bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple signals in STEM
Authors: Burge R. E. , Browne M. T. , Charalambous P. , Clark A. , Wu J. K.
Published: August 2011

A brief description is given of the eight image signals that can be simultaneously recorded in digital form using an appropriate data recording system and high resolution STEM; this involves a set of detectors covering bright and dark field. Details are provided of the collection and manipulation of image data for STEM, including on‐line estimation of off‐focus distance, on‐line assessment of astigmatism, on‐line signal to noise ratio in any or all detectors; examples of all three procedures are given. The use of the data recording system is discussed together with some aspects of image display, including the use of colour. A method is introduced of providing, using the appropriate hardware, a prescribed colour map (image intensity converted to colour) using replaceable PROMS.

Two off‐line procedures are considered. One is the Karhunen‐Loeve transform and its application to bringing together the information contained within the multiple signals, and the other is the discrete cosine transform by which the storage problem for digital image storage may be reduced, according to the image, by between 15 and 30 times.
An integrated approach to scanning microscope data acquisition
Authors: Jones A. V. , Unitt B. M.
Published: August 2011

This paper describes an integrated data acquisition system built for a cryogenic STEM which is currently under construction in the authors' laboratory. Advantage was taken of modern digital and microprocessor techniques to construct a versatile and expandable system in the minimum time. The system is able to operate in conjunction with an external image processing computer or as an independent system with built‐in image processing facilities.
Topographical analysis in the SEM using an automatic focusing technique
Authors: Holburn D. M. , Smith K. C. A.
Published: August 2011

A novel technique for automated topographical analysis in the SEM has been investigated. It utilizes a 16‐bit minicomputer arranged to act as an automatic focusing unit. The computer is coupled to the objective lens of the microscope, by means of a digital to analogue converter, and may regulate the excitation of the lens under program control. Further digital‐to‐analogue converters allow the computer to act as a programmable scan generator by applying ramp waveforms to the scan amplifiers, permitting the beam to be swept over a small sub‐region of the field of interest. The video signal is sampled and applied to an analogue‐to‐digital converter; the resultant binary numbers are stored in computer memory as an array of values representing relative image intensities within a subregion. A differencing algorithm applied to the collected data allows the level of objective lens excitation to be found at which the sharpness of the image is optimized, and the excitation may be related to the working distance for that subregion through a previous calibration experiment. The sensitivity of the method for detecting small height changes is theoretically of the order of 1 μm.
A review of on‐line processing of chemical analysis data using a conventional CTEM/STEM
Authors: Nicholls A. W. , Jones I. P.
Published: August 2011

A review of the on‐line processing of X‐rays and the energetically complementary electron intensities to yield chemical compositions is followed by a discussion of X‐ray mapping, energy filtered images and some extended uses of conventionally supplied equipment associated with these analytic techniques.
Glycol methacrylate embedding of bone and cartilage for light microscopic staining
Authors: Cole Madison B.
Published: August 2011

A method is described for embedding bone and cartilage in glycol methacrylate (GMA) for light microscopy. Dehydration‐infiltration of the hard tissue is with aqueous GMA solutions minimizing solvent and dehydration artefact, and polymerization is by UV light in the cold to minimize thermal damage. Over fifty stains, enzyme localizations and related histochemical methods for 0·5–3·0 μm thick sections of GMA embedded tissue are listed. The increased resolution plus the localization of cellular and extracellular chemical moieties is now easier and more accurate providing an improved method for the study of the musculo‐skeletal system by light microscopic histochemistry.
An improved double‐tilt stage for the AEI EM7 high‐voltage electron microscope
Authors: Turner J. N. , Ratkowski A. J.
Published: August 2011

Vibration transmitted by the specimen rod of a side‐entry stage frequently decreases image resolution, and the length of the rod in the high‐voltage electron microscope can make the problem severe. A detachable tip clamped to the translation stage minimizes the effect, but eliminates the rod as a means of tilting. Furthermore, the second tilt mechanism is usually built into the rod. Thus the sample is coupled not clamped to the rod, increasing the effect. The image resolution attainable with our double‐tilt stage was 1·5 nm along the rod's axis and 5·0 nm perpendicular to it before modification.

An isotropic resolution of 0·5 nm was achieved by attaching the specimen tip to the rod with a flexible coupling and clamping both ends of the tip to the translation ring. The couplings used transmit the torque to rotate the specimen holder but dampen vibration. Ion‐pumping the vacuum system with all other pumps off also improved specimen stability.
Improved handling of embedding plastics for electron microscopy
Authors: Shannon W. A.
Published: August 2011

An improved, safer, rapid method for preparing embedding plastics for electron microscopy is described. The method consists of contained storage and dispensing of individual plastic components on an automatic tare balance. The proportions are based on weight measurements and may be calculated from volume or proportion recipes. The usual problems in and resulting from embedding plastic handling have been eliminated.
Automated cell counting in tissue sections: a new approach by ‘multiple grey‐level analysis‘
Authors: Wynford‐Thomas D. , Garrahan N. , Jasani B. , Williams E. D.
Published: August 2011

A system is described for the automated quantitation in tissue sections of the follicular and stromal cell populations of the thyroid gland, using a computer‐linked TV image analyser. The paper illustrates how the problems of variable staining intensity and clustering of images (nuclei) may be overcome by a novel approach, in which multiple analyses of each field are made at increasing grey‐level thresholds, and then ‘synthesized’ by computer to give a composite image. Discrimination between cell (nuclear) types was by a single minimum width criterion.

