bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunofluorescence and protein A‐gold techniques in localization of plant pathogen antigens in Lowicryl K4M embedded tissue
Authors: Rey M. E. C.
Published: August 2011

This paper investigates the use of Lowicryl K4M in the embedding of apple tissue for immunocytochemistry. The localization of the extracellular protease of the apple pathogen, Nectria galligena Bres., in infected apple tissue by immunofluorescence and protein A‐gold immunoelectron microscopy is also described. Infected apple tissue was fixed in 0.5% glutaraldehyde and 4% paraformaldehyde and embedded in Lowicryl K4M at 313 K. The protease was isolated and purified from rotted apple tissue by gel filtration and ion‐exchange chromatography. Monospecific antibodies against the protease were raised in rabbits and purified by protein A‐affinity chromatography. Incubation of apple tissue sections, infected with N. galligena, with the mono‐specific antibody and tetramethylrhodamine isothiocyanate (TRITC)‐conjugate, resulted in specific fluorescence of fungal hyphae and cytoplasm of apple cells. Similar localization of colloidal gold particles over hyphae and host cell cytoplasm was demonstrated employing protein A‐gold immunochemistry. The low temperature embedding resin Lowicryl K4M appears to provide adequate morphological preservation of apple tissue and excellent retention of antigenicity. TRITC conjugates and protein A‐gold may prove useful in immunocytochemical investigations of plant‐pathogen interactions.
The unbiased estimation of number and sizes of arbitrary particles using the disector
Authors: Sterio D. C.
Published: August 2011

A three‐dimensional counting rule and its integral test system, the disector, for obtaining unbiased estimates of the number of arbitrary particles in a specimen is presented. Used in combination with ordinary and recently developed stereological methods unbiased estimates of various mean particle sizes and the variance of particle volume are obtainable on sets of two parallel sections with a known separation. The same principle allows the unbiased estimation of the distribution of individual particle volumes in sets of serial sections.
Effects of fixing and staining on images of tropomyosin Mg‐paracrystals
Authors: Lamvik Michael K. , White Linda R. , Eik‐Nes Kristen B.
Published: August 2011

Tropomyosin Mg‐paracrystals have been studied by positive and negative staining with uranyl acetate and sodium phosphotungstate, either unfixed or fixed with glutaraldehyde. Fixation causes changes in some of the paracrystal bands. Some of the bands are more intense when a positively‐charged staining ion is used than when a negatively‐charged ion is used, in both negative and positive‐staining techniques. This result rules out the suggestion that negative staining is not affected by the charge of the stain in tropomyosin, and agrees with findings in other specimens. Therefore it is unlikely that any simple parameter based on residue size can be used to predict the image intensity from the amino acid sequence for tropomyosin. These staining patterns cast doubt on the earlier interpretation of a prominent white band in the paracrystal as an overlap of alpha‐helical molecular ends.
Diffusive motions in living cytoplasm probed by laser Doppler microscopy
Authors: Steer Martin W. , Picton Jill M. , Earnshaw John C.
Published: August 2011

We have examined cytoplasmic motions in growing pollen tube tips using laser Doppler microscopy, a non‐invasive and non‐destructive technique. The observed correlation functions were consistent with the existence of diffusive motions in the tip cytoplasm. Further analysis of the data revealed the existence of at least two major and a third, minor, diffusing species in the cytoplasm. Information from quantitative electron microscopy and from published literature values of cytoplasmic viscosity was used to tentatively identify the organelles contributing to the observed diffusive motions. It is concluded that motions of mitochondria and secretory vesicles dominate the observed correlation functions with a minor component detected from either ribosomes or the microfilament network. Sources of variation in the observed data are discussed, as are the physical limitations that prevent a more positive analysis of the present data.
An automatic pulsed laser microfluorometer with high spatial and temporal resolution
Authors: Docchio F. , Ramponi R. , Sacchi C. A. , Bottiroli G. , Freitas I.
Published: August 2011

The paper describes an automatic pulsed laser microfluorometer with high spatial and temporal resolution, developed in our laboratories. The instrument consists of: (i) a nitrogen‐laser‐pumped dye‐laser for the excitation of the fluorescence, (ii) a microscope with additional optics to focus the excitation beam on the sample and to collect the fluorescence, (iii) filters or monochromators to select the output wavelength, (iv) a fast photomultiplier tube to detect the signal, and (v) a dual time‐scale microprocessor‐controlled signal averager for the acquisition and processing of the signal.

