Biology Commons^™

Scanning Tunneling Microscopy Of Biological Macromolecular Structures Coated With A Conducting Film, M. Amrein, H. Gross

Scanning Microscopy

We have studied the capability of scanning tunneling microscopy (STM) to reveal the three-dimensional structure of biological macromolecular structures that have been rendered conductive by metal-coating. The sample preparation used has been derived from a well established method in transmission electron microscopy (TEM). It includes adsorption, freezing and dehydration by vacuum-sublimation, followed by metal-shadowing of the specimen. As an alternative to adsorption and coating, fluid biomaterials can be replaced by conductive freeze-fracture replica.

We give an introduction into the sample preparation of metal-coated specimens and discuss how each step can affect the structural preservation and thereby the quality of the …

Visualizing Cells In Three Dimensions Using Confocal Microscopy, Image Reconstruction And Isosurface Rendering: Application To Glial Cells In Mouse Central Nervous System, Frank Morgan, Elisa Barbarese, John H. Carson

Scanning Microscopy

This paper describes a general method for visualizing individual cells in intact tissue in three dimensions. The method involves immunostaining intact tissue to label specific cells, "optical sectioning" the stained tissue by laser scanning confocal microscopy, computationally reconstructing a three dimensional image data set from the digitized confocal optical sections, delineating isosurfaces of specific intensity within the reconstructed image by a "marching cubes" algorithm to generate polygon meshes defining boundaries of cells, and displaying individual cells, identified as three dimensional objects enclosed by contiguous polygon meshes, using computer graphics techniques. Each of the components of this method has been described …

Growth Of Yba2cu3o7-Δ Thin-Films–Nucleation, Heteroepitaxy And Interfaces, M. Grant Norton, C. Barry Carter

Scanning Microscopy

A major research effort in high-temperature oxide superconductors is the growth of high-quality thin-films. The physical properties of polycrystalline thin-films are controlled by their microstructure which is influenced by the early stages of film growth and the establishment of epitaxy. In this article, the nucleation and heteroepitactic growth of YBa₂Cu₃O_7-δ thin-films is reviewed. Many of the examples illustrating film growth will be taken from studies using transmission electron microscopy. The importance of the substrate surface in controlling film growth will be discussed, in particular the growth of YBa₂Cu₃O_7-δ films on …

The Interpretation Of X-Ray And Electron Signals Generated In Thin Or Layered Targets, R. H. Packwood, G. Remond

Scanning Microscopy

This work outlines the development of a comprehensive theory for the electron probe microanalyser and scanning electron microscope or SEM, that is intended to serve as a framework of understanding for those employing electron beam methods and as a basis for improved correction procedures. There is particular emphasis on applications to layered and non-uniform specimens. Starting from a simple Gaussian depth distribution of electrons and making assumptions about the X-ray production, a series of predictions of X-ray and electron signals are made for various target configurations. When compared with experimental measurements a series of interesting discoveries follow, which, taken altogether, …

The Stability Of Adsorbates Imaged With A Scanning Tunneling Microscope Using Hopping Versus Constant Current Scanning, Terje G. Vold

Scanning Microscopy

We have studied the stability of various adsorbates, including gold, a platinum-iridium alloy, and DNA, on monoatomically flat gold imaged with a scanning tunneling microscope. We find that adsorbates are generally more stable, sometimes dramatically so, if imaged with a hopping trajectory of the tip rather than with the conventional constant-current scanning technique. Gold pits and associated debris formed on flat gold surfaces under saline solution by mechanical impact of the tip with the surface are always much more stable when imaged with hopping. Samples of thin, sub-monoatomic layers (0.1 nm and 0.2 nm thick) of a platinum-iridium alloy evaporated …

Image Processing Procedures For Analysis Of Electron Back Scattering Patterns, N. C. Krieger Lassen, D. Juul Jensen, K. Conradsen

Scanning Microscopy

At present, computer-aided Electron Back Scattering Pattern (EBSP) analysis often requires large amounts of operator time if statistically reliable measurements are needed. This paper presents ways to automatically detect and localize bands in EBSPs and thereby enable fully automatic EBSP analysis. The main focus will be on a procedure using a modified Hough transform by which more than 12 bands in a typical EBSP can be detected and localized. This procedure seems as effective and reliable for extracting the bands of EBSPs as any human operator. The performance of this procedure is compared with that of other image processing procedures.