The results were highly reproducible and correlated well with counts obtained by a comparable manual method.
Freeze‐substitution
Authors: Harvey Diana M. R.
Published: August 2011

Freeze‐substitution is a technique suitable for the preparation of unicellular and multicellular plant and animal specimens for conventional light microscopy, TEM and SEM. It is also widely used as a means of preparing animal and plant tissues for the localization of water soluble substances by analytical electron microscopy, autoradiography or visual detection of precipitates.

The technical requirements of preparation, together with an evaluation of the procedures, are presented for various applications. Careful selection and evaluation of freezing technique, substitution solvent and regime are required for meaningful results.
A gripping stub* for the examination of multilaminous biological specimens in the scanning electron microscope
Authors: McTurk G. , Bulman S. , Ockleford C. D.
Published: August 2011

A simple specimen holder has been developed which expedites study of multilaminous biological specimens in the scanning electron microscope. It has the interesting additional property of allowing examination of the top and bottom of a single sample.
Applications of secondary ion mass spectrometry (SIMS) in biological research: a review
Authors: Burns Margaret S.
Published: August 2011

Secondary ion mass spectrometry (SIMS) is a potentially valuable but not fully exploited technique for problems in biological research. It is valuable because of: (1) detection of all elements and isotopes from mass 1, hydrogen; (2) high sensitivity for physiologically important elements, Na, K, Mg, Ca; (3) ion imaging of elements in areas as large as 250 μm in diameter with a lateral resolution of 0·5 μm; (4) promising efforts at quantitation at levels as low as 0·1 mmol/kg; (5) ability to analyse sequential layers to form a three‐dimensional analysis. The problems which complicate its use are primarily variations in ion emission, presence of polyatomic interferences and tissue preparation. Examples are cited of SIMS analysis of biomineralization, botany, toxicology and physiology, mostly by ion imaging techniques. SIMS has not yet been fully exploited for any single biological problem. In particular, studies using isotope discrimination, quantitative analysis and depth profiling will enhance the usefulness of SIMS as a technique for biological research.
A simple device for recording serial optical sections
Authors: Haarlem R. Van , Lagerweij C. , Horn L. C. J. E. M. Ten
Published: August 2011

A stepper motor was attached by way of a speed reducer to the coarse focusing spindle of a light microscope. After exposure of a film frame the stepper motor displaces the microscope stage according to the programmed instructions laid down in the electronic control unit. In this manner the focal plane is changed and serial optical sections of the developing fish blastoderm were recorded on 16 mm film. This design facilitates the analysis of cell behaviour during early development of annual fishes, of either large numbers of cells simultaneously or of individual cells in detail.
Coalignment of the muscle cell and nucleus, cell geometry and Vv in the tunica media of monkey cerebral arteries, by electron microscopy
Authors: Canham Peter B. , Henderson Ruth M. , Peters Mark W.
Published: August 2011

We undertook to ascertain how well aligned is the rod‐shaped nucleus within the spindle‐shaped cell of vascular muscle in order that we might use the darkly staining nucleus in histological sections to indicate precisely the directional alignment of the cell. We fixed cerebral arteries from five monkeys under physiological pressure and embedded portions of the tissue so that mid‐plane longitudinal sections of the arteries were obtained; the circumferentially arranged muscle cells were cut in cross‐section. From the electron micrographs we obtained the cross‐sectional profile of the cell and its nucleus, determining that the centre of the nucleus was on average 9·5 ± 5·8% (SD) away from the centre of the cell (expressed as a ratio of the cellular diameter). We calculated the alignment between the cell and nucleus to be from 0 to 3°, and obtained a volume fraction of 59% for muscle tissue in the tunica media of these arteries.
Water in malignant tissue, measured by cell refractometry and nuclear magnetic resonance
Authors: Ross K. F. A. , Gordon R. E.
Published: August 2011