Examples are given that show the potential of the time‐resolved fluorescence microscopy in studying, quantitatively and qualitatively, the properties of fluorescent molecules.
The appearance in TEM of proteoglycan predentine is fixation dependant
Authors: Goldberg M. , Escaig F.
Published: August 2011

The appearance of proteoglycans visualized on thin sections with alcian blue was compared in rat incisor predentine fixed either in aqueous aldehyde solutions or by anhydrous methods (acrolein vapours or rapid freezing‐freeze substitution). The granular or branched chain‐like structures detected after aqueous fixation probably resulted from precipitation or shrinkage of an expanded amorphous gel observed only after anhydrous fixation. In predentine, morphological interactions between proteoglycans and collagen fibres may be also artefactual.
Demonstration of mitochondrial mineral deposits in osteoblasts after anhydrous fixation and processing
Authors: Manston J. , Katchburian E.
Published: August 2011

The fine structure of early developing bone was examined using a modification of a new non‐aqueous processing method in which anhydrous glutaraldehyde dissolved in dimethyl‐sulphoxide (GLUT‐DMSO) is used as a fixative.

Preliminary results show that the basic morphological features of the cellular and extracellular compartments of developing bone were preserved. The cytoplasm of osteoblasts revealed numerous well‐preserved mitochrondria and other membranous organelles. Inside the mitochrondria there were large and conspicuous electron‐opaque granules of mineral which were more prominent and numerous than in specimens prepared by conventional aqueous procedures. The collagenous matrix also revealed electron‐opaque deposits of mineral. These results support the evidence for the existence of mitochondrial calcium phosphate in osteoblasts in the form of granules.
Pitting and bubbling artefacts in surface replicas made with silicone elastomers
Authors: Gordon Kathleen D.
Published: August 2011

Pitting or bubbling on the surface of epoxy resin replicas is a frequently encountered artefact associated with the use of silicone rubber impression materials. Several variants of this type of artefact are illustrated. It is suggested that bubbles or pits form at the mould: epoxy interface during curing of the epoxy, and that they are caused by gas escaping from the mould material itself. Duration of silicone rubber polymerization, base‐catalyst ratios, and surface porosity of the object may all influence the occurrence of mould degassing.
Internal medulla of hair revealed by scanning electron microscopy
Authors: Gabra‐Sanders Thea , Ryder M. L. , Williams A. E.
Published: August 2011

The standard scanning electron microscope method for examining surface structure using the secondary electron mode has unexpectedly revealed the internal ladder‐type medulla of some animal hairs.
Modification of the anticontamination device of a Reichert‐Jung freeze fracture apparatus
Authors: Allain Manuel , Ardonceau Jacques
Published: August 2011

We describe a modification in the cooling system of the anticontamination shield of a Reichert‐Jung freeze fracture apparatus. We replaced the flexible pipes by a flexible copper braid in order to increase reliability and keep the high quality of replicas.
The relative efficiency of cryogenic fluids used in the rapid quench cooling of biological samples
Authors: Bald W. B.
Published: August 2011

The heat transfer characteristics of various cryogenic fluids used in the rapid quench cooling of biological samples are examined. It is concluded that cryofixation should be achieved during the initial plunge stage of the cooling. Liquid nitrogen maintained near its melting point of 63·1 K at a pressure in excess of the critical value of 33·5 atm will produce the quickest cooling. Nitrogen could also provide the best ultrastructure at atmospheric pressure if the minimum plunge depth and velocity criteria are satisfied. Plunging bare thermocouples into cryogenic fluids will lead to erroneous conclusions about the relative cooling efficiencies of various liquids for cryofixation. A qualitative explanation for the results obtained by Silvester et al. (1982) during the rapid cooling of water drops in various fluids is proposed.
Quantitative analysis of pore patterns on rat prostate nuclei using spatial statistics methods
Authors: Pedro Nuno , Carmo‐Fonseca M. , Fernandes Pedro
Published: August 2011

Spatial statistics methods have been used to analyse the nuclear pore pattern in rat ventral prostate nuclei isolated from adult animals. The observed results show that: (1) pores on prostate nuclear membranes are not randomly distributed; (2) the data sets obtained from different micrographs are consistent with the same statistical model thus suggesting the existence of a typical pore distribution.
Selective demineralization of hard tissues in organic solvents: retention or extraction of proteoglycan?
Authors: Scott J. E. , Burton Shirley M.
Published: August 2011