Quantitative Imaging Ion Microscopy: A Short Review, G. A. Valaskovic, G. H. Morrison

Scanning Microscopy

A short review of recent efforts being made in the quantification of images in ion microscopy is given. Special aspects of instrumentation, detection and acquisition, which are unique to direct imaging secondary ion mass spectrometry, are discussed in relation to the successful application of traditional empirical quantification schemes. Application of such quantification schemes requires proper sample preparation, standardization, analysis, and quite often, special techniques in image processing and the correlation of ion microscopy with other microscopies. Quantification within this technique is a difficult goal which can only be realized if the analyst pays strict attention to every step of the …

High Precision, High Spatial Resolution Analysis Of Sulfur Isotopes By Laser Combustion Of Natural Sulfide Minerals, S. P. Kelley, A. E. Fallick, P. Mcconville, A. J. Boyce

Scanning Microscopy

Laser induced combustion provides a powerful new technique for sulfur isotope measurements in natural sulfides. A high power continuous laser, focused through a modified microscope system onto a sulfide mineral target, produces highly localized heating. The laser beam is focused to a spot 25 μm in diameter at the sample surface. In the presence of low pressure oxygen, temperatures at the centre of the spot are sufficiently high to produce localized oxidation. Resultant SO₂ is transferred to a gas source isotope ratio mass spectrometer, where the 𝛿³⁴S can be measured to a precision better than 0.25‰. Sulfur …

Cathodoluminescence Spectroscopy: An Accurate Technique For The Characterization Of The Fabrication Technology Of Gaalas/Gaas Heterojunction Bipolar Transistors, A. C. Papadopoulo, C. Dubon-Chevallier, J. F. Bresse

Scanning Microscopy

Cathodoluminescence (CL) spectroscopy and imaging performed at low temperature have been used to qualify the heterojunction bipolar transistor fabrication technology, particularly the etching and ion implantation steps. CL has been used to optimize low defect technological processes. The protection of the active region during the insulation process has been optimized. The best result is obtained when using a bilayer of silicon nitride and photoresist. In order to minimize it, the damage induced by the etching process has also been studied. The best result is obtained when combining Ar ion beam etching and chemical etching. The possibilities to perform localized spectroscopy, …

Influence Of Bragg Scattering On Plasmon Spectra Of Aluminum, P. Schattschneider, D. -S. Su, P. Pongratz

Scanning Microscopy

Plasmon spectrometry is an important method to obtain information on many-body effects in the solid state. The plasmon halfwidth and the dispersion coefficient are well investigated for a number of materials, and compare well with quantum mechanical predictions. The excitation strength of the coherent double plasmon has been investigated to a lesser extent. Experimental results are at variance with one another and with theory. This is partly due to the plural scattering which masks the coherent double plasmon.

Accurate analysis of plasmon spectra requires not only to remove the inelastic plural processes but also to take into account the coupling …

The Distribution Of Impurities In Antarctic Ice, W. T. W. Potts, K. Oates, E. W. Wolff, R. Mulvaney

Scanning Microscopy

Antarctic snow and ice contain very low concentrations of ions, < 10 μmole/l Na, Cl and SO₄ but these ions may play a significant part in determining the physical properties of the ice and atmospheric chemistry. At the temperatures pertaining in the Antarctic sulphuric acid would still be liquid and information on the distribution of these ions, whether they are disseminated through the crystals or concentrated in a liquid phase at the triple junctions between the crystals, is essential in order to model some of the physical properties of the ice, including electrical conductivity, rigidity and transparency to radar.

Samples of 125 year old ice from …

Levels Of Complexity In The Microstructure Of Mammalian Enamel And Their Application In Studies Of Systematics, W. V. Koenigswald, W. A. Clemens

Scanning Microscopy

Description and analysis of the complex structure of enamel can be facilitated through the application of a system of hierarchical levels of structural complexity. Five interdependent levels are distinguished. These are the levels of:

1) crystallites,

2) prisms,

3) enamel types,

4) schmelzmuster, and

5) dentition.

This system provides a basis for analysis of both variation of particular structures and variation of structural types throughout a mammal's dentition. Optimally, in wide ranging systematic and biomechanical studies, all levels of structural complexity should be considered, but lack of information about one level does not prevent significant analyses at other levels.