It has long been known that tumour‐bearing tissues often have a significantly higher water content than the normal tissues from which they have been derived. Most of the evidence suggesting this in recent years has been obtained from methods employing nuclear magnetic resonance (NMR), which, although undoubtedly indicative of hydration, cannot at present be precisely quantified. Furthermore it has not been possible by these means to determine whether this overall increase in the water content of the tissue is principally an increase in the extracellular fluid or whether the water content of the tumour cells and the cells immediately adjacent to the tumours increases also. In this investigation, which is the first of its kind, a combination of NMR and immersion refractometry techniques have been used to examine the water content of normal and tumour bearing tissues. NMR measurements were made on pieces of normal and tumour bearing tissue from rat livers: intact living cells were also isolated from these pieces and their refractive indices measured by immersion refractometry from which the water content of their cytoplasm was calculated. It was found that all the cells so measured obtained from hepatomas had more water in their cytoplasm (usually over 5% more water) than any of the cells from normal livers; and that normal‐looking cells taken from the vicinity of hepatomas also all had more water in them than those of normal liver cells, although the differences in this case were less. These results were closely parallel to those obtained by NMR measurements. It is therefore concluded that an appreciable proportion of the increase in the water content of the tissue as a whole that occurs during carcinogenesis, must, in this tissue, be an increase in intracellular water.
Autoradiography: its methodology at the present time
Authors: Williams M. A.
Published: August 2011

The autoradiographic methods used in biological and medical research are reviewed from an historical viewpoint. It is pointed out that early methodological research was directed at the selection of suitable detector substances for radiation (nuclear emulsions) and at the preparation and application of suitable layers of these detectors to biological samples. On the other hand, later research was more concentrated on aspects of the preparation of the biological components of the specimens. In particular, the retention of biochemicals in the tissue sample was studied in detail. Research in the quantification of the emulsion response to α and β radiation paved the way for certain quantitative applications of autoradiography. This has been followed by the derivation of models for the measurement of image spread and the evolution of image analysis methods which allow refined determinations of the specific radioactivity of fine structures taking into account losses and gains of silver grains by image spread.

The sum of all these advances is a methodology capable of localizing and quantifying radioactivity and hence marking chemical processes from an organism and organ level down almost to the present limits of anatomical delineation.
The distribution of sodium and potassium in toad (Bufo bufo) oocytes estimated by a stereological method
Authors: Ibrahim K. B. , Dick D. A. T.
Published: August 2011

As the toad oocyte grows, the intracellular Na concentration rises and the K concentration falls. By stereological analysis using a point counting method in a projection apparatus, it is shown that as the oocyte grows the volume density of the nucleus, endoplasmic reticulum and mitochondria falls, while that of the yolk platelets and cytoplasmic vesicles rises. A multiple regression analysis applied to the sodium and potassium concentrations and to the volume densities of the subcellular components indicates that sodium is most concentrated in cytoplasmic vesicles while potassium is most concentrated in a component composed of the cell cytoplasmic ground substance, the mitochondria and the cell nucleus.
Frequencies of n‐polygons in planar sections of polyhedrons
Authors: Voss Klaus
Published: August 2011

In geology and metallography thin or polished sections of crystalline aggregates are a main subject of microscopic investigations. It is important to know what kinds of polygons are generated by planar sections through polyhedrons. In recent years computer simulation was the common method to determine the frequencies of n‐polygons. In this paper an integral geometric approach to derive exact formulas for these frequencies is described. For some families of simple polyhedrons results are given in analytical form and as numerical values.
Labelled‐replica techniques: post‐shadow labelling of intramembrane particles in freeze‐fracture replicas
Authors: Rash John E. , Johnson Timothy J. A. , Hudson C. Sue , Giddings F. Dennis , Graham William F. , Eldefrawi Mohyee E.
Published: August 2011

Three methods are described for direct post‐fracture, post‐shadow labelling of individual classes of intramembrane particles (IMPs) in freeze‐fracture replicas of biological membranes. The P‐face IMPs corresponding to the acetylcholine receptor complexes (AChRs) of vertebrate neuroeffector junctions are identified by post‐replication labelling with ferritin‐antibody complexes and with neurotoxin‐biotin‐avidin‐colloidal gold affinity ligands. (The freeze‐etch nomenclature of Branton et al., 1975, is used in this report.) These post‐shadow labelling techniques resemble conventional en bloc labelling techniques except that the labelling reagents must penetrate a thin but discontinuous layer of platinum superimposed on the molecules of interest. In the ‘sectioned labelled‐replica technique’, the replicated and labelled tissues are stained, embedded in plastic and sectioned parallel to the replica‐tissue interfaces. In the direct ‘labelled‐replica techniques’, the replicated and labelled samples are freeze‐dried or critical point dried, the labelled surfaces are stabilized by carbon coating, and the underlying tissues are dissolved, allowing the labelled‐replicas to be examined as conventional freeze‐fracture replicas. The unshadowed side of each AChR IMP is shown to retain sufficient biochemical information to permit both immunospecific and neurotoxin specific labelling despite formaldehyde fixation, freezing, fracturing, platinum shadowing, and thawing in aqueous media. A new mixed ferricyanide‐osmium staining method reveals electron opaque structures spanning the membrane bilayer in the same size, number and distribution as the labelled IMPs. These experiments demonstrate the feasibility of identifying individual IMPs in freeze‐fracture replicas and may allow the identification of specific membrane lesions in human disease.
Dynamic focusing in the confocal scanning microscope
Authors: Wilson T. , Hamilton D. K.
Published: August 2011