Ideally, demineralization of tissues, fossils, etc., should not result in removal of other chemical species along with the mineral components. Organic solvents containing alkylammonium salts of EDTA are good demineralizing media (Scott & Kyffin, 1978), offering great flexibility and a variety of solvent properties which can be chosen either to extract or to retain tissue polymers in situ. The properties of the alkylammonium cation are crucial in determining whether a given solvent will extract tissue polyanions (i.e. nucleic acids, proteoglycans, acid glycoproteins, etc.) or not. The situation is analysed for the general case, using the fact that a simple ion‐exchange reaction determines the outcome. Some solvent‐alkylammonium combinations, e.g. ethanol‐trimethylammonium, are very poor solvents for proteoglycans, leaving them behind in the tissue, whereas by increasing the size of the organic portion of the alkylammonium salt (e.g. to tri‐n‐butylammonium) or changing the solvents to e.g. dimethyl sulphoxide, the proteoglycan can be extracted from cartilage, etc. The aninic half of the alkylammonium salt plays a relatively minor role. The exception is EDTA itself, which sequestrates inorganic ions (e.g. Na+) in organic solvents, thus driving the ion‐exchange towards completion, with efficient conversion to alkylammonium salts of the polyanion.

These results, and the chemical background to them, constitute a general framework within which various strategies for demineralization (with or without extraction of tissue polyanion such as proteoglycans) can be formulated.
A thermoelectric microscope stage for the measurement of the supercooling points of microscopic organisms
Authors: Wharton D. A. , Rowland J. J.
Published: August 2011

The design and construction of a thermoelectric cooling stage and its use for the determination of the supercooling points of microscopic organisms is described. Stage cooling is produced by a Peltier unit with a refrigerated circulator as a heat sink. Control is achieved by automatically comparing the temperature input to a reference ramp and varying the power input to the Peltier module accordingly.
A standardized technique for the in toto epoxy resin embedding and precipitate‐free staining of small specimens covered by strong protective outer surfaces
Authors: Mothes‐Wagner U. , Wagner G. , Reitze H. K. , Seitz K. A.
Published: August 2011

A method is described for the in toto embedding and precipitate‐free staining of small invertebrates covered by strong protective outer surfaces.

The difficulties of tissue infiltration in undissected animals with cuticles were overcome by the use of ethanol and 1,4‐dioxane as dehydrating agents, and Spurr's medium as the embedding resin. (1) Fixation as appropriate, followed by embedment in aqueous agar; (2) dehydration: 50% ethanol, 70% ethanol, 2 times 100% 1,4‐dioxane, 15–20 min for each step; (3) infiltration: dioxane:resin 1:1 90 min, dioxane:resin 1:3 90 min, pure resin overnight, changed once for an additional 10 h, all steps with constant shaking; (4) polymerization 16 h at 343 K; (5) sectioning; (6) staining: 0·9% cacodylate buffered KMnO4 (pH 6·5) 30–60 s, washing, Reynolds' lead citrate 5 min, washing by grid floating.

The advantage of the method is discussed in relation to serial sectioning of entire invertebrates which could not be dissected prior to embedding.
The use of touch‐sensitive screen in interactive morphometry
Authors: Silage Dennis A. , Gil Joan
Published: August 2011

We propose the use of a touch‐sensitive screen as a tool to facilitate acquisition of data both in point counting and morphometric planimetry. It obviates the use of a ‘correspondence image’ required with many planimetric devices in which the means for data acquisition and display are separate. The touch‐sensitive screen offers operational advantages over the light pen. The characteristics of this new graphical input device, its assembly and basic operational considerations are introduced and discussed.
Review of the principal contrast effects observed at interphase boundaries using transmission electron microscopy
Authors: Pond R. C.
Published: August 2011

This review is concerned with the interpretation of contrast features observed at interphase interfaces using conventional transmission electron microscopy. The principal features which arise are interfacial dislocations and steps, four types of fringes, namely thickness, Δw, displacement and moiré fringes, and lattice images. The contrast mechanisms leading to these features are discussed in the framework of the dynamical theory of electron diffraction. In particular, the influence on image contrast of the cyrstal composition and structure, the interfacial configuration and the crystallographic relationship of the two phases in the specimen is considered. In general, electron diffraction in bi‐crystalline specimens is complicated, and reliable interpretation can only be carried out when the diffraction conditions have been carefully established. The diffraction conditions most widely used are discussed, and their relative merits compared.
Optimized acquisition parameters and statistical detection limit in quantitative EELS
Authors: Pun Thierry , Ellis James R. , Eden Murray
Published: August 2011