Correlative Transmission Electron Microscopy And High Resolution Scanning Electron Microscopy Studies On The Fine Structural Organization Of The Chicken Pituitary Gland, Susan Tai

Scanning Microscopy

The present study employs transmission (TEM) and high-resolution scanning electron microscopy (HR-SEM) to examine the inter- and intra-cellular organization of the pars distalis of the chicken anterior pituitary gland. The overall view of the cryofractured surface of the par distalis illustrates the arrangement of different pituitary cells and tissues in the follicles. Fine structural examination by HR-SEM shows that the membrane of the mitochondria has a similar configuration to that of the rough endoplasmic reticulum. Granule-like structures, on the surface of the mitochondrial membrane, are similar in size to ribosomes. Standard imaging and three-dimensional imaging demonstrated the formation of developing …

The Ionic Composition Of Ion Transporting Cells In Stonefly Nymphs, W. T. W. Potts, I. Bricknell

Scanning Microscopy

Freshwater stonefly larvae take up sodium and chloride ions from low concentrations in water and concentrate them into haemolymph. The ions are transported by mitochondria rich cells (MRITCs) which are easily identified by their windows in the cuticle. Sodium uptake is generally inhibited at low pH but a few species of stoneflies live in acid waters. Acid tolerance is associated with presence of caviform or bulbiform MRITCs while nymphs possessing only the more common coniform cells are sensitive to acid conditions.

The intracellular concentrations of Na, K, Cl and P in coniform cells of Perla bipunctata and bulbiform cells of …

Simulating Ion Microtomography Data For Improving Reconstruction Quality, A. J. Antolak, A. E. Pontau

Scanning Microscopy

Ion Microtomography (IMT) provides quantitative, fine resolution density imaging of samples for materials characterization. Reconstructed tomographic images are obtained by application of a filtered backprojection algorithm to the collected data. The attainable resolution and data acquisition rate are affected by several parameters. These include the number of ions measured per spot, using either the mean or median residual energy, utilizing Bragg additivity, changing the number of rays or the number of projections and oversampling the data. A tomography simulation computer program is described and used to study the contributions from these effects on the numerical reconstruction of an array of …

A Light, Transmission And Scanning Electron Microscope Study Of Snuff-Treated Hamster Cheek Pouch Epithelium, S. H. Ashrafi, A. Das, R. Worowongvasu, B. Mehdinejad, J. P. Waterhouse

Scanning Microscopy

The effects of smokeless tobacco (snuff) on hamster cheek mucosa were studied by light microscopy, transmission (TEM) and scanning electron microscopy (SEM). Two grams of commercially available smokeless tobacco were placed into the blind end of the right cheek pouch of each experimental animal, once a day and five days a week for 24 months. The control animals did not receive smokeless tobacco. After 24 months treatment with smokeless tobacco, hamster cheek mucosal epithelium lost its translucency and had become whitish in color. By light microscopy hyperorthokeratosis, prominent granular cell layers with increased keratohyalin granules and hyperplasia were seen. At …

Integrated Single Crystal Detector For Simultaneous Detection Of Cathodoluminescence And Backscattered Electrons In Scanning Electron Microscopy, Rudolf Autrata, Josef Jirák, Jiří Špinka, Otakar Hutař

Scanning Microscopy

The design of the majority of the available high efficiency cathodoluminescence (CL) detection systems allows the secondary electron (SE) and backscattered electron (BSE) image modes to be detected simultaneously to a limited degree only. The described CL-BSE detector is based on the single crystal YAG scintillator shaped as a spherical mirror which reflects photons emitted from the CL specimen and focuses them on to the entrance surface of a fibre optic light guide connected to the photomultiplier tube (PMT I). The backscattered electrons emitted from the CL specimen move through the reflecting layer of the mirror into the YAG scintillator …

Cathodoluminescence Applied To The Microcharacterization Of Mineral Materials: A Present Status In Experimentation And Interpretation, G. Remond, F. Cesbron, R. Chapoulie, D. Ohnenstetter, C. Roques-Carmes, M. Schvoerer

