plate, a florescent plate which receives multiplied secondary electrons and generates a visible image, and a visible image sensor. However, its limited dynamic range and non-linearity in the ion-electron-to-photon conversion process make a quantitative measurement difficult. The proposed system using a stacked CMOS APS has several advantages over the conventional system such as high spatial resolution, no insensitive time, high S/N, wide dynamic range, nondestructive readout capability, high robustness, and low power consumption.
3-D Reconstruction of Thick IVEM Samples Using Tuned Aperture Computed Tomography
Authors: Gray Jerome W , Grant Ken , Al Gailany A M , Yancey Patricia G , Betterman Wolfram , Webber Richard
Published: July 2020

The development of high and intermediate voltage electron microscopes provides the ability to investigate the ultrastructure of thick biological samples. This produces a unique view of the 3-D interrelationships of cells and organelles. However, the analysis and display of this 3-D information has presented challenges, particularly to quantitative biologists. Major advances in adapting computed tomography (CT) to electron microscopy have provided the major means of 3-D analysis of cellular ultrastructure (1,2). In particular, tremendous gains in determining the structure of macromolecules have occurred with advances in electron microscopy CT (3). However, CT remains an arduous undertaking and one not well suited to analysis of the structural alterations within large populations of cells requiring many reconstructions. We have adapted an alternative approach, Tuned Aperture Computer Tomography (TACT®), for use with high and intermediate voltage electron microscopes. Although with EM specimens TACT® does not usually produce as high Z axis resolution as CT, the simplicity of the TACT® algorithms lend themselves to the rapid analysis of multiple samples. TACT® is already gaining acceptance in radiology (4, 5). Like CT, TACT® uses a series of images of the sample taken from different angular perspectives. However, unlike CT, for TACT® only a few projections are required and thus good reconstructions of beam sensitive material are possible. The quality of the reconstruction is determined by the quality of the input images, the total angular spread, and the individual tilt increments between images. Better images, a greater angular spread, and smaller angles between images will produce more accurate results.
Novel High Resolution Measurement of Bone Mineralite Size and Shape
Authors: Eppell S J , Tong W , Katz J L , Kuhn-Spearing L , Glimcher M J
Published: July 2020

Study of the microscopic structure of bone dates back to the earliest use of the optical microscope. Understanding the complexities of the structure of this material has roughly paralleled advances in microscopic technique. Relatively recently a new powerful microscopic technique, atomic force microscopy (AFM), has been invented. So it should not be surprising to expect a new step in our understanding of bone. There have been several recent publications applying AFM to bone and its constituents. None of these studies have examined the shape of naturally occurring mineralites in bone. The issue we addsgss in this work is the elucidation of the three dimensional structure of the apatite mineralites in bone. Chiefly, two methods have been used previously to ascertain this structure: transmission electron microscopy (TEM) and X-ray diffraction (XRD,). The major difficulty with TEM is that it is a projection technique. Each image only yields information about geometry in a single plane. Landis et al. showed how to circumvent this problem using tomographic TEM. AFM provides a corroborative and perhaps more gentle method of directly measuring the structure of very small bone mineralites. It is possible that the interaction energy of the electron beam with very small mineralites is sufficient to vaporize them. The problem with XRD is that the line broadening used can have contributions from sources not directly related to mineralite size. In particular, if a single mineralite contains more than one crystalline domain, then the line broadening yields a measure of the domain size, not the overall mineralite size.
Early Stages of Bone Bonding to HA-Coated Prostheses
Authors: Porter Alexandra , Benezra Valarie , Spector Myron , Hobbs Linn W
Published: July 2020

The need to optimize adhesion between the femoral stem and the surrounding tissue has led to the development of plasma sprayed HA-coated implants as attachment veichles for hip prostheses. The strength of attachment of bone to the HA surface cannot be fully explained by mechanical bonding at the macroscopic or microscopic level; thus, molecular interactions must contribute significantly to bone bonding, facilitating adhesion at the nanometer level. Observations of apatite crystallites and cell adhesion proteins in the direct vicinity of coated implants indicates that these play a role in modifying the surface for osteoblast attachment and subsequent collagen deposition.