Optimizing the acquisition parameters for EELS recording has to be accomplished simultaneously from the physical and the statistical points of view; the statistical aspect of the question is covered here. Approximate probability density functions of the variables of interest are derived, which provide a global measure of signal‐to‐noise ratio taking into account every step of the EELS edge area estimation process. Qualitative and quantitative advice is given regarding the critical choice of the estimation and integration energy regions. The notion of visual contrast is presented; it permits the introduction of the concept of statistical detection limit. It is found that for typical experimental conditions, when other factors are equal, the required analysis time for the sample varies approximately as the inverse square of the concentration.
Geometric model of the rat intestinal mucosa for stereological evaluation of villus amplification factors
Authors: Mayhew T. M.
Published: August 2011

This study was prompted by a desire to compare villus amplification factors at various sites along the small intestine of the laboratory rat. The paper describes a precise and efficient stereological method for achieving this objective. The method relies on conventional intersection counts performed exclusively on transverse sections.

Analyses are based on six adult rats. Pieces of small bowel fixed by in situ perfusion were taken from proximal, middle and distal regions. Intersection counts on villus and primary mucosal surfaces were performed on transverse and longitudinal sections through the same regions. These counts revealed that intestinal architecture can be reasonably approximated by the simple geometric model of an isotropic villus surface on an anisotropic (cylindrical) primary mucosal surface. As a result, intersection counts may be confined to transverse sections (to reduce sampling variance) and the appropriate ratio estimator multiplied by the numerical coefficient 4/¶ (to obtain an estimate of surface amplification due to villi).
Flash X‐ray microscopy with a gas jet plasma source
Authors: Feder R. , Pearlman J. S. , Riordan J. C. , Costa J. L.
Published: August 2011

A novel flash X‐ray source, the gas jet plasma source, has been used for contact X‐ray microscopy. Using a wavelength range of 2–7 nm a resolution of the order of 30 nm can be obtained. The gas jet plasma source provides a new and unique tool which should allow future imaging of wet live cells.
Quantitative assessment of radiation damage in a thin protein crystal
Authors: Jeng Tzyy‐Wen , Chiu Wah
Published: August 2011

Radiation damage is a limiting factor for high resolution structural determination of protein crystals. We have used the median and quartile values of Ne exposures and the structural disordering factor (ΔB) to describe the crystalline disordering due to radiation damage in thin crystals of crotoxin complex embedded in glucose at room and low temperatures and in ice embedded crystals. By approximating the radiation damage to follow first order kinetics, we applied a correction to the diffraction intensities from patterns with high accumulated exposures. We used the structural similarity factor (R) to show that, for some data, the effectiveness of the correction can be as good as those in protein X‐ray crystallography.
A cryoembedding method for cutting ultrathin cryosections from small frozen specimens
Authors: Steinbrecht R. A. , Zierold K.
Published: August 2011

A technique is described which allows one to embed small frozen specimens for easy sectioning in a cryoultramicrotome. n‐Heptane is recommended as a cryoembedding medium due to its suitable melting point (182.4 K), high vapour pressure, chemical inertia, and good sectioning properties when solid. Freeze dried cryosections of cultured muscle cells, slime moulds, insect antennae and unicellular algae are presented as examples. There is no evidence that the preparation disturbs the distribution of soluble elements in the specimen, since X‐ray spectra of muscle cells exposed to heptane do not differ from those of samples not exposed.
Representation of three‐dimensionally reconstructed objects in electron microscopy by surfaces of equal density
Authors: Radermacher M. , Frank J.
Published: August 2011

A surface representation algorithm has been developed to visualize the results of three‐dimensional reconstructions from projections in electron microscopy. It produces an easily recognizable image of the reconstructed particle and facilitates comprehension of the three‐dimensional structure. Thus building of physical models can be avoided in many cases. If such a model is needed the algorithm can be used to establish the correct particle boundary in advance. Some further effort has been made to represent long fibres and visualize inner structures of objects, which are obscured by outer high‐density regions. Shading may be used to enhance the depth perception from single views.
Formaldehyde and glutaraldehyde in the fixation of chromatin for electron microscopy
Authors: Sewell B. Trevor , Bouloukos Catherina , von Holt Claus
Published: August 2011

Glutaraldehyde and formaldehyde have been used to fix chromatin core particles for electron microscopy. Glutaraldehyde crosslinks protein only, whereas formaldehyde crosslinks protein and DNA. This is confirmed by the observation that the detergents sodium dodecyl sulphate, Sarkosyl NL 35 and benzylalkyldimethyl ammonium chloride separate the DNA from the protein in the case of glutaraldehyde fixed core particles but not in the case of formaldehyde fixed core particles. The fixative used in the preparation must therefore be considered as a further variable when evaluating electron microscopic images of chromatin.
Double‐axis rotary replication for deep‐etching
Authors: Shibata Y. , Arima T. , Yamamoto T.
Published: August 2011