The previously reported depth discrimination property of the confocal scanning microscope, whereby detail outside the focal plane is effectively suppressed from the image, is exploited to give a high resolution system with in principle an indefinitely large depth of focus. The object is scanned axially, and the image built up as each section of the object passes through the focal plane.
Reliable use of resistance evaporation of Pt and C for high resolution freeze‐fracturing and a crystal surface image complementary to the E‐face of yeast plasma membranes
Authors: Steere Russell L.
Published: August 2011

Freeze‐fracture specimens of bakers yeast plasma membrane faces were prepared in both a modified Denton DFE‐2 freeze‐etch module and a modified Balzers BAF‐301 freeze‐etch unit. Each unit was equipped with a liquid nitrogen cooled shroud, resistance evaporators with PT‐C and C sources 7 cm from the specimens and with a resistance monitor to control PT‐C shadow film thickness. Optical diffraction patterns of specimens prepared in these units have fourth, fifth or sixth order spots. Therefore, on the basis of optical diffraction patterns, resolution of yeast plasma membrane specimens prepared in these units is equivalent to or better than that obtained by others with an ultrahigh vacuum system equipped with specially redesigned electron guns. A new image with tube‐like particles in hexagonal arrays, each surrounded by six substructure particles, nearly perfect high‐resolution complement to the hexagonal array of ring‐like depressions and the six surrounding subunit depressions of the E‐face, has been revealed on the surfaces of cubic crystals (presumably ice) which formed in the gap between the P‐ and E‐faces within fissures that occurred when the samples were frozen in liquid Freon 22. When the samples were subsequently freeze‐fractured at 77 K at a chamber vacuum of 13 μPa in which the specimens were protected from surface contamination by a liquid nitrogen cooled shroud, these crystals remained attached to the P‐face but pulled away from the E‐face against which they had apparently made molecular contact.
Ice formation in aqueous systems
Authors: Rasmussen Don H.
Published: August 2011

A recent treatment of the energetics of homogeneous nucleation brings classical nucleation theory into conformity with classical thermodynamic limits for supersaturated phase stability called spinodals. This energetic treatment, when extended to crystal nucleation from the liquid state, predicts that nucleation of ice cannot be avoided simply by rapid quenching. Nucleation will occur on cooling an aqueous system to a temperature called the physical spinodal because at and below this temperature icelike clusters in the aqueous system can grow without any free energy barrier whatsoever. Interpretation of microscopic observations of rapidly quenched biological systems must account for the fact that ice nuclei are certain to have been present throughout.
The relative efficiency of various fluids in the rapid freezing of protozoa
Authors: Silvester N. R. , Marchese‐Ragona S. , Johnston D. N.
Published: August 2011

The cooling efficiencies of various fluids at low temperature were compared by measuring the temperature decay in 3 μl water samples plunged into them. A simple model of cooling was used in order to discuss the results. Liquid ethane was found to produce a cooling rate of 660 K s−1, about twice that of liquid propane, while ethanol was almost as effective as ethane between 273 to 223 K. The heat‐transfer coefficient of liquid ethane was estimated to be between 1500 and 5000 W m−2 K−1, depending on the physical state assumed for the water sample. Samples of flagellated organisms, after being frozen rapidly in the above way, were freeze‐substituted by the method of Barlow & Sleigh (1979). Although this fixation did not give good definition of the microtubules of the flagellar axoneme, it exhibited reasonable tissue preservation in thin sections of the cell body. The fixation method resulted in preserved flagellar wave shapes, which were observed under the light microscope and in critical‐point dried cells examined by scanning and conventional electron microscopy. It was concluded (a) that methods for preserving the wave shape of the flagellum and for preserving its internal structure may not be compatible, and (b) that although the present cooling method (with ethane) approaches the speed required to arrest a flagellar wave, further improvements in the speed of the method are desirable.
A procedure for selecting specific areas of sections for comparative light and electron microscopic studies
Authors: Nilsen Rune , Dalen Helge , Røli Jakob
Published: August 2011

The paper describes a method which enables correlative light and electron microscopical studies to be made of specific tissue structures or of individual cells. For this purpose a new specimen marker has been constructed. The device, which is attached to the objective of a light microscope, permits the operator to examine and exactly encircle the selected area without changing the objective. The technique is illustrated by two examples using immunoelectronmicroscopy.
Preparing the liverwort Ricciocarpus natans for scanning electron microscopy
Authors: Kronestedt Eva , Echlin Patrick
Published: August 2011

Freeze‐drying, and chemical preparations followed by critical point drying, were tried on the aerenchymatous plant Ricciocarpus natans, which was to be studied by scanning electron microscopy. Promising results were obtained by the freeze‐drying technique.
Correlated optical and electron microscopy of Leeuwenhoek's elder‐pith sections
Authors: Ford Brian J.
Published: August 2011

A section of elder‐pith cut from Sambucus nigra in May 1674 by the pioneer Dutch microscopist, Antony van Leeuwenhoek, has been imaged under SEM, a modern Leitz photomicroscope, and the original Leeuwenhoek microscope at the University of Utrecht. The coordinated findings are examined.
Sampling of particulate suspensions for morphological evaluation: a simple method for routine and quantitative use
Authors: Aaronson Robert Peter
Published: August 2011

A general method for obtaining representative samples of particulate suspensions for morphologic evaluation is presented. The key to the method is the orientation of a suitable substrate for sample collection normal to an applied centrifugal field. Precise orientation is achieved by floating the substrate on a dense water‐immiscible fluid of low viscosity. Centrifugation may be performed in common angle‐head centrifuge rotors.