An understanding of the mechanism of “bone bonding” could lead to improvements in the design of HA coatings, leading to enhanced performance of orthopedic implants. Transmission electron microscopy (TEM) has been used to elucidate a sequence of early bone bonding events at the interface of PSHA-coated titanium implants.

The deposition of biological apatite and subsequent formation of a bone/HA bond seem to depend on the dissolution of the coating and the reprecipitation of carbonated apatite. Previous investigations in vitro have shown that the degree of dissolution and reprecipitation decreases as the coating crystallinity increases. Thus, coatings which had undergone heat treatment were used to look at the effect of solubility of the coating on mechanism of bone bonding.
An In Vitro System for the Simulation of Enamel Growth
Authors: Heidel Dan , Burman Blair , Fong Hanson , Sarikaya Mehmet
Published: July 2020

The enamel of human teeth is composed of parallel rods (bundles) of long, closely-packed, ribbonlike hydroxyapatite (HAP) Ca10(PO4)6(OH)2 crystallites oriented normal to the tooth surface. Both the unique structure of the crystallites and the higher order arrangement of the crystallites are believed to be a consequence of interactions between proteins secreted by the enamel forming tissues and the growing crystallites. A better understanding of the precise molecular mechanism that controls enamel crystallite formation could potentially lead to both novel enamel repair strategies and crystal growth control systems. It has been previously reported that the unidirectional flow of calcium ions across a membrane can be used to form ribbon-like structures of a related form of calcium phosphate. We have created a similar model system which utilizes the unidirectional flow of calcium ions to generate ribbon-like HAP crystallites. Ultimately, this system will be modified so that enamel proteins can be introduced into the crystallization milieu to better characterize their role in enamel formation.

SEM and TEM samples were prepared using the experimental setup shown in Fig. 1. Several tubes containing a 50mM HAP solution dissolved in 0.1M HC1 were placed upside down into a 50mM MOPS pH buffering solution at pH 7.0.
Characterization and Analysis of Highly Hydrated, Three Dimensional Cell-Matrix Constructs
Authors: Bellamkonda Ravi V , Balgude Amit , Yu Xiaojun
Published: July 2020

Neural Tissue Engineering involves the use of three-dimensional biomaterial scaffolds for transplantation of neural tissue or for induction of regeneration of severed peripheral or central nerves. There is a paucity of information regarding the optimization parameters for the design of 3D constructs from the mechanical, morphological, or charge perspective such that neurite extension is maximized. In addition, cytoskeletal organization and response of neurons in 3D matrices to various stimuli is not well understood. Our laboratory has been using a combination of electron, force, light, video and confocal microscopy to address these issues. Custom surface rendering image reconstruction techniques were used to reconstruct the three-dimensional structure of neurons extending processes in 3D as visualized by confocal microscopy. We have previously reported that low concentration gels of hydroxyethylated agarose are capable of supported neurite extension from a variety of neural cells in vitro.

Agarose hydrogels were fabricated in concentrations ranging from 0.75%-3.0% (w/v). These highly hydrated gels were prepared for electron microscopy using a variety of sample preparation protocols including freeze drying, and critical point drying and their morphological structure and pore radius was computed using scanning electron microscopy.
Multimodal Imaging of Biomaterials: Correlative Nano-Indentation and Elasticity Measurement with an Atomic Force Microscope
Authors: Parbhu Ashok , Bryson Warren , Lal Ratnesh
Published: July 2020

Nano-mechanical properties of composite biomaterials are governed by the morphological features and chemical distribution of heterogeneous multiple subunits. However, very little is known about such structure-function correlation at the nanoscale. We have used wool fibers as a model of composite biomaterials for such study. Wool fibers provide an ideal two component cylindrical model for such study as their ultrastructure and chemical composition have been well characterized, they have a core cellular component surrounded by an outer cell layer. The core is 16-40 micron in diameter and rich in axially aligned keratin microfibrils. Outer cells have multiple laminar layers, 60-600 nm thick and are distinctly rich in disulfide bonds. In this study, we have used an atomic force microscope (AFM) to examine the micromechanical properties of the different structural components using complementary techniques of nano-indentation and force-volume imaging. AFM images of transverse sections of wool fibers were obtained in ambient environment (Figure 1).