Scanning Microscopy

Experimentation and interpretation of cathodoluminescence (CL) microscopy and spectroscopy applied to the microcharacterization of material minerals are reviewed. The origins of the intrinsic (host lattice) and extrinsic (impurities) luminescence emissions in crystals are briefly discussed. Merits and limitations of the available techniques are illustrated. CL emission changes as a function of the incident electron dose are illustrated for the case of natural quartz and sphalerite (ZnS) crystals. These effects are discussed in terms of the development of bulk charging, production of heat, diffusion of impurities, and creation of lattice defects induced by the incident ionizing particles. Although CL emission is …

Nuclear Microprobe Application In Semiconductor Process Developments, Mikio Takai

Scanning Microscopy

Scanning nuclear microprobes using Rutherford backscattering (RBS) with light ions have been applied to semiconductor process steps, in which minimum feature sizes of several microns down to submicron and multi-layered structures were used. Two or three dimensional RBS mapping of processed semiconductor layers such as multi-layered wiring, semiconductor-on-insulator (SOI), focused ion implanted layers, and laser processed layers, has clearly revealed process failures and inhomogeneity in buried layers without layer removal processes. Radiation damage due to the probe beams was found to be induced by high probe doses at and above 10¹⁷/cm², in which the degradation of …

Structural Artifacts And Advantages Of Cytocentrifugation Of Cells As Viewed By Scanning Electron Microscopy, Mário N. D. Peão, Artur P. Águas, Carlos Moreira De Sá, Nuno R. Grande

Scanning Microscopy

Cytocentrifugation of cell suspensions onto glass slides is a widely used procedure in contemporary cytology. We employed here scanning electron microscopy (SEM) to investigate putative morphological changes induced in cells submitted to cytocentrifugation. The fine structure of murine pleural exudate cells (macrophages mainly) processed by spinning was compared with that of similar cells treated without centrifugation (poly-L-lysine attachment of the cells to glass slides at 1 g). Cells of cytocentrifuged preparations showed a significant increase in diameter and smoothening of the cell surface as compared with the morphology of non-centrifuged cells. Cytocentrifugation also induced the formation of thin elongations coming …

Architecture Of Mixed Calcium Oxalate Dihydrate And Monohydrate Stones, Hidenobu Iwata, Shouzo Iio, Shunji Nishio, Masafumi Takeuchi

Scanning Microscopy

Calcium oxalate dihydrate (COD) and monohydrate (COM) are the most frequent constituents of urinary stones, and there still exist some questions about the interrelation between the two hydrates. Architecture of mixed COD and COM stones was observed by electron microscopy to solve the questions.

The fractured surface of a stone is composed of the fractured face of the crystals. In this situation a morphological criterion of typical dipyramid shape is useless to identify COD. But we could identify COD using the partial dissolution method, which etched square pits on COD crystals.

COD and COM formed distinctly separate layers. COD was …

Idiopathic Calcium Oxalate Nephrolithiasis: A Cellular Disease, Giovanni Gambaro, Bruno Baggio

Scanning Microscopy

Physico-chemical, metabolic and hormonal theories regarding the pathogenesis of calcium oxalate nephrolithiasis do not sufficiently explain many features of this disease. The recent findings of an abnormally faster oxalate self-exchange and higher phosphorylation of band 3 in erythrocytes of idiopathic calcium oxalate stone formers suggest the hypothesis that nephrolithiasis may be a cellular disease, characterized by a defect in the function of the anion-exchange. The cellular anomaly seems genetically controlled. Band 3 anion exchanger function seems to be biochemically regulated through modulation of band 3 phosphorylation, which depends on cyclic AMP- and phospholipid-sensitive Ca²⁺ independent protein kinases. In this …

Factors Affecting Fasting Urinary Calcium Excretion In Stone Former Patients On Different Dietary Calcium Intake, Piergiorgio Messa, Giuseppe Mioni, Rossana Franzon, Michele Messa, Aldo Cruciatti, Sandro Giannini, Angela D'Angelo

Scanning Microscopy

The effects of variable calcium content on daily and fasting urinary calcium and other lithogenic solutes excretion, on the bone turnover index (fasting hydroxyproline urinary excretion) and on the calciotropic hormones were studied in 312 stone former patients with an outpatient protocol and 15 stone former patients in an inpatient study. Furthermore in 60 of these patients, 30 while on a low calcium diet (LCD) and 30 on a free calcium home diet (FCD), the effects of an oral calcium load (OCL) on bone turnover index, calciotropic hormones and calcium excretion were evaluated. The results demonstrate that an LCD is …