Two variations of the method are described which permit direct visualization or embedding of the particulate sample. Both variations are simple, can be performed routinely, and require only small amounts of material. They can also be performed simultaneously with centrifugation of the bulk samples.
Video‐enhanced microscopy with a computer frame memory
Authors: Allen Robert Day , Allen Nina Stromgren
Published: August 2011

Video‐enhanced microscopy combined with the use of a computer frame memory extends considerably the useful range of our video enhanced contrast (AVEC) methods for polarizing, double‐beam interference and differential interference contrast microscopy. Increased visual contrast is achieved by two stages of amplifications: the first optical, by using high bias retardation settings, and the second electronic. These steps are followed by a reduction of background brightness by means of a clamp voltage applied to a DC restoration circuit of the video camera. One of the limitations of the AVEC method alone is the inevitable appearance under high gain conditions of a pattern of mottle due to inaccessible dirt and defects in the lenses even of high quality. This limitation has been circumvented by storing the mottle pattern in the frame memory (frame store) and continuously subtracting it from each succeeding frame to clear the image. A major gain in image quality has resulted. In polarizing microscopy, the frame memory can be used also to subtract the image at one compensator setting from that at the equivalent setting of opposite sign, thus removing from the final image not only most of the mottle pattern but also the contrast due to the bright‐field contrast. In the polarizing microscope, these manipulations of the raw video image make it possible to observe and measure the birefringence of various organelles and elements such as microtubules, intermediate filaments and bundles of as few as a half dozen actin filaments. Since scattered light is also removed from the image, features hidden from view in the unprocessed image become visible. In differential interference microscopy, the AVEC method makes visible (i.e. detectable) many linear elements and particles that are an order of magnitude smaller than the resolution limit and not visible in the optical image. Such features are inflated by diffraction, however, to Airy disk size.
Total internal reflection fluorescent microscopy
Authors: Axelrod Daniel , Thompson Nancy L. , Burghardt Thomas P.
Published: August 2011

This review discusses applications of fluorescence microscopy using totally internally reflected excitation light. When totally internally reflected in a transparent solid at its interface with liquid, the excitation light beam penetrates only a short distance into the liquid. This surface electromagnetic field, called the ‘evanescent wave’, can selectively excite fluorescent molecules in the liquid near the interface. Total internal reflection fluorescence (TIRF) has been used to examine the cell/substrate contact regions of primary cultured rat myotubes with acetylcholine receptors labelled by fluorescent α‐bungarotoxin and human skin fibroblasts labelled with a membrane‐incorporated fluorescent lipid. TIRF examination of cell/substrate contacts dramatically reduces background from cell autofluorescence and debris. TIRF has also been combined with fluorescence photobleaching recovery and correlation spectroscopy to measure the chemical kinetic binding rates and surface diffusion constant of fluorescent labelled serum protein binding (at equilibrium) to a surface.
Scanning acoustic microscopy: a review
Authors: Wickramasinghe H. K.
Published: August 2011

The main features of the scanning acoustic microscope are reviewed with particular reference to applications in biology and non‐destructive evaluation. Recent developments on imaging the interior of solids are discussed. Proper interpretation of recorded images can lead to maps of velocity and density via the V(z) response. Further improvements in resolving power (using cryogenic liquids or high pressure gases) should make the acoustic microscope a powerful tool in many areas of science and technology.
Semi‐automatic analysis of microscopic images of the human cerebral cortex using the grey level index
Authors: Sauer Bernward
Published: August 2011

A new semi‐automatic method is introduced which makes possible the quantitative examination of cresyl‐fast‐violet stained sections of the cerebral cortex. These sections are divided into measuring fields of 20 × 20 μm in a well‐defined area. A scanning procedure then automatically determines the grey level index of every single field. The image of the histological section can be compared with the grey level index data matrix by means of a plotted image, the density of the plotted points of which is proportional to the measured grey level index. By overlaying this plotted image with the original section, the boundaries of the laminae can be fixed in the data matrix. The grey level index profiles—the grey level data plot as a function of the cortical depth—after smoothing and standardization are typical of special cortical areas. The mean thickness and the mean portion of the grey level index profile of the different laminae of the striate area are compared with data from other publications.
Electron microscopy of frozen biological suspensions
Authors: Lepault J. , Booy F. P. , Dubochet J.
Published: August 2011