The exo-cuticle showed a significantly higher mechanical stiffness than the internal cellular core region (Figure 2).
Tubulin Structure and the Development of Electron Crystallography
Authors: Downing Kenneth H
Published: July 2020

The identification of tubulin as the major protein component of microtubules (1) occurred at about the same time, a little over thirty years ago, as the first demonstration of the ability to obtain 3- dimensional data from electron micrographs. DeRosier and Klug's paper on image reconstruction of the bacteriophage tail (2) marks what many consider the beginning of electron crystallography - determining protein structures by electron microscopy. Microtubules have long been the target of active study by light and electron microscopy, and even before the discovery of tubulin played a significant role in driving technical developments in EM. The demonstration that tubulin could form two-dimensional crystalline sheets (3) seemed to pave the way for high resolution.structural studies. The discovery of the sheet polymer of tubulin came shortly after Unwin and Henderson's first publications on bacteriorhodopsin showing secondary structure resolved by EM (4). Application of their methods to tubulin in the late 1970's resolved low resolution features of the protein, but technical problems prevented achieving much more than showing the molecular envelope. Many developments in both tubulin studies and electron crystallography followed this work over the next 15 years. As techniques were refined for purifying, labeling and visualizing tubulin and microtubules, the dynamic properties of microtubules and their role in the cell cycle were characterized, and some hints about the mechanism of polymerization and disassembly were derived from biophysical and microscopic investigations.
Insulin Receptor: 3D Reconstruction from Darkfield Stem Images, Structural Interpretation and Functional Model
Authors: Ottensmeyer FP , Luo RZ-T , Fernandes AB , Benia D , Yip CC
Published: July 2020

We have reconstructed the three-dimensional quaternary structure of the complete 480 kDa insulin receptor (IR), complexed with NanoGold-labelled insulin, via sets of electron micrographs obtained by low-dose low-temperature dark field scanning transmission electron microscopy (STEM).

Insulin binding to IR in mammalian cell membranes is essential for its manifold effects such as glucose homeostasis, increased protein synthesis, growth, and development. IR belongs to the superfamily of transmembrane receptor tyrosine kinases that include the monomeric epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR). In contrast, IR and its homologues IGF-1R (insulin-like growth factor 1 receptor) and IRR (insulin receptorrelated receptor) are sub-types of this family that are intrinsic disulfide-linked dimers of two αβ heterodimers. Monomeric receptor TKs are inactive, but are activated by ligand-induced dimerization that results in autophosphorylation. IR-like TKs are also inactive even though they are already dimeric, and are activated by ligand binding without further oligomerization. Insulin binding to the extracellular domain of IR results in autophosphorylation of specific tyrosines to initiate an intracellular signal transduction cascade. However, because the quaternary structure of IR is not known, the structural basis for the mechanism of IR activation by extracellular insulin binding has not been elucidated.

The insulin receptor was purified from human placenta. Bovine insulin was derivatized with NanoGold at the B chain Phel, a location not directly involved in receptor binding. Binding of derivatized insulin to the purified receptor was reduced only slightly compared to binding of the native insulin.
High-Resolution Analysis of Rapidly Frozen Biological Specimens: Capabilities and Limitations
Authors: Andrews SB , Pivovarova NB , Hongpaisan J , Leapman RD
Published: July 2020

The past decade has seen major advances in the analytical capability and utility of both fixed beam and scanning beam electron microscopes. In particular, scanning transmission electron microscopy (STEM) and energy-filtering transmission microscopy (EFTEM) have benefited from the development of devices and techniques—including improved electron optics, sensitive solid-state detectors and new software for imaging and electron energy loss spectroscopy (EELS)—that optimize detection of weak spectroscopic signals arising from biological specimens while minimizing specimen damage. Here we discuss and illustrate some of these advances, especially in the context of structural imaging, detection limits and mapping techniques for the biologically important elements phosphorus and calcium. Analytical microscopy of biological tissues is absolutely dependent on cryotechniques. It is generally agreed that rapid freezing and subsequent low-temperature processing, e.g., cryosectioning or direct cryotransfer of frozen-hydrated specimens, is the most reliable way to preserve the native distribution and organization of biological structures. Equally important, however, as an adjunct to spectroscopic analysis is the use of established low-temperature, low-dose techniques for recording optimized images. By limiting beam exposure, low-dose methods greatly improve the quality of images from fragile, freeze-dried preparations. In this case, the quality and information content of, e.g., cryosections are virtually as good as conventional preparations (Fig 1).
Electron Microscopic Tomography of Whole, Frozen-Hydrated Rat-Liver Mitochondria at 400 kv
Authors: Mannella CA , Hsieh C-E , Marko M
Published: July 2020

