bluuebunny/crossref_metadata_2025 huggingface dataset:
Microscopic Detection of Early Cancerous Changes in Bladder Tissue by Autofluorescence
Authors: T.Arendt Joseph , M.Cothren Robert
Published: July 2020

The intrinsic fluorescence achieved without dyes (autofluorescence) of bladder tissue, benign mucosa and the various stages of transitional cell carcinoma (TCC), is being characterized. D'Hallewin, et al., and Koenig, et al., are testing autofluorescence spectroscopy for detecting bladder carcinoma using an optical fiber probe inserted down the auxiliary channel of an endoscope. These studies do not include fluorescent microscopy to directly examine the fluorescing structures (fluorophores) within the tissue. Therefore, frozen unstained sections are being imaged on a fluorescence microscope to identify and reveal the morphology and location of the fluorophores. Tissue biopsies taken at endoscopy are immediately frozen in liquid nitrogen. Later, the tissue is cut into 5 μm sections. Serial sections are alternately imaged frozen on the fluorescence microscope or stained with hematoxylin-and-eosin (H&E). The H&E stained slides are reviewed by a pathologist.

A 75-W xenon arc lamp fitted with a 380-nm narrow-band interference filter is coupled to microscope slides on the stage of an Olympus MT-2 inverted microscope.
Confocal Raman Line-Scanned Imaging of Complex Geological and Histological Thin Sections
Authors: Adar Fran , Bernard Roussel , Wang Alian , Hawi Shari , Nithipathikom Kasem
Published: July 2020

Chemical imaging of complex multi-component materials has important potential for the analyst in many fields of research. Raman imaging is of particular interest for several reasons. The Raman spectra contain detailed information on chemical species and crystalline phase. Because the Raman effect is excited by optical radiation, the spatial resolution, which is proportional to the wavelength of the light, is better than 1 μm. and with near field optical techniques currently under development, there is potential for even higher spatial resolution in the chemical image.

The methods used to produce an image fall into essentially two categories - global imaging and confocal mapping. When creating global images, a large area of the sample is bathed in laser light. The light scattered by the sample is filtered to select a Raman band, and then that light is used to create an image of the sample on a two-dimensional detector.
Volumetric Raman Spectral Imaging With a Confocal Raman Microscope: Image Modalities and Applications
Authors: Brenan Colin JH , Hunter Ian W , Korenberg Michael J
Published: July 2020

Confocal Raman microscopy is an invaluable tool for non-invasive three-dimensional (3-D) analysis and visualization of chemically heterogeneous samples. The confocal Raman microscope (CRM) combines focused laser illumination with spatially filtered detection to define in the sample a localized volume element (voxel) from which scattered or reflected light is detected. Attenuation of light from outside the voxel by the pinhole spatial filter increases image contrast and results in voxel sizes typically <10−18 m3. Moving the specimen relative to the voxel and recording the scattered light Raman spectrum at each scan position generates a Raman spectrally-encoded 3-D sample image for non-invasive in situ differentiation between optically similar yet chemically distinct materials.

The motivation for the present work stems from our application of continuum modeling techniques as the mathematical framework for development of physiologically realistic biosystem models. A Type 2 correlation-based confocal scanning laser microscope (CSLM) integrated with a high performance, parallel-drive micro-motion robot (MR-1) was built for spatially-distributed acquisition of optical and mechanical data within a < 1 mm3 workspace volume.
Characterising the Morphology of Polymer Articles Using Polarised FTIR Reflectance and Raman Microscopies.
Authors: Everall NJ , Chalmers JM
Published: July 2020

The optical and mechanical performance of polymer articles is often very strongly influenced by the crystallinity and degree of molecular orientation in the polymer. It is therefore important to have available techniques for measuring and mapping these parameters over article surfaces and through wall thicknesses, on scales from centimetres to microns. It is also desirable that these techniques do not in themselves perturb the morphology of the polymer, for example by introducing excess orientation or crystallinity through sectioning.

In this paper we will show how Raman microscopy can provided a useful approach for mapping polymer crystallinity on the micron scale, particularly when coupled with multivariate calibration techniques for data analysis. The acquisition and treatment of data to obtain crystallinity information will be described in detail. We will also describe the development and application of polarised FTIR specular reflectance and attenuated total reflection (ATR) microbeam techniques for quantifying and mapping surface orientation of polymer articles.
Quantitative Analysis of Metal Nanoparticles
Authors: Lyman Charles E
Published: July 2020

Analysis of nanosize particles by x-ray emission spectrometry in the analytical electon microscope (AEM) is limited by the level of electron current that can be placed in a 1-2 nm excitation beam and by the consequent electron beam damage. The current in the beam limits the number of x-ray counts generated in the particle, while the beam damage may cause changes in particle chemistry, structure, orientation, and location. The objective of “ultimate AEM analysis” is to make useful measurements right up to the limit where beam damage prohibits analysis.