The methodology for preparing specimens in the frozen, hydrated state has been assessed using crystals and T4 bacteriophages. The methods have also been demonstrated with lambda bacteriophages, purple membrane of Halobacterium halobium and fibres of DNA. For particles dispersed in an aqueous environment, it is shown that optimum structural preservation is obtained from a thin, quench‐frozen film with the bulk aqueous medium in the vitreous state. Crystallization of the bulk water may result in solute segregation and expulsion of the specimen from the film. Contrast measurements can be used to follow directly the state of hydration of a specimen during transition from the fully hydrated to the freeze‐dried state and permit direct measurement of the water content of the specimen. By changing the concentration and composition of the aqueous medium the contrast of particles in a vitreous film can be controlled and any state of negative, positive or zero contrast may be obtained. At 100 K, frozen‐hydrated, freeze‐dried or sugar embedded crystals can withstand a three‐ to four‐fold increase in electron exposure for the same damage when compared with similar sugar‐embedded or freeze‐dried samples at room temperature.
The preparation of leaf surfaces for scanning electron microscopy: a comparative study
Authors: Sargent J. A.
Published: August 2011

Cryopreservation is the superior technique for viewing leaf surfaces in the SEM. Epidermal cells become distorted when freeze dried and disrupt the orientation of epicuticular wax structures. The latter are largely lost during critical point drying. Nevertheless, the appearance of surface structures after subjecting them to each drying method is valuable in interpreting the features observed by cryopreservation.
A double lead stain method for enhancing contrast of ultrathin sections in electron microscopy: a modified multiple staining technique
Authors: Daddow L. Y. M.
Published: August 2011

A modification of the conventional method for the staining of ultrathin sections resulted in an increase in contrast of ultrastructural detail in tissues. Tissues embedded in Spurr's low viscosity embedding medium were stained with freshly centrifuged Reynolds' lead citrate for 1–5 min, rinsed in double distilled water and dried prior to staining with a saturated solution of uranyl acetate for 40 min, and freshly centrifuged Reynolds' lead citrate for 20 min.

Sections treated by this procedure showed enhanced staining of cellular organelles and cytoplasmic matrix. This procedure is recommended for tissues with poor staining qualities resulting from either prolonged fixation or from inadequacies in the buffer or embedding medium used.
The characterization of the arrangement of feature centroids in planes and volumes
Authors: Schwarz Helmut , Exner Hans Eckart
Published: August 2011

The quantitative characterization of the arrangement of microstructural features is important for understanding the properties or function of materials or tissues. In this paper, the well‐known analysis of nearest neighbour distances is modified in order to separate clusters from a background of randomly disposed features and to characterize significant parameters of such sets. In addition, simple procedures are indicated to detect and to characterize regular arrangements, and computer simulated examples are used to demonstrate the efficiency of the suggested algorithms. Each step of the procedures can be used for three‐dimensional arrangements as well.
An expeditious procedure for the accurate alignment and assembly of stereo micrographs
Authors: Fotino Mircea
Published: August 2011

A simple procedure is described for the reproducible and convenient assembly of stereo micrographs, particularly as transparencies, with the help of an apparatus developed for this purpose. Technical features and applications are discussed.
Freeze substitution sample preparation for ion microscopy of plant tissue
Authors: Ross Gary D. , Morrison George H. , Sacher Robert F. , Staples Richard C.
Published: August 2011

Tomato leaf sections were freeze substituted with either ethanol, n‐propanol, 20·0% acrolein: diethyl ether or acetone and then examined with an ion microscope to determine Na, K and Ca distributions. Thin sections were also examined for ultrastructural preservation using a transmission electron microscope. Generally, the 20·0% acrolein: diethyl ether and to a lesser degree the n‐propanol substituted materials demonstrated better ultrastructural preservation than the ethanol or acetone substitued materials. Ion micrographs for Na, K and Ca2+ of the 20·0% acrolein:diethyl ether and n‐propanol substituted leaves revealed a low contrast, well‐graded response with more informational content while tissue substitued with ethanol gave a high contrast response with little or no gradation. Ion losses from the leaf sections after substitution were monitored with neutron activation analysis (NAA) and initial experiments revealed significant contamination by molecular sieve desiccants. Empirically derived correction factors compensated for these contaminants; however, further experimentation utilizing silica gel as a solvent desiccant demonstrated ion retention values in excess of 80%.
Vertical sections of cultivated anchorage‐dependent cells for electron microscopy
Authors: Pentz S. , Amthor S. , Vergani G.
Published: August 2011

A method is described for electron microscopic preparation of cultivated cells for vertical sections using the test chamber system TCSC‐1. The cells are cultivated on a special foil. They can be fixed and embedded directly in the chamber. After polymerization of the resin, the foil can be easily taken off and a second resin layer is poured upon the embedded cells. Then distinct cells can be marked under an inverted light microscope. The bilayer of the resin allows optimal conditions for vertical sections of anchorage‐dependent cells.
An investigation of order/disorder in a chromia‐doped rutile by high‐resolution electron microscopy
Authors: Wood G. J. , Bursill L. A. , Smith David J.
Published: August 2011