Acquisition Hardware For Imaging, Peter Rez, J. K. Weiss, W. J. De Ruijter

Scanning Microscopy

In electron microscopy images can either be recorded in parallel (Transmission Electron Microscopy) or acquired as the variation in a signal as a probe is scanned over the specimen (Scanning Electron Microscopy). To extract the most information from an image requires that the best possible systems are used for acquiring image data. Ultimately, the limit to information capture is achieved when every electron from the scattering event of interest is recorded. The ideal system can be realised both for parallel recording with scientific grade CCD cameras, and for scanning microscopy with single electron counting electronics. The data rates from these …

Simulation For Scanning Electron Microscopy, G. R. Anstis, X. S. Gan

Scanning Microscopy

Simulations of images of surface steps obtained by high energy reflection electron microscopy are presented. It is shown that double images of simple steps, with no associated strain field, may occur when surface resonance conditions are established. Accurate calculation of image intensity requires large calculations and care is needed in relating the computed wave functions to those occurring for a semi-infinite incident wave. Estimates of the time to compute accurate wavefunctions are given and it is shown that they are reasonable for modem fast computers.

Atomic Resolution Electron Holography, K. Ishizuka, T. Tanji, A. Tonomura

Scanning Microscopy

It has been demonstrated that electron holography is a very powerful tool to investigate an electromagnetic potential in medium resolution, since the phase of an electron wave is approximately proportional to the potential. Now, electron holography is at the second stage of development: to establish holography at atomic resolution and further to realize Gabor's idea to improve the resolution restricted by the spherical aberration of the objective lens. We investigate the possibility of electron holography to get information at atomic resolution by computer simulations as well as by digital processing of electron holograms. We show that the phase distribution has …

Image Delocalisation And High Resolution Transmission Electron Microscopic Imaging With A Field Emission Gun, W. Coene, A. J. E. M. Jansen

Scanning Microscopy

The high spatial and temporal coherence of a field emission gun (FEG) increases the information limit of high-resolution transmission electron microscopes (HRTEM), but has also its implications on the localisation of the high resolution information in the image. In this paper, we present the results of a combined theoretical and experimental study of delocalisation in HRTEM. First, we derive a spatial frequency analysis of the delocalisation for crystal defects. Next, the delocalisation is studied from a real-space point of view, in terms of the impulse-response function, for which an instructive asymptotic mathematical analysis has been set up. Finally, we present …

Image-Matching As A Means Of Atomic Structure Evaluation In High Resolution Transmission Electron Microscopy, John C. Barry

Scanning Microscopy

Two of the major applications of the high resolution transmission electron microscope (HRTEM) image-matching technique are: a) to refine the structure of defects and interfaces in crystals, and b) to identify minority phases in multi-phase materials. An important advantage of the technique is that it provides spatial information at the atomic level; but an important disadvantage is that the results are often hard to quantify. There are many factors which affect the precision of the image-matching technique, and methods for measuring the pertinent experimental parameters are discussed in this paper. It is shown that if the experimental parameters are known, …

Sub-Angstrom Microscopy Through Incoherent Imaging And Image Reconstruction, S. J. Pennycook, D. E. Jesson, M. F. Chisholm, A. G. Ferridge, M. J. Seddon

Scanning Microscopy

Z-contrast scanning transmission electron microscopy (STEM) with a high-angle annular detector breaks the coherence of the imaging process, and provides an incoherent image of a crystal projection. Even in the presence of strong dynamical diffraction, the image can be accurately described as a convolution between an object function, sharply peaked at the projected atomic sites, and the probe intensity profile. Such an image can be inverted intuitively without the need for model structures, and therefore provides the important capability to reveal unanticipated interfacial arrangements. It represents a direct image of the crystal projection, revealing the location of the atomic columns …

Segmentation Of Pores In Backscattered Images Of Sediments And Soils And Their Relationship To Domain Structure, M. W. Hounslow, N. K. Tovey

Scanning Microscopy

A new technique to quantify variations in pore structure across an image is described in this paper. The method involves several stages as the original images are rarely suitable for simple segmentation by the selection of a single threshold. The processing first involves the application of a Wiener filter to remove imaging artefacts and to sharpen the edge of void/particle contacts. This image reconstruction is followed by the selection of a global threshold based on the relative contrast method, to create a binary image separating the voids from the particles. Such an image can be used to evaluate pore structure …