Elemental analysis of sub-10nm metal particles supported on a ceramic support requires a field-emission source operating at 100-300 kV to generate a significant number of x-ray counts from the area under the beam in a reasonable time [1]. The high current density in the beam of such an instrument can cause damage to the particle or to the underlying support material.
Capabilities and Limitations of Biological Electron Energy-Loss Spectroscopy
Authors: Leapman RD , Andrews SB
Published: July 2020

Perhaps the ultimate aim of analytical electron microscopy in biology is to detect single atoms or ions bound to isolated macromolecular assemblies and small cellular organelles rapidly frozen in their native state. Although the meaningful spatial resolution in such analyses is limited to ∽10 nm or more by radiation damage, the high intrinsic sensitivity of electron energy-loss spectroscopy (EELS) coupled with recent developments seems to make this rather ambitious goal—originally proposed some twenty years ago—within reach. Here we describe examples where EELS has been able to detect surprisingly small numbers of atoms in biological specimens and discuss some fundamental limits that are encountered as well as some possible strategies for circumventing these difficulties.

In our laboratory we have used a scanning transmission electron microscope (STEM) equipped with a field-emission source and parallel-detection EELS because the probe size and collection efficiency of this instrument are optimized to give the lowest detectable number of atoms and lowest detectable atomic fraction.
X-Ray Microscopy Projects In Aarhus
Authors: Medenwaldt R , Abraham J , E.UggerhØj E , Vollrath F , SØgaard E
Published: July 2020

X-ray microscopy with soft X-rays is well suited for investigations of aqueous samples of some microns thickness when the resolution is required to be better than in visible light microscopy an not better than 30 nm. Sample preparation is as simple as for light microscopy, i.e. no fixation or metal coating is needed.

In Aarhus, an X-ray microscope is used for investigations in fields as biology, medicine and soil sciences. A ray diagram of the Aarhus X-ray microscope is shown in Fig.l. Synchrotron radiation at a wavelength of 2.4 nm from the Aarhus Storage Ring is focused by a condenser zone plate onto an object. Another zone plate as an objective behind the object forms the image on a CCD camera. Objects are located under atmospheric pressure. For dry samples, almost any kind of holder can be mounted in the microscope. Wet samples are placed between thin silicon foils in a sealed chamber. With typical liquid layer thickness of 5-15 µm, samples can be kept in the chamber for many hours without drying out.
What X-Ray Microanalysis Can Tell Us About The Regulation of Intracellular Calcium
Authors: Bailey GW , Dimlich RVW , Alexander KB , McCarthy JJ , Pretlow TP , Andrews SB , Pivovarova NB , Pozzo-Miller LD , Leapman RD
Published: July 2020

Many cell functions, including such critical processes as secretion, neuronal communication, muscle contraction, and even gene expression, are regulated by spatial and temporal changes in the intracellular concentration of Ca2+ ions. The high spatial resolution and analytical sensitivity of energy dispersive x-ray (EDX) microanalysis are ideally suited to characterizing biologically relevant changes in total concentration of cellular Ca. However, EDX results have not yet had a wide impact in the field of Ca regulation, with most laboratories preferring to focus on optically measured changes in free Ca concentrations as the important determinant of biological activity. This paper advocates a more positive perspective for the role of EDX microanalysis in studies of Ca regulation.

It is well known that the large majority of cellular Ca is in the bound form, with typical bound/free ratios on the order of l03-104 5. However, the most dramatic changes during Ca2+ -dependent cell activation occur as transients in the free Ca2+ pool.
Elevated Cytoplasmic Sodium and Chloride Is Associated With Purinergic Receptor Stimulation of Rat Ventricular Myocytes: Implications For Myocardial Ischemia
Authors: Bailey GW , Dimlich RVW , Alexander KB , McCarthy JJ , Pretlow TP , Bond Meredith , Zhang Bin-Xian , Desnoyer Russell W
Published: July 2020

During myocardial ischemia, several factors have been identified which contribute to the development of cardiac arrhythmias. These include membrane depolarization, increased cytosolic Na+ and decreased intracellular pH. This increase in intracellular Na+ has been shown to be associated with the development of ventricular arrhythmias. Changes in intracellular Cl- may also be involved in development of cardiac arrhythmias since decreasing extracellular Cl- concentration in the perfusion medium can prevent development of arrhythmias during myocardial ischemia in vitro. Extracellular ATP is known to increase in the heart during myocardial ischemia. ATP activation of p2 purinergic receptors on cardiac myocytes has been proposed to contribute to the initiation of the arrhythmias and ventricular fibrillations which characterize myocardial ischemia. Activation of p2 purinergic receptors results in membrane depolarization and decreased intracellular pH, thus reproducing some of the changes that occur during ischemia. We recently showed that activation of p2 purinergic receptors in both quiescent and electrically stimulated ventricular myocytes triggers spontaneous oscillatory contractions and Ca2+ transients.
Recording, Processing and Extracting Information From Sequences of Spatially Resolved Eels Spectra
Authors: Brun N , Colliex C , Suenaga K , Tencé M , Bonnet N
Published: July 2020

The sophisticated acquisition procedures now available in time or space resolved spectroscopies, also known as spectrum-imaging modes, produce large amounts of data which require specific developments for efficient processing and information extraction. For instance, in electron energy-loss spectroscopy (EELS), a line-spectrum consists of typically one hundred spectra recorded at regular intervals when scanning the subnanometer incident electron probe across the feature of interest: interfaces, multilayers or nanostructures of various shapes and dimensions. The useful information in any of these spectra depends on many factors such as the problem under investigation, the involved energy-loss range or the signal-to-noise ratio of the different features. However it is generally contained in the spectral changes, as well in energy channel as in position along the sequence.