Electron microscopic tomography is providing important new insights about the internal structure of the mitochondrion. In particular, the infoldings of the mitochondrial inner membrane (cristae), which are usually rendered as lamelliform baffles, are revealed to have considerable tubular nature. Rather than opening wide to the peripheral compartment (between the inner and outer membranes), the cristae connect to the outside and to each other through narrow (20-30 nm) tubular segments, which can be hundreds of nanometers long. This suggests that diffusion of ions, metabolites and proteins between the intracristal and intermembrane spaces may be restricted.

The earlier tomographic reconstructions were done on conventionally prepared, plastic-embedded specimens, which raises the usual concerns about structural preservation. More recently, we have undertaken tomography of isolated rat-liver mitochondria that have been embedded in vitreous ice (by plunge-freezing in iso-osmotic buffer without chemical fixatives or stains). These frozen hydrated specimens are imaged with a JEOL 4000FX equipped with a Gatan cryo-transfer holder and a Tietz automated data collection system, with a Ik × Ik CCD. For 3D reconstructions, images were recorded at a dose of 5 e−Å2 at 2° increments over the range +/− 60°.
High Pressure Freezing and Freeze Substitution Maintain Structural Details and Protein Antigenicity in Protein-Storing Plant Cells
Authors: Lonsdale JE , Jones RL , McDonald KL
Published: July 2020

The cells of protein-storing plant tissues, including the aleurone of monocots and the cotyledons of dicots, have been widely used to study biological phenomena. The cells of protein-storing tissues share many characteristics including the presence of protein storage vacuoles (PSV) which act as reservoirs for the proteins, lipids and minerals required for germination. Many proteins synthesized in these tissues during germination are transported to the PSV. As a result, protein-storing tissues have become a model system for the study of protein transport.

Research in our laboratory focuses on the protein-storing barley aleurone layer. The aleurone layer is notoriously difficult to prepare for transmission electron microscopy (TEM). Aleurone cells are surrounded by thick cell walls that are difficult to penetrate with fixatives and resins. To increase penetration of chemicals, most of our studies are performed on protoplasts, aleurone cells that have had their walls enzymatically removed. Protoplasts, however, are sensitive to the osmolality of the fixation medium and solutions must be closely monitored during specimen preparation. The development of cells in the aleurone layer adds to the difficulty of preparation for TEM.
Preservation of Biomembranes by High Pressure Freezing ?
Authors: Müller Martin , Listemann Jens , Shimony Eyal , Walther Paul
Published: July 2020

Sample preparation techniques for electron-microscopy (EM) dictate our perception of the microworld: any structural information which is lost or distorted during preparation can not be regenerated later and might lead to wrong interpretation of the observed micrograph.

Cryofixation based procedures for specimen preparation can avoid most of the structural alterations associated with standard techniques based on chemical fixation. The ultrastructure can be represented in a near “native state” thanks to the high time resolution for dynamic cellular events (1).

High pressure freezing (2) permits to cryoimmobilize biological samples up to approx. 200μm thick, in contrast to rapid freezing procedures at ambient pressure that are useful to cryoimmobilize samples up to 10-20 μm thick. The actual samplethickness that can be adequately frozen (=without visible damage due to ice crystal formation in freeze-substituted or freeze-fractured specimens) depends on the concentration of naturally occuring substances that exhibit cryoprotective activities.
Micro- and Cryo-Techniques Prevent the Loss of Structural Information. EM-Studies on High-Pressure Frozen Tissues, Suspensions and Cell Monolayer.
Authors: Hohenberg H
Published: July 2020