Computer image simulations predict that the detailed atomic arrangements along extended CS defects in slightly‐reduced rutile may be identified in 500 kV high‐resolution electron micrographs. Experimental images from a chromia‐doped rutile have generally confirmed these predictions although also indicating that considerable disorder remains. The need for very careful control of sample preparation, including heat treatments, is emphasized.
The use of moiré techniques to measure rigid body displacements
Authors: Ecob R. C. , Stobbs W. M.
Published: August 2011

The relative advantages and disadvantages of a variety of experimental electron microscopical techniques for the measurement of rigid body displacements are considered. The principles of the application of a moiré method of great simplicity and high potential accuracy are then further discussed as are the experimental difficulties associated with the superposition of suitable reference latices on a substrate. The method is then applied to obtain an upper limit for the rigid body displacement at {111} Σ3 twin boundaries in stainless steel.
Intergrowths and defect structures in Bi9Ti3Fe5O27 revealed by high‐resolution electron microscopy
Authors: Smith David J. , Hutchison John L.
Published: August 2011

A high‐resolution 500 kV study of the long‐period (n = 8) perovskite Bi9Ti3Fe5O27 has revealed a variety of order/disorder effects, in marked contrast to low‐n materials, as well as the occurrence of a new two‐dimensional superlattice structure corresponding to a 3 × d110 ordering along the bismuthate layers. Intergrowths of the 2‐D SL phase with the ‘usual’ perovskite/bismuthate phase, as well as sharp interfaces between these two phases, were observed and the common occurrence of steps along these interfaces suggested ledge migration as one possible transformation mechanism. Coherent intergrowths of structural elements rather similar to tetragonal‐tungsten‐bronze (TTB)‐type were also found and these sometimes appeared to be relics of the super‐lattice phase. The chemical implications of the sevarious observations are briefly discussed.
Are the core structures of dislocations and grain boundaries resolvable by HREM?
Authors: Bourret A. , Thibault‐Desseaux J. , D'Anterroches C. , Penisson J. M. , De Crecy A.
Published: August 2011

Linear or planar defect structures have been recently studied by high resolution electron microscopy (HREM). Defects are generally difficult to solve for several reasons: they are non‐periodic and their images are limited to a very few projections along low index axes. For edge dislocations in materials having a high lattice friction, image analysis is possible and relatively fine details are now available about core radii and location of the atom pairs. The accuracy, however, of the proposed structures is within ± 0·025 nm if a priori information is known. This is not always sufficient to distinguish between several models. For a high angle tilt grain boundary the situation is comparable. A discussion of the errors introduced by several factors (object orientation, optical axis alignment) and measurement of experimental parameters is presented. At present all the information content of an image is not used: intensity profiles and quantitative comparison of simulated and experimental images should provide better ways for solving an unknown structure. However, the improvement of the point resolution is the key factor for further progress in defect analysis.
Microstructure of Sm5Ni19 intermetallic compound observed by high‐resolution electron microscope
Authors: Takeda S. , Horikoshi H. , Komura Y.
Published: August 2011

The method of high‐resolution electron microscopy is applied to study the microstructure of the Sm–Ni intermetallic compound whose composition is near the Sm5Ni19. Several new polytypic structures (5T, 7T, 15R and 18R) are found and their crystal structures are analysed by high‐resolution imaging method combined with electron diffraction method. Various kinds of defects are observed and the atomic arrangements near the defects are deduced; the defects include stacking faults in Sm5Ni19, intergrowth of the Sm2Ni7 or other complex structures in between the matrix of Sm5Ni19, complicated defect structure which changes the stacking sequence of Sm5Ni19, and lattice defects in the region of terminating the intergrowth.
Melamine resins, a new class of water‐soluble embedding media for electron microscopy
Authors: Bachhuber Karlheinz , Frösch Dieter
Published: August 2011

Melamine‐formaldehyde, a universally used technical aminoplast, can be modified into a water‐compatible electron microscopic embedding medium. The complete processing of tissues—from fixation to sectioning—is described in this paper. It produces stable sections showing remarkable ultrastructural detail.
Automated techniques for the study of lung alveolar stereological parameters with the IBAS image analyser on optical microscopy sections
Authors: Rigaut J. P. , Berggren P. , Robertson B.
Published: August 2011

In the case of lung alveoli, the good contrast of the features allows the use of a fully automated image analysing procedure, yielding many of the desirable stereological parameters which can be estimated on optical microscopy slides: alveolar volume and surface densities, a form factor and average mean integral curvature.

With our program, specially designed for the IBAS image analyser (Kontron Bildanalyse, Munich, B.R.D.), the whole automated process takes an average of 28 s per field. The image analyser‐obtained stereological estimates compare well with those given by point and intersection counting methods.

Some difficulties inherent in image analysers arise when measuring alveolar boundary lengths: a scheme is therefore proposed which eliminates the image‐edge errors, with no sampling bias, by comparing three different frames inside each field. A solution to the problem of estimating automatically the positive and negative tangent counts, necessary for the curvature calculations, is also proposed.