To detect, measure and identify these variations, new methods have to be developed and the accompanying algorithms to be implemented. A first category encompasses all the routines which apply successively to all spectra in the sequence the well-known software which have been elaborated for processing individual spectra.
Energy-Filtered Imaging and Spectrum Imaging: Which One Should I Choose?
Authors: Crozier PA
Published: July 2020

There are now several commercially available methods for performing spatially resolved electron energy-loss spectroscopy in a transmission electron microscopy. In the energy-filtering electron microscope (EFEM), an Omega filter (Zeiss 912) or a post-column magnetic-prism filter (Gatan Imaging Filter) forms an electron image with electrons of a selected energy loss. A series of such images are acquired to provide spatially resolved information on sections of the energy-loss spectrum. These images can be processed to yield elemental maps from areas of interest in the material. In the spectrum imaging method (EMISPEC System), a small focused electron probe is rastered over the sample and a parallel energy-loss spectrum acquired from each point in the selected area. These spectra are then batch processed and information like elemental distributions extracted and displayed in the form of a map. Each of these two approaches has advantages and disadvantages and the appropriate choice is often dictated by factors such as time constraints, sample stability and the nature of the information of interest.
Electron Energy-Loss Spectrum Profiling of Polymer-Polymer Interfaces
Authors: Siangchaew K , Arayasantiparb D , Libera M
Published: July 2020

The spatially-resolved structure and composition of amorphous or semicrystalline polymer-polymer interfaces are poorly understood. The absence of atomic-level periodicity precludes many of the transmission electron-optical techniques that have been so fruitful in studying interfaces in polycrystalline inorganic materials. Similarly, there has been relatively little work done on spatially-resolved spectroscopic analysis of polymer-polymer interfaces to determine chemical widths, largely because of concerns over electron-beam damage. Digital microscope control and parallel spectral acquisition provide for low-dose exposure, quantification of dose, and efficient data collection which open new windows to study polymer interfaces.

This research is studying various multiphase polymer systems by focused-probe spectroscopic analysis to identify appropriate spectral features, data acquisition protocols, and background-modeling/data-processing algorithms in order to establish chemical widths characteristic of specific polymer-polymer interfaces (1). This research uses a 200keV Philips CM20 FEG TEM/STEM with a Gatan 666 PEELS spectrometer and an Emispec digital data acquisition/control system.
Eels Study of Valence State and Oxygen Vacancies In Magnetoresistive Oxides
Authors: Wang ZL , Yin JS
Published: July 2020

Colossal magnetoresistive (CMR) has been observed in a class of oxides, La1-xAxMnO3 (A = Ca, Sr, or Ba). Transition and rare earth metal elements with mixed valences are usually present in these materials for stimulating structural and chemical evolutions, leading to specific functionality. The partial substitution of trivalent La3+ by divalent element A2+ is balanced by the conversion of Mn valence states between Mn3+ and Mn4+ and the creation of oxygen vacancies as well, since the ionic structure of La1-xAxMnO3-y is proposed to be

in which the valence conversion is the key for determining the material's properties. In practice, quantifying of oxygen vacancies is a challenge to existing microscopy techniques particularly for thin film specimens because of the strong effect from the defects at the substrate-film interface and the surface disordering.
Intermediate Voltage Aem's: Have They Fulfilled Their Potentials?
Authors: Zaluzec Nestor J
Published: July 2020

For the last few years analytical developments in intermediate voltage AEM's have remained relatively quiescent. We have instead seen a number of improvements in the basic microscope mainly along the lines of microprocessor controls, automation, and of course improvements in image resolution and electron sources. Since the spectroscopic technologies (XEDS, EELS) have seemed to reach a mid-term plateau it is appropriate to consider whether or not we have achieved the expectations in these methodologies that we predicted nearly a decade ago.

In XEDS, there are three topics to consider: signal, sensitivity and quantification. In AEM's employing conventional electron sources we expected that as a function of accelerating voltage (E0) we should realize a continuous increase in intensity even though the ionization cross-section decreases with kV, and this has been realized (figure 1). It occurs simply due to the fact that the increase in probe current with E0 is more rapid than the more slowly decreasing ionization cross-section.
Rocking-Beam Spectrum Images and Alchemi of Ni50Al40Fe10
Authors: Anderson Ian M , Bentley Jim
Published: July 2020