We have developed a simple new method of rotary shadowing, double‐axis rotary replication, for observing three‐dimensional structures in deep‐etched, rapid‐frozen tissues. The technical details of this method are described and compared with the conventional fixed‐angle rotary shadowing procedure.
Quantitative morphological analysis in a SEM‐microcomputer system—I. Quantitative shape analysis of single objects
Authors: Sergeev Y. M. , Spivak G. V. , Sasov A. Y. , Osipov V. I. , Sokolov V. N. , Rau E. I.
Published: August 2011

The combination of a scanning electron microscope and a microcomputer can be used to carry out automatic, quantitative, morphological analysis of a wide range of samples. A system implemented around a commercially available 8‐bit computer and interface hardware is described. Computerized analysis of shape is most accurately carried out by the use of numerically determined Fourier transforms of the high contrast bright field transmitted electron image; however, Walsh functions offer very significantly faster processing times on smaller computer systems.
Quantitative morphological analysis in a SEM‐microcomputer system—II. Morphological analysis of complex SEM images
Authors: Sergeev Y. M. , Spivak G. V. , Sasov A. Y. , Osipov V. I. , Sokolov V. N. , Rau E. I.
Published: August 2011

The application of automatic image analysis image techniques in the SEM to massive samples with complex morphology requires a careful choice of the imaging mode and detector geometry used. Thus the quantitative analysis of conjugated surfaces requires summary images, derived from backscattered, secondary, transmitted electron and cathodo‐luminescent signals, as well as dedicated processing algorithms. Careful attention must also be paid to the problems of obtaining and matching these various images.
High resolution structure images around the pole pattern of a bent crystal. Application: deformation of a crystal near a defect
Authors: Cesari C. , Nihoul G. , Yangui B. , Boulesteix C.
Published: August 2011

The variation of high resolution structure images with the tilt of the incident electron beam has been studied on bent crystals. By systematically computing images for different situations we show that the aspect of the image can be linked to the angle of tilt of the incident beam, and to the direction of tilt. Good agreement is obtained between micrographs and computed images. As one possible application, we analyse the surroundings of a planar defect.
A unidirectional temperature gradient stage for solidification studies in aqueous solutions
Authors: Kourosh Sohrab , Diller Kenneth R.
Published: August 2011

A low temperature light microscope stage has been designed and fabricated for quantitative analysis of the solidification of aqueous solutions. Thermal boundary conditions are controlled during freezing so as to enable direct comparison of data with complementary theoretical models. Evolution of the temperature distribution within the system is monitored by a one‐dimensional thermocouple array, and growth of the phase interface is measured via a displacement transducer. The two‐dimensional solution concentration field is assessed by computer densitometric analysis of the grey scale distribution for a light absorbing solute. Experimental and theoretical analyses of freezing processes with the stage correlate well, and the data base is shown to be internally consistent by independent thermal and mechanical measures of the interface velocity.
A study of the effect of solid particle impact and particle shape on the erosion morphology of ductile metals
Authors: Rao P. Veerabhadra , Young Stanley G. , Buckley Donald H.
Published: August 2011

Studies were conducted to identify the differences in the modes of erosion between impulsive and steady‐jet glass bead erodent particle impingement at normal incidence. A 6061‐T6 aluminium alloy, copper and 1045 steel were used as test materials. A small bore or calibre gas gun apparatus was used for impulse testing and a commercial sand blasting facility was used for steady‐jet impingement testing. Crushed glass was also used as an erodent in the steady‐jet apparatus and the erosion patterns from crushed glass were compared with those of glass bead impingement erosion.

Morphological features and material removal mechanisms for the specimens were studied using weight, loss measurements, scanning electron microscope, energy dispersive X‐ray spectroscopy (EDS), and surface profilometry. Impulsive conditions induced more severe damage and results in embedment and fragmentation of glass beads, likely due to more intense pressure pulses and longer loading times on impact. Patterns from steady‐jet glass bead impingement experiments exhibited overlapping of plastically formed craters with very little evidence of particle fragmentation.

Recorded profiles and micrographs of the specimens subjected to steady‐jet impingement indicate that crushed glass induced deeper and wider pits than glass beads. The volume loss of 1045 steel was almost half that observed on aluminium and copper specimens with both types of erodent particles. The material removal process for glass bead impingement appears to be in the form of deformation induced fatigue failure with flake‐like debris visible at high magnification. With crushed glass, material appears to have been removed as small chips, leaving a jagged, angular, faceted surface characteristic of ‘cutting wear’.