Our program has been applied routinely to the study of premature newborn rabbit lung alveoli, as part of a work dealing with an evaluation of the possibility of preventing the respiratory distress syndrome by the administration of heterologous surfactant.
The correlation of light and electron microscope observations
Authors: Perrie W. T. , Webb D. M.
Published: August 2011

A technique is described to allow electron microscopic investigation of a specific feature of a section on a glass slide. A section on a glass slide (previously treated with a silicone release agent) is processed as required for light microscopy. The section is then impregnated with Araldite and cured with an epoxy resin block on top of it. The section and block are removed from the slide and viewed with a light microscope. The selected area for ultrastructural study remains under continuous observation while the block is trimmed. Semi‐thin (1 μm) sections retain the original staining for light microscopy and ultra‐thin sections are stained with heavy metals in the normal manner. We show how an inflammatory lesion in a large area of muscle in a case of polymyositis may be quickly located and studied at the ultrastructural level.
A simple and efficient method for objective discrimination between pseudostratified, metaplastic and dysplastic nasal epithelium
Authors: Boysen M. , Marton P. F. , Pilström L. , Solberg L. A. , Torp T.
Published: August 2011

Morphologic features may sometimes be interpreted differently by different pathologists. By introducing objective methods in the histological evaluation the subjectivity of the judgement is reduced and a higher degree of consistency achieved. In the present study three linear parameters of nasal epithelial basal cells: the largest transversal nuclear diameter, the sum of the longitudinal and transversal nucleolar axes and the basal cell width, i.e. the width of the attachment face to the basement membrane, were classified by means of three‐class rulers. From this classification weighted indices were made which were then given equal weight and transformed, so that when added up their sum, the joint index (Q) to the largest possible extent was consistent with pseudostratified/stratified cuboidal, stratified squamous and dysplastic epithelium when Q ≤ 1, 1 < Q ≤ 2 and Q > 2 respectively. Tested prospectively we found 91% agreement between the morphometric and histological classification.

The method presented is extremely simple and rapid to perform, and appears to have diagnostic potential in distinguishing various types of epithelial alterations of the nasal mucosa. The method may also be useful in approaching similar problems in other epithelia.
An investigation of the degree of randomness in the dispersion of point features (triple junctions) in a plane
Authors: Stock S. R. , Hilliard J. E.
Published: August 2011

The nearest‐neighbour distance distribution was used as a criterion to investigate the dispersion of triple junctions. Comparison of the expected and measured distributions by the χ2‐test indicated that the triple junctions were probably not randomly located, contrary to an earlier conclusion based on an areal probe of the same specimen. The present results suggest that areal probes are insensitive to large deviations from randomness in the location of points in a plane.
A loop for the transfer of thin tissue sections
Authors: Deer B. C.
Published: August 2011

The construction and use of a simple wire loop for the transfer of thin tissue sections are described. The loop picks up, carries, and releases a drop of liquid containing the sections. The loop is opened and closed by manipulation of the forceps to which it is attached. This controls the attraction of the loop for the water drop and ensures reliable pickup and release of the sections.
The application of high resolution electron microscopy to the study of oxidation
Authors: Newcomb S. B. , Smith David J. , Stobbs W. M.
Published: August 2011

High resolution electron microscopy has been used to examine both the similarities and differences in the growth morphologies of Fe2O3, Cr2O3 as formed on iron alloys, and MgO as formed by burning Mg in moist air. A general discussion is also given of the relative advantages and disadvantages of a variety of TEM techniques as applicable to the study of oxidation phenomena.
ALCHEMI: a new technique for locating atoms in small crystals
Authors: Spence J. C. H. , Taftø J.
Published: August 2011

Atom Location by Channelling Enhanced Microanalysis (ALCHEMI) is a quantitative technique for identifying the crystallographic sites, distribution and types of substitutional impurities in many crystals. The method involves no adjustable parameters, can be applied to areas as small as a few hundred Angstroms and to impurity concentrations down to about 0·1 atomic per cent. It is capable of distinguishing neighbours in the periodic table. The method uses the incident electron beam orientation dependence of characteristic X‐ray emission and uses an energy dispersive X‐ray microanalyser fitted to a transmission electron microscope. The method does not require the specimen thickness or precise orientation to be known, and makes few assumptions about the form of the dynamical electron wavefunction, which need not be calculated or predicted. The classical problems of cation ordering in spinels, feldspars and olivine have now been studied by this method.
The relative importance of multiple inelastic scattering in the quantification of EELS
Authors: Bourdillon A. J. , Hall D. J. , Morrison C. J. , Stobbs W. M.
Published: August 2011

A comparison is given of energy loss results obtained for the L and K edges of aluminium as a function of specimen thickness, crystallographic orientation and collection angle. It is demonstrated that as the thickness is increased post‐loss elastic scattering is generally important in reducing the fraction of electrons collected. The implications for the quantification of EELS data are discussed while a comparison of the Fe/C ratio in cementite demonstrates the improved consistency which can be obtained when comparing K and L losses at lower energy separation than are the losses for aluminium.