A spectrum image can be acquired with a scanning transmission electron microscope (STEM) by translating a focused probe over a two-dimensional area of a specimen and acquiring a spectrum at an array of positions of the probe. Analogously, a rocking-beam spectrum image is formed when the incident electron beam is tilted relative to the specimen and a series of spectra is acquired from a single volume of the specimen. The orientation-dependence of such a series of spectra can be used to extract the atomic site-distributions of an ordered alloy or compound. One of the drawbacks of spectrum image acquisition is that the raw images are typically files of many megabytes, whereas the actual information of interest may be reducible to a fraction of this size. The dimension of the data set may be reduced by a number of methods. For example, Rossouw and coworkers have acquired rocking-beam X-ray maps for the characterization of atomic site-distributions in titanium aluminide intermetallic alloys.
Elemental Mapping at Grain Boundaries in Alloy X-750 by EFTEM
Authors: Bentley J , Evans N D , Kenik E A
Published: July 2020

Alloy X-750 is a γ -strengthened, nickel-base alloy used in demanding high-temperature applications. The HTH commercial heat treatment condition provides excellent strength and good corrosion resistance. However, the resultant precipitate structure is complex with fine (˜20 nm) intragranular γ' phase and copious intergranular precipitation of at least four phases (γ', M23C6, M23B6, and TiN). The intergranular precipitation causes localized grain boundary migration and results in a convoluted grain boundary structure. Such complex grain boundary microstructures increase the difficulty of phase identification and interfacial composition measurements by traditional analytical electron microscopy methods. Elemental mapping by EFTEM is a useful additional or alternative technique for characterizing such structures. A Gatan Imaging Filter (GIF) on a Philips CM30 (LaB6) was used in the current investigation. Experimental details have been summarized elsewhere.

Elemental maps of Cr, Ti, and Ni (net L23 intensities) are presented in Fig. la-c for a typical grain boundary region of a HTH heat treated specimen.
Practical Factors Affecting the Quantification of Elemental Concentrations in Beam-Sensitive Ceramics by EFTEM
Authors: Rice P M , Alexander K B
Published: July 2020

In order to obtain high spatial resolution microchemical data from beam-sensitive materials, alternative techniques which avoid the use of fine probes, such as energy-filtered imaging techniques must be used. Robust quantification procedures are currently under development. The quantification procedure which yields images whose intensities are proportional to the elemental concentrations (atoms per unit volume) has already been determined. The net core-loss intensity images, Si, can be corrected for diffraction contrast by normalization with low loss images, whereas thickness variations are corrected by normalization with t/λ, maps. This procedure has been shown to give the correct value for the concentration ratio between two phases for a single elemental map.1 Determining concentration ratios between two elements in the same phase can actually be simpler, in theory, than the above procedure since it only requires dividing the net core-loss intensity images of two elements and correcting for the difference in cross-section (Cx/Cy = [Sx/Sy][(σy/(σx])- However, there are a few practical factors that must be considered in practice.
Enhanced Esi-Detection of Metal Using the Core-Edges at Energy-Loss Below 100 Ev
Authors: Troester H , Haking A , Richter K , Probst W , Trendelenburg M F
Published: July 2020

ESI (electron spectroscopic imaging)-detection methods are mainly based on the core edges above 100 eV (see, as an example, Ref. 1). However, it would be of major interest to use the high intensity values in the low-loss region below 100 eV. There are at least two elements of general interest for biological applications showing core edges in this region: Au (O2,3 ) and Fe (M 2,3 )• Since colloidal gold and ferritin are used to label targets in biological samples at high spatial resolution we started our study aiming at iron-detection (iron-maximum at 62 eV comparable to gold-maximum at 65 eV).

Ferritin (Ref. 2) contains about 200 to 2000 iron atoms in a densely packed core representing an ideal prerequisite for ESI-detection. Furthermore, ferritin used to be a common marker in conventional transmission electron microscopy in the past. The contrast produced by ferritin in a conventional transmission electron microscope (CTEM) is not specific for iron but only for a dense region in the specimen.
Quantitative Convergent Beam Electron Diffraction (CBED) Measurements of Low-Order Structure Factors in Nickel
Authors: Saunders M , Fox A G , Midgley P A
Published: July 2020

The introduction of quantitative CBED techniques in recent years has led to experimental studies of charge densities in crystalline materials with unprecedented accuracy. Despite these successes, the development process is still on-going with the aim of gaining a better understanding of the techniques. With this in mind, we have undertaken a systematic study of the low-order structure factors of nickel using the ZAPMATCH zone-axis pattern matching technique of Bird and Saunders. In this approach, a set of low-order structure factors (both elastic and absorptive components) is adjusted until a best-fit is obtained between a many-beam simulation and an elastic filtered zone-axis pattern. Additional higher-order structure factors are included to converge the scattering potential but are kept fixed at neutral atom values during the fit. The questions we set out to address are (i) how to choose the correct number of structure factors to refine from a given data-set, (ii) are the absorptive structure factor components we refine of any use, and (iii) can we determine Debye-Waller factors at the same time as we measure the charge density? The first two points are discussed here.
The Relation Between the Phase of the Electron Wave (Which is Lost When an Em Image is Recorded) and the Preserved Crystallographic Structure Factor Phases
Authors: Hovmöller Sven , Zou Xiaodong
Published: July 2020

The phase problem in X-ray crystallography is one of the most interesting and most studied mathematical problems that exist. There is still today no general solution of the phase problem, but partial solutions have resulted in at least 8 Nobel Prizes, even though one of the most prominent solutions, the Patterson function, was not awarded the Prize.