EDS analyses indicated traces of silicon (from the erodent particles) on all three materials. Silicon amounts were maximum at the pit bottom.
A simple and inexpensive liquid helium cooled ‘slam freezing’ device
Authors: Heath I. Brent
Published: August 2011

A liquid helium cooled copper block device has been designed for the rapid freezing of biological material. The apparatus differs from previous designs in being easily constructed from readily available components. It incorporates a novel and simplified specimen carrying system. Construction of this apparatus is possible for any laboratory and makes the advantages of freeze substitution reliably available for many cell types at minimal cost.
The use of Hanks'‐pipes buffers in the preparation of human, normal leucocytes for TEM observation
Authors: Haviernick S. , Lalague E. D. , Corvellec M.‐R. , Pelletier L. , Calamba A. D. , Derghazarian C. , Cousineau G. H.
Published: August 2011

It is suggested that the use of Hanks' + pipes + sucrose buffers, in combination with glutaraldehyde and osmium tetroxide fixatives, represent an excellent mode of preparation of fresh and cultured peripheral blood leucocytes, not only for transmission electron microscopy observation of these cells but also for scanning electron microscopy.
Image processing in a general purpose microcomputer
Authors: Russ John C. , Russ J. Christian
Published: August 2011

Video images from light and electron microscopes can be acquired in ‘digital’ form within a personal computer, using standard peripherals and interfaces. In addition to providing for measurement of such images (counting of features by size class, integration of density, etc.) the stored image can be processed in a variety of ways. Using techniques known in the image processing field but only recently practical in such small computers, smoothing of the grey‐scale information, sharpening of edges, extraction of texture information, directional derivatives, and shifts of grey scale can be applied. Combination of images with previous ones stored on disk is especially useful for electron and X‐ray images from SEM. The system described carried out all operations in software, typically requiring times of 1–20 s per image which should be practical in many microscopy laboratories.
The use of a hybrid diffraction unit in the measurement of dislocation densities by electron microscopy
Authors: Ecob R. C.
Published: August 2011

The expression used in the electron microscopical measurement of dislocation densities is ϱ=2N/Lt, where N is the number of intersections between the dislocation lines in a TEM image, L the total length of a set of sampling lines superimposed upon that image (corrected for magnification) and t the thickness of the thin foil containing the dislocation structure. During the estimation of ϱ it is therefore important to reduce errors in the measurements of all three variables. It is shown how calibration of the microscope magnification, careful consideration of diffraction conditions and use of the hollow cone and rocking beam facilities on the hybrid diffraction unit fitted to the Philips EM 400T can assist in increasing the speed and accuracy of the measurement procedure.
A simple chamber for observing microscopic specimens in both top and side views
Authors: Boocock C. A. , Brown A. F. , Dunn G. A.
Published: August 2011

A method is described which permits the observation of microscopic specimens in both top and side views, a rapid change between the two views being possible without disturbing the specimen. The system requires the introduction of simple optical components into a culture chamber, does not restrict the types of microscopy that may be used, and does not necessitate modification of the microscope itself. The technique is discussed in relation to the study of tissue cells in culture, but may find applications in other areas.
A tannic acid based preparation procedure which enables leucocytes to be examined subsequently by either SEM or TEM
Authors: McCarthy D. A. , Pell B. K. , Holburn C. M. , Moore S. R. , Perry J. D. , Goddard D. H. , Kirk A. P.
Published: August 2011

A modification of the glutaraldehyde‐osmium tetroxide‐tannic acid‐uranyl acetate (GOTU) fixation procedure is described which allows human leucocytes to be examined subsequently by either transmission electron microscopy (TEM) or scanning electron microscopy (SEM).
A new method for obtaining scanning electron microscopic images of the reorganization process of human dermal microvascular endothelium in vitro
Authors: Tsuji Takuo , Karasek Marvin A.
Published: August 2011

A new method for obtaining scanning electron microscopic images of the reorganization process of endothelial cells has been developed. When covered with a collagen‐coated disk, all the cultured endothelial cells reorganized on the collagen of the disk, which was easily taken out from the dish to process for SEM.