Cells are information driven systems. Cellular information is stored in certain molecules, at certain places, in a certain concentration, at a particular time and under given physiological conditions. The goal of biological electron microscopy is to provide this information network, to correlate the cellular ultrastructure and its function. In this sense, it is essential to combine the high resolution of our electron optical instruments with a high information density of the biological system. Most of the structural information is lost in the course of the different preparation steps prior to electron microscopy. For this reason it is necessary that all preparation steps such as: 1. sampling: e.g. excision of tissues, 2. cryoimmobilisation, 3. follow-up procedures: e.g. freeze-fracturing, freeze-substitution and embedding, should have identical high quality levels preventing or minimizing the loss of structural information. To this aim our methodological activities focus on the development of special micro-techniques for the sampling of: a) native tissues, with an automatic fine-needle biopsy technique (1), of b) suspensions, with a special cellulose capillary technique (2), of c) cell monolayer, with a thin film cultivation technique (3) and the application/perfection of cryotechniques (high-pressure freezing (HPF) and freeze-substitution). In particular, the high-pressure freezer (HPM 010, Bal-Tec) has proven to be a highly useful tool for successful cryoimmobilization of almost any kinds of cells and tissues, bulk specimens (< 200 μm in thickness) being included. This freezing technique does not require any cryoprotection, and if combined with our micro-techniques the risk of inducing artefacts as a result of specimen preparation prior to freezing is minimized.
A New Concept and Machine for High Pressure Freezing
Authors: Studer Daniel
Published: July 2020

To date, high pressure freezing is the only reliable cryofixation method for vitrifying bulk biological samples up to 150μm in thickness (1). This proves the enormous potential of the method. Nevertheless, the procedure is not well accepted by the scientific community. It seems that people not completely convinced by the method have reservations to use high pressure freezing machines. The main reasons seem to be that the commercially available machines are noisy, large, very heavy, expensive and consume a lot of liquid nitrogen. Last but not least, a relatively large volume of cryogen is pressurized to 2000bar representing a potential risk of accident. Considering high pressure freezing to be a very potent method, it was tried to establish a new machine avoiding the above mentioned disadvantages.

The first functional high pressure freezing apparatus was freezing fine metal rods with a boring of 0.2mm in diameter (2). This boring was filled with suspensions, the rod was connected to a pressure generator thus pressurizing the suspension, and simultaneously the metal rod was frozen from the outside by a low pressure jet of liquid nitrogen. The big disadvantage of the method was that only freeze-fracture experiments could be performed at the time because the sample could not be removed from the boring. As a consequence, this approach was never commercialised. In the mean time sample preparation has improved. Cellulose capillaries can be used to freeze suspensions and tissue samples (3,4). To freeze these capillaries in the new machine, they are introduced in a sample holder which consists of a copper tube with an inner diameter of 0.3mm and a length of 16mm. This tube fits into a concentrical stainless steel tube which stabilises the copper rod and helps to fix the sample in the cryofixation device.
High Pressure Freezing/Freeze Substituion: Comparison of Chemical Fixation Versus Cryoimmobilization of Candida Albicans Cultured in Cellulose Tubing
Authors: Erlandsen S , Holzer A , Gavin M , Frethem C , Wells C
Published: July 2020

In the interactions of Candida albicans with host cells, the cell wall of the yeast may play important roles in the adhesion of yeast cells to tissues. The outer cell wall of yeast (e.g. Saccharomyces cerevisiae, C. albicans) has been shown to consist of a dense network of radially projecting fibrils composed of mannoproteins that are known as fimbriae and which previously have required cryopreservation either by jet propane freezing or by plunge freezeing for their visualization. High pressure freezing provides an advantage over jet or plunge freezing in terms of the higher consistancey in the quality of freezing, and the minimization of formation of ice I with this method. Hohenberg et al reported a method utilizing cellulose capillary tubes to cryoimmobilize suspensions of microoganisms by high pressure freezing (HPF) and freeze substitution (FS), and herein, we describe an adaptation of this method by culturing microorganisms within the tubing to increase cell density prior to high pressure freezing and freeze substution.
A Pumping Station for Cryo-Transfer Specimen Holders
Authors: Melvin G , Talbot T
Published: July 2020

Cryo electron microscopy has become an important technique for studying the structure and composition of biological specimens in their frozen hydrated state. Many modern transmission electron microscopes can be modified for cryogenic analysis by incorporating liquid nitrogen (2) cooled anti-contaminator blades and a special 2 cooled cold stage specimen holder. These specimen holders are used routinely for the examination of frozen hydrated specimens, and many laboratories have several such specimen holders to permit efficient handling of multiple specimens. Typically, these devices are cooled conductively via intimate contact with a reservoir of 2 inside of a small-evacuated dewar. The periodic evacuation of these dewars is one of the primary purposes of the pumping station. In addition, a means of storing the cryo holders under low vacuum is an important design feature of the pumping station.