It has frequently been claimed that there should be a phase problem also in electron microscopy. It may be more correct to say that there is no phase problem, only a phase confusion problem in electron microscopy. The phase confusion problem has arisen since the word phase is used for (at least) two very different physical entities in the field of electron microscopy. When crystallographers speak about phases, they mean the crystallographic structure factor phases, while physicists mean the wave front phases. In order to resolve the phase confusion problem, it is necessary to define clearly which phases are meant.
Direct Phasing Determination of Si(111)-(3X1)-Ag Surface Reconstruction
Authors: Bailey GW , Dimlich RVW , Alexander KB , McCarthy JJ , Pretlow TP , Grozea D , Collazo-Davila C , Marks L D
Published: July 2020

The Si(111)-(3x1) reconstruction, observed for submonolayer coverage of all alkali metals and Ag, has been an attractive subject to study. Their almost identical low-energy electron diffraction (LEED) I-V curves suggest a similar surface structure, adsorbate size-independent. Moreover, filled-state scanning tunneling microscopy for Li, Na, Ag show similar features, double rows of maxima, resembling zigzag chains, and a pi surface unit cell symmetry. The atomic structure of this reconstruction is still controversial. Several models, most of them based on a π-bonded silicon chain, such as the Seiwatz or extended Pandey chain, have been proposed. This study attempts to determine this structure using direct phasing of transmission electron diffraction (TED) patterns.

Transmission electron microscopy silicon samples were cleaned and the Si(111)-(3x1)-Ag structure obtained in a surface science system, SPEAR, under ultrahigh vacuum conditions. Using the attached Hitachi UHV H-9000 microscope, through exposure series diffraction patterns were recorded and reduced with a cross-correlation technique.
Structure of the TiO2(100)-1X3 Surface Determined by Direct Methods
Authors: Bailey GW , Dimlich RVW , Alexander KB , McCarthy JJ , Pretlow TP , Landree E , Marks L D , Zschack P
Published: July 2020

An atomic model of the TiO2(100)-1x3 surface has been determined based on Grazing Incidence X-Ray Diffraction (GIXD) data analyzed by direct methods coupled with χ2 minimization. The final model has every surface octahedral site occupied, and resembles the NaCl structure, in contrast to the normal structure of bulk rutile which has half of the sites occupied (Fig. 1). This structure is non-stoichiometric which is consistent with the observed spectroscopic data that indicates that the surface is titanium rich. The structure contains edge and face sharing octahedra, present in other known defect structures in TiO2, and appear consistent with what has been observed in Scanning Tunneling Microscopy.

The data consisted of 19 measured structure factors acquired from GIXD. The phases for the measured beams were calculated using a Sayre equation with Unitary Structure factors and a Genetic Algorithm global search technique. Based upon the calculated phases, charge density maps were constructed and used to determine the positions of titanium atoms within the unit cell (Fig. 2).
Current State of the Art of Digital Imaging in TEM
Authors: Fan G Y , Ellisman M H
Published: July 2020

The increasingly wide use of digital computers, the world-wide-web and electronic publishing has had a fundamental impact on the way scientists conduct research in every discipline of science. Electron microscopy is no exception. A considerable amount of effort has been devoted to the development of digital imaging acquisition systems for transmission electron microscopy (TEM). Digital image acquisition systems for TEM, including complete systems, have been produced by several companies, including: Advanced Microscopy Techniques (Rowley, MA), JEOL (Peabody, MA), Gatan (Warrendale, PA), Princeton Instruments (Trenton, NJ) and Tietz-Video (Herbststrasse, Gauting, Germany). While most systems are CCD-based, JEOL has also offered a system which is based on the Imaging Plate technology.

The Imaging Plate has the same size as, and is compatible with the camera system for, the 8.09 cm × 99.6 cm electron microscope film. As with film, a latent image is formed on the plate when exposed to electrons. A stack of exposed imaging plates are then taken out of the microscope and scanned by a laser beam in a readout device which converts the latent image to a digital form.
Measurement of Point-Spread Function of IP-System
Authors: Oikawa T , Hosokawa F , Taniyama A , Shindo D , Kersker M
Published: July 2020

For the resolution estimation of the Imaging Plate (IP)-system, several methods were carried out. One of them is the measurement of the modulation transfer function (MTF) from the phase contrast transfer function taken with an amorphous Ge film specimen. One of another is the measurement of the point-spread function (PSF) from the auto-correlation of the Poison noise obtained by the IP-system.

In this experiment, the PSF of the IP-system were measured from the intensity distribution recorded with a 1.6 μm-diameter electron probe on the IP.