The reorganization process could be divided into four stages: endothelial cell growth (Stage 1), reticular network formation (Stage 2), tubular structure formation (Stage 3), and cytolysis of the tube (Stage 4). Between Stages 1 and 2 the endothelial cells transformed from a cobblestone to a spindle‐shaped pattern and fused each other, forming a board‐like structure. Between Stages 2 and 3 break up of parts of the board‐like structure and outflow of a necrotic mass from the centre of the structure occur. At Stage 3 a tubular structure is formed following enwrapping of the cleared centre by the surrounding endothelial cells. This method produces a means to study the angiogenesis in a variety of disorders including tumours and wound‐healing process using SEM.
Penetration rate of glutaraldehyde in various buffers into plant tissue and gelatin gels
Authors: Coetzee J. , van der Merwe C. F.
Published: August 2011

The choice of buffer vehicle during fixation with glutaraldehyde influences not only the rate of crosslinking in a bovine serum albumin model system, but also the penetration rate of the glutaraldehyde into plant tissue and gelatin gels. The rate of penetration into plant tissue and the crosslinking rate of bovine serum albumin by glutaraldehyde are independent of each other in any one buffer. Buffer penetration into gelatin gels is not as fast as glutaraldehyde penetration into the same gels. The highest rate of crosslinking of bovine serum albumin by glutaraldehyde is obtained with a buffer vehicle consisting of Na2HPO4 and NaH2PO4. Glutarldehyde concentrations of 2–3% are advised for routine fixation of plant material.
Image analyser measurements of coal reflectance
Authors: Lee James B.
Published: August 2011

The application of an image analyser to the rapid measurement of coal reflectance histograms is described. The ability of an image analyser to utilize a two‐dimensional image allows the coal only to be represented in the reflectance histogram, with contribution from the resin used in particulate block specimens eliminated. While the accuracy of reflectance measurement with an image analyser is not as great as can be obtained with a spot photometer system, sufficient accuracy is obtainable for rank determination.
Simple procedures for the transfer of grid images onto glass coverslips for the rapid relocation of cultured cells
Authors: Small J. V.
Published: August 2011

Procedures are described whereby electron microscope‐grid images may be transferred onto the surface of glass coverslips. By using coordinate grids, patterns are obtained that are ideal for the rapid relocation of cultured cells. Two basic procedures are presented. In the first, metal is evaporated, in a vacuum unit, onto glass coverslips carrying electron microscope grids to produce an exposed, glass replica of the grid pattern. The grid pattern is then etched in the glass surface with hydrofluoric acid and the metal subsequently dissolved away. In the second method silicone monoxide is evaporated onto coverslips carrying a ‘negative grid’ or a negative grid image, to produce a pattern directly visible in the phase contrast or interference microscope. Modifications for scanning electron microscopy are also described.
Choosing the appropriate section thickness in the melamine embedding technique
Authors: Frösch Dieter , Westphal Christel
Published: August 2011

When biological materials are infiltrated by a water‐soluble melamine resin and hardened, they become as hard as glass. This is a prerequisite for extreme thin‐sectioning.

In this paper, the structural information from unsupported transparent thin sections of beef liver catalase, calf thymus DNA, horse spleen ferritin, insect muscle and rat microtubules is compared to that of normal thin sections. While ferritin molecules (12 nm diameter), microtubule subunits (8 nm long axis) and catalase crystals (8 nm subunit diameter) appear to become mechanically damaged in a 10 nm section (as measured by resectioning), DNA‐molecules (3 nm diameter) are satisfactorily preserved during sectioning. Remarkably, for electron phase contrast imaging of unstained cross‐sectioned insect muscle, a minimum section thickness of about 30–40 nm is required.
Computer‐based determination of size and shape in living cells
Authors: Marsh Donald J. , Jensen Peter Koch , Spring Kenneth R.
Published: August 2011

Measurement of cell volume in living epithelial cells has become an important technique in studies of membrane transport processes that function in cell volume regulation. Planimetry of video images of optical sections enables the measurement of the cross sectional area of each section. Cell volume is calculated from the measured area of each section and the known focus displacements. In the past the measurement of cross section area has been done by manual positioning of a cursor superimposed on the video image. Each experiment generates approximately 200 images in which two or more cells may be analysed. We have developed a computer‐based method that uses one image as a template, and allows automated area determination of successive images by template matching and digital image processing. This new method is comparable to the older method in speed and accuracy, but requires much less effort from the experimenter.
Prolonged ethanol replacement by CO2 increases splits on articular cartilage surface after critical point drying
Authors: Helminen H. J. , Jurvelin J. , Tammi M. , Pelttari A. , Svartbäck C.‐M. , Kiviranta I. , Säämänen A.‐M. , Paukkonen K.
Published: August 2011

