bluuebunny/crossref_metadata_2025_split huggingface dataset:
An In Vitro System for the Simulation of Enamel Growth
Authors: Heidel Dan , Burman Blair , Fong Hanson , Sarikaya Mehmet 
Published: July 2020

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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 (&lt; 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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.