When recording the electron intensity with a small enough electron probe as compared with the pixel size of the IP (25 μm × 25 μm), a single pixel detects the electron signal in case of ideal IP-resolution which is limited only by the pixel size. However, practical IP-resolution is limited by the electron diffusion, x-ray emission and the laser beam diffusion in the IP, and so on. Therefore, it is possible to measure the PSF from the intensity distributions of the patterns recorded with a small enough electron probe.
Approaches for High Resolution Cryo-Electron Tomography of Biological Specimens
Authors: Agard DA , Braunfeld MB , Chen Hans , McQuitty Rebecca , Sedat John
Published: July 2020

Electron tomography is a powerful tool for elucidating the three-dimensional architecture of large biological complexes and subcellular organelles. Use of intermediate voltage electron microscopes extended the technique by providing the means to examine very large and non-symmetrical subcellular organelles, at resolutions beyond what would be possible using light microscopy. Recent studies using electron tomography on a variety cellular organelles and assemblies such as centrosomes (Moritz et al.,1995a,b), kinetochores (McEwen, 1993) and chromatin (Woodcock, 1994), have clearly demonstrated the power of this method for obtaining 3D structural information on non-symmetric cell components. When combined with biochemical and molecular observations, these 3D reconstructions have provided significant new insights into biological function.

Although the information that tomography provides is unique, its use as a general tool in the biological community has been limited due to the complexities involved in data collection and processing.

We are simultaneously trying to make this approach accessible through automation as well as trying to extend the resolution and accuracy of the reconstructions. Significant, has been the use of low-dose cryo-electron microscopic automated data collection methods.
Automated CCD Data Collection and 3D Reconstruction of Aleutian Mink Disease Parvovirus
Authors: Olson NH , Chipman PR , Bloom ME , McKenna R , Agbandje-McKenna M , Booth TF , Baker TS
Published: July 2020

Acquisition of CCD image data from frozen-hydrated specimens provides several advantages compared with the use of photographic film. CCD cameras possess excellent linearity, sensitivity and dynamic range. The CCD also allows one to pre-check the quality of a cryo sample much easier than with a TV rate camera or on film. However, the limited field of view (IK2 pixels) of the CCD makes data collection difficult. Gatan, Inc. has produced a LowDose software package, in conjunction with their DigitalMicrograph™ environment, that automates this process and allows for efficient collection of larger amounts of data. This software, along with our own image analysis software, has facilitated the computation of a three-dimensional reconstruction (3D) from images of baculo-expressed, VP2 particles of Aleutian mink disease parvovirus (ADV).

ADV has a capsid (∼28.5 nm diam.) that contains two proteins (VP1-2) of which ∼90% is VP2. ADV infections lead to plasmacytosis in mink and are a significant problem worldwide.
Three-Dimensional Time-Lapse Digital Movie Analysis of the Developing Fruit Fly Eye in Organ Culture
Authors: Archie Pollock John , Maneckshana Bejon T , Leonardo Teresa E
Published: July 2020

The compound eye of the fruit fly, Drosophila melanogaster, is composed of a highly ordered array of facets (FIG. 1), each containing a precise set of neurons and supporting cells. The eye arises during the third larval instar from an undifferentiated epithelium, the eye imaginai disc, which is connected to the brain via the optic stalk (FIG. 2). During eye development, movement of the morphogenetic furrow, progressive recruitment of specific cell types and the growth of photoreceptor axons into the brain are each dynamic processes that are routinely studied indirectly in fixed tissues. While stereotyped development and the ‘crystalline’ like structure of the eye facilitates this analysis, certain experiments are hindered by the inability to observe developmental processes as they occur. To overcome this limitation, we have combined organ culture with advanced microscopy tools to enable the observation of eye development in living tissue.
Bioimage: Towards a New Data Base of Biological Volumes
Authors: José María Carazo
Published: July 2020

This presentation introduces the current work of the Bioimage Corsortium and its international collaborators in the setting up of a new data base containing volumes of biological specimens.

The scope of the data that we will be organizing during the next three years is very broad, ranging from macromolecules to larger structures or even entire cells. The approach is designed to combine the desire to break many undesired formal barriers between the type of volume information produced at the different resolution levels whith, at the same time, the clear need of implementational flexibility required to appropriately handle different types of data.

The internal organization of the Bioimage Consortium clearly reflects the objectives presented above. In the first place, partners in the consortium have the scientific knowledge and projection in areas ranging from macromolecular structure to cell dynamics. These partners are then nucleated around two well-defined “servers”, one in Madrid, responsible for the work on macromolecular structures, and the other in Heildeberg, responsible for data more in the area of cell biology.
3D Reconstruction from Single Particles: Improved Orientation Determination from Noisy Images
Authors: Ottensmeyer FP , Timmer M , Allen K , Fernandes AB , Moremen KW
Published: July 2020

The correct reconstruction of the 3D structure from electron micrographs of sets of individual macromolecules or macromolecular assemblies depends crucially on the alignment and orientation determination of the individual images. The lateral alignment from one image to the next is generally easily accomplished by calculating the centre of mass of the individual molecular micrographs. For relatively noise-free pairs of images of unknown structures the sinogram correlation function provides common axes which can determine a correct angular orientation for any set of three images, building consecutively to a complete orientation determination for all images [1]. Low-dose images, however, are inherently noisy. For noisy images the angle determination becomes more problematical, since any fourth noisy image will have three different orientations, each with respect to one of the three possible pairs of three previously oriented noisy images. As more noisy images are added to the set, the number of possible orientations for any given image grow factorially.
Inorganic Crystal Structures Solved from EM Images and Refined to 0.02 À Accuracy Against Electron Diffraction Data
Authors: Hovmöller Sven , Zou Xiaodong , Weirich Thomas
Published: July 2020