In our experience the commercially available unit for pumping and storing the cryo holders proved unsatisfactory in two important aspects. The commercial unit made no provision for isolating the pump vibrations from the fragile cryo holders. Occasionally, this resulted in damage to the devices and subsequent costly repairs. Economically, the mechanical arrangement of the ports and associated valves were not as convenient to operate as they could have been. The purpose of this paper is to describe a custom pumping station that was designed and built with off-the-shelf components that overcomes the shortcomings of the commercial unit.
Three-Dimensional Structure of Neurospora Mitochondria: New Insights Provided by Electron Tomography of the Frozen-Hydrated Organelles
Authors: Nicastro D , Frangakis A S , Nickell S , Baumeister W
Published: July 2020

Electron tomography is the most widely applicable method for obtaining three-dimensional information by electron microscopy. It is, in fact, the only method suitable for investigating pleomorphic structures, such as many supramolecular assemblies, organelles and cells. With the recent development of automated low-dose data-acquisition schemes, it is now possible to study molecules and cells embedded in vitreous ice. This opens up new horizons for investigating the functional organization of cellular components with minimal perturbation of the cellular context.

In the present study we used automated electron tomography in conjunction with cryomicroscopy to reveal the internal organization and ultrastructure of mitochondria. The whole isolated cell organelles from Neurospora crassa were quick-frozen and examined in vitrified ice. Single-axis tilt series were recorded with a Philips CM 120 Biotwin under low-dose conditions; the estimated total exposure was 10,000 e−nm−2. The tilt increment was 1° and the tilt series ranged from −65° to +65°. In order to enhance the contrast of the rather thick samples zero-loss energy filtering was employed. The images were recorded with a Ik × Ik CCD camera. The experimental setup has been described by Koster et al2 Three-dimensional reconstructions were performed by weighted backprojection. Prior to the three-dimensional visualization of 3-D data sets a denoising technique based on nonlinear anisotropic diffusion was applied (Figs. 2, 3).
Comparative TEM of Freeze-fractured Colloidal Liquid Aphrons and Conventional Emulsions
Authors: Srivastava P , Heckman JW , Worden RM
Published: July 2020

Colloidal Liquid Aphrons (CLAs) are composed of surfactant doped non-aqueous inner cores surrounded by thin surfactant films. These are created by intense stirring of the non-aqueous phase in an aqueous surfactant solution. Large interfacial areas, due to small size, and high stability (much greater than conventional emulsions) make aphron dispersions attractive for a variety of mass transfer limited processes. The unusual stability of CLA dispersions has been attributed to the multi-layered structure that includes an entrapped water shell around the non-aqueous core as postulated by Sebba. However, no direct evidence for the structure of the surfactant-stabilized interface surrounding the CLAs has been previously published.

CLAs were prepared as previously reported. Comparative conventional emulsions were made similarly, with the lipid phase free of the Tergitol®(15-S-3) surfactant used in the CLAs. Aliquots (≈30 μl) of the dispersions were spread on 0.15 mm thick, 10 mm diameter copper supports These supports were ultra-rapidly frozen via plunging in liquid propane(−190°C). The frozen samples were inserted onto a pre-cooled rotary stage of a freeze-etch unit (modified Balzers BA-510) and fractured by knife at 150°C with a residual pressure of approx. 6.5 x 10−5 Pa. The replicas were shadowed with ≈2nm of Pt at 30° followed by 10-15 nm of carbon by arc evaporation. The resulting replica was washed in an 10% graded isopropanol series, rehydrated, washed in chromic acid, rinsed in de-ionized H2O, and then mounted on 300 mesh grids. TEM was performed on a JEOL 100CX operated at 120keV.