Single crystal X-ray diffraction is the traditional method for accurate crystal structure determination. A major difficulty in X-ray crystallography is the phase problem; diffracted intensities contain amplitude information but no phases. In order to solve a structure, the phases of at least the strongest reflections must be estimated by Patterson techniques, so-called direct methods or in any other way. Once the structure has been solved (i.e. the atoms found to within about 0.2 Ångström of their correct positions), then refinement is rather straight-forward for single crystal data. Typically, single crystals diffract to about 1 Â resolution. After refinement, the atomic co-ordinates are obtained with an accuracy of about 0.01 Â for organic molecules and down to 0.001 Â for inorganic structures. One limitation of single crystal X-ray diffraction is that the crystals need to be at least about 10μm in all dimensions in order to diffract, even if the radiation source is a synchrotron.
Quantitative Information from Image Processing in ADF Stem
Authors: Nellist PD , Pennycook SJ
Published: July 2020

Annular dark-field (ADF) imaging in the scanning transmission electron microscope (STEM) at atomic resolution can be regarded as being almost perfect incoherent imaging, which has two major advantages over conventional high-resolution transmission electron microscopy (HRTEM), which is close to being perfectly coherent: Firstly, the images formed are direct structure images of the projected atomic structure, with regions of intensity located at the positions of the atomic columns. Secondly, the images can be written as the convolution between two real and positive functions: a point-spread function given by the intensity of the STEM probe, and an object function that consists of narrow, almost <5-function-like, peaks located exactly over the nuclei of the atomic columns. Thus there is no phase problem in ADF STEM imaging, unlike in conventional HRTEM where phase retrieval techniques such as holography are often employed.

The lack of a phase problem in ADF STEM imaging creates immediate opportunities for image processing.
Extracting Structure Information from Cbed Patterns
Authors: Zuo JM
Published: July 2020

The advance of digital recording and energy filtering now makes it possible to extract quantitative crystal structure information in an accuracy close to or better than the x-ray method. The extraction of structure information employs the refinement technique by comparing theory and experiment using chisquare criteria of

For the unit cell or crystal orientation determination, the experiment yx is the line equation of Kikuchi or HOLZ lines, and/or positions of convergent beam disks or diffraction spots. Lines, or disks, can be measured directly from the digitally recorded two-dimensional patterns using techniques of image processing and transformation such as Hough. A practical method for measuring unit cell parameters was described by [1]. For the determination of atomic positions, the experiment yx is the diffracted intensities of medium and high order reflections. For the measurement of charge density, the yx is the energy-filtered diffracted intensities of low order reflections or high order reflections coupled with a low order reflections.
Electron Holography at Atomic Dimensions
Authors: Lichte Hannes
Published: July 2020

Many of the problems of transmission electron microscopy (TEM) are due to the fact that wave optics which governs the interaction of electrons with the specimen and the imaging process definitely is brought to an end with the detection of the final electron image. Unfortunately, resolution is limited by an increasing number of aberrations. Furthermore, wave optical tools in the electron microscope which are needed for example to produce phase contrast better than that given by the phase contrast transfer function, for distinction of amplitude contrast and phase contrast, or to measure phases in Fourier space, are only poorly developed. Since subsequent image processing of electron diffraction patterns or real space images can never compensate for the loss of phase information, the phase has to be recorded also, i.e. one has to work holographically to collect and reconstruct all data of the object structure.
On-Line Aberration Measurement and Correction in STEM
Authors: Krivanek Ondrej L , Dellby Niklas , Spence Andrew J , Camps Roger A , Michael Brown L
Published: July 2020

Aberration correction in electron microscopy is a subject with a 60 year history dating back to the fundamental work of Scherzer. There have been several partial successes, such as Deltrap's spherical aberration (Cs) corrector which nulled Cs over 30 years ago. However, the practical goal of attaining better resolution than the best uncorrected microscope operating at the same voltage remains to be fulfilled. Combining well-known electron-optical principles with stable electronics, versatile computer control, and software able to diagnose and correct aberrations on-line is at last bringing this goal within reach.

We are building a quadrupole-octupole Cs corrector with automated aberration diagnosis for a VG HB5 dedicated scanning transmission electron microscope (STEM). A STEM with no spherical aberration will produce a smaller probe size with a given beam current than an uncorrected STEM, and a larger beam current in a given size probe.
Field of View and Image Distortion : A Review of Low Magnification Imaging In the Environmental and Conventional Scanning Electron Microscopes (SEM)
Authors: Griffin Brendan J
Published: July 2020

Most scanning electron microscopy is performed at low magnification; applications utilising the large depth of field nature of the SEM image rather than the high resolution aspect. Some environmental SEMs have a particular limitation in that the field of view is restricted by a pressure limiting aperture (PLA) at the beam entry point of the specimen chamber. With the original ElectroScan design, the E-3 model ESEM utilised a 500 urn aperture which gave a very limited field of view (∼550um diameter at a 10mm working distance [WD]). An increase of aperture size to ∼lmm provided an improved but still unsatisfactory field of view. The simplest option to increase the field of view in an ESEM was noted to be a movement of the pressure and field, limiting aperture back towards the scan coils1. This approach increased the field of view to ∼2mm, at a 10mm WD. A commercial low magnification device extended this concept and indicated the attainment of conventional fields of view.
Comparison of Amplification and Imaging Behaviours of Several Gases In The Environmental Sem
Authors: Thiel BL , Fletcher AL , Donald AM
Published: July 2020

We have investigated the amplification properties of several imaging gases in the Environmental SEM (ESEM) with the intent of forming a set of general guidelines for the selection of gases. In the ElectroScan ESEM, a gas ionisation cascade is used to amplify the secondary electron signals emanating from the specimen surface. The presence of gas in the chamber also gives rise to a pressure dependent background signal derived from ionisation events between gas molecules and high energy primary beam and backscattered electrons. The fraction of secondary electron signal decreases as the pressure is raised. This point is illustrated in figures la and lb which show the calculated fraction of signal contributed by secondary, backscattered, and primary electrons as a function of pressure in helium and water vapour. Helium yields a very pure secondary electron signal over the entire range of pressures shown. Unfortunately, helium does not provide a great deal of signal amplification compared to water vapour (figure 2).
A New Mechanism for the Imaging of Crystal Structure in Non-Conductive Materials: An Application of Charge-Induced Contrast in the Environmental Scanning Electron Microscope (ESEM)
Authors: Griffin Brendan J
Published: July 2020

The mechanism of the contrast in ‘environmental’ or ‘gaseous’ secondary electron images in the environmental scanning electron microscope is at best poorly understood. The original theory suggested a simple gas amplification model in which emitted secondary electrons ionise the chamber gas, leading to signal amplification and finally measurement at a biased detector. This theory is being advanced but little attention has as yet been paid to the factors which influence the actual secondary emission, although unusual contrast effects have been noted in one case. The conven-tional view is that the positive ion product of the gas-electron interaction results in charge neu-tralisation at the sample surface.

The implantation and trapping of charge in non-conductive materials was recently described, in reference to electron range measurements. This work demonstrated that trapped charge influ-enced the secondary electron yield, with enhanced secondary electron emission above the region of trapped charge. The consequence is that the distribution of the trapped charge is seen as a bright circle on the surface of the specimen, centred on the point of beam exposure (Fig.l).
Application of ESEM/Eds to Pharmaceutical Synthesis
Authors: Carlton R A , Orton E , Lyman C E
Published: July 2020

Solid phase synthesis (SPS) is used for the discovery and optimization of biologically active substances for the pharmaceutical and agricultural industries. Large numbers of novel organic compounds are generated by successively adding functional groups onto a solid substrate - usually polystyrene. Several techniques have been applied to the qualitative and quantitative analysis of these polymer-bound compounds with varying success. Environmental scanning electron microscopy (ESEM) with energy dispersive x-ray spectrometry (EDS) offers a unique approach to the qualitative analysis of SPS resins. Heteroatoms (such as Cl, Br, and S) are added either as a part of the synthesis or as a marker for analysis. Heavier atoms, such as these, are easily detected by EDS. The advantages of EDS are speed, sensitivity, and small required weight of the sample. The advantages of the ESEM are that it eliminates the need for a conductive coating and the vacuum conditions are not as severe on the specimen as with a conventional SEM.
Environmental SEM Electron Damage Imaging of Self Assembled Monolayers with SIMS
Authors: Wight Scott , Gillen Greg , Herne Tonya
Published: July 2020

The scattering of primary beam electrons has several implications for energy dispersive X-ray analysis on the environmental scanning electron microscope (ESEM). One of the most basic problems is determining the spatial distribution and fraction of primary beam electrons scattered under typical high pressure analysis conditions A method for studying the scattering of the primary electron beam in the ESEM has been demonstrated. The method involves the use of self-assembled alkanethiol monolayers (SAM) which are sensitive to damage by primary beam electrons. After irradiation, the electron damaged molecules can be exchanged out of the monolayer by immersion in a second alkanethiol solution. The spatial distribution of this second marker compound can then be imaged directly by static secondary ion mass spectrometry (SSIMS). Alternatively, only a single fluorinated thiol is used and the decrease in fluorine secondary ion signal is used to map the electron damage.
The Low Voltage Scanning Electron Microscope
Authors: Joy David C
Published: July 2020

A majority of the scanning electron microscopes (SEMs) now in use are probably employed as low voltage SEMs (LVSEMs), that is to say they are operated to produce beams with energies below 5keV. This trend away from the more conventional mode of operation at 20 or 30keV has gathered momentum over the past decade and has been driven by both theoretical and practical considera-tions.

Firstly, the distance travelled by an electron falls rapidly (in fact as about E1.6 ) as the incident ener-gy E is reduced. Images generated by low energy electron beams therefore contain enhanced surface information compared to those images recorded at higher energies. Since surfaces are of great inter-est in both the life sciences and in materials science this has been a persuasive factor. Secondly, both the secondary and the backscattered electrons now come from essentially the same interaction volume, rather than from volumes which are widely different in size and shape.