Ethanol replacement by CO2 of glutaraldehyde‐fixed and ethanol‐dehydrated rabbit articular cartilage specimens was monitored with both gas chromatograph and alcometer prior to critical point drying (CPD). The surface structure of the patellar specimens was also systematically registered with a semiquantitative scanning electron microscopic method. After a 2 h interval, when about 28 μl of ethanol/15 min CO2 extract was removed, the articular surface was smooth, although small areas of striated surface and superficial splits were present. A long‐term CO2 treatment (16 h) removed ethanol completely, but increased superficial splitting of the articular surface after CPD. Air‐drying of the specimens gave rise to inferior preservation of the cartilage: large areas with pitted and leafy surface qualities, but no superficial splits, were present on the surface. It was evident that prolonged ethanol replacement by CO2, prior to CPD, degraded surface structure of the articular cartilage which should be taken into consideration in the planning and design of experiments. Ethanol removal by CO2 could conveniently be monitored by an alcometer.
Models and observations of fcc/bcc interfaces
Authors: Ecob R. C.
Published: August 2011

Whereas models of the structure and periodicity of CSL or near CSL high angle grain boundaries are relatively well developed and have been to some extent verified by experiment, the nature of more general (e.g. fcc/bcc) interfaces is very imperfectly understood. One of the major differences between homo‐ and heterophase boundaries is the occurrence, in the latter case, of reproducible orientation relationships due to the crystallographic requirements of phase transformation (e.g. a glissile interface for martensitic growth, low energy interfaces for classical nucleation). A review is given of the relationships commonly observed to obtain between fcc and bcc crystals (as well as ordered phases based upon these structures).

Within a given relationship, it is possible to use models of the available interfaces to predict their dislocation content; the success of the various approaches is considered. It is shown that the dislocation arrays identified on fcc/bcc boundaries are consistent with the occurrence of a primary structural relaxation, but that the latter does not appear to be complete. Possible reasons for this are discussed, together with the extent to which secondary structural models may be applicable.

Attempts to rationalize the observed orientation relationships and morphologies are discussed, and it is pointed out that these all rely on searches for situations of minimum primary misfit, but that the way in which the latter is quantified determines the results of the analyses. Nevertheless, the implication is that the primary misfit does appear to determine the actual behaviour, though no structural or mechanistic conclusions can safely be drawn from this observation.
Optical fluorescence microscopy in three dimensions: microtomoscopy
Authors: Van Resandt R. W. Wijnaendts , Marsman H. J. B. , Kaplan R. , Davoust J. , Stelzer E. H. K. , Stricker R.
Published: August 2011

A unique property of confocal fluorescence microscopy is utilized to obtain scanned images of a number of samples in three dimensions. This kind of microscopy for the first time enables the direct study of the 3‐D arrangement of fluorescently labelled materials in living cells. The technique is based on a home build, confocal, scanning laser microscope. The entire volume of the object is scanned mechanically through the well‐defined focus of a laser illuminated, high numerical aperture objective. An identical optical path, arranged confocally, is used to detect the fluorescence emission from the focal volume. A pinhole in the image plane of the detection channel prevents fluorescence light, generated outside the focal volume, to reach the detector. Consequently a true resolution is obtained along the optical axis (identified as z‐axis) and three‐dimensional imaging without the need for mathematical operations is possible. A high resolution along the z‐axis (0.9 μm) is shown as well as in the object plane (0.25 μm) using Rhodamine as the fluorescent dye. The resolution along the z‐axis is explained and experimentally demonstrated using an object which contains only a z‐axis dependent structure. Pictures of sections through living cells are shown, both parallel to the object plane and parallel to the optical axis.
Cryo‐electron microscopy of tropomyosin magnesium paracrystals
Authors: Stewart Murray , Lepault Jean
Published: August 2011

Magnesium paracrystals of tropomyosin have been examined in thin films of vitreous ice by electron microscopy. This material is radiation sensitive, with damage being located near the molecular ends. However, low‐dose images show a repeating pattern of pairs of dark lines every 40 nm which indicates that the structure has been well preserved. These dark lines are separated by about 15 nm and probably represent extra protein density at the molecular ends. It has not been possible previously to see this feature by electron microscopy. We also demonstrate that the mercurial p‐chloro‐mercuribenzoate (PCMB) attached to the tropomyosin thiol at residue 190 can be detected in unstained paracrystals embedded in vitreous ice.
Quantitative assessment of chemical fixation, including the use of malachite green, for electron microscopy of vacuolate mesophyll cells from leaves of a grass
Authors: Lawton June R.
Published: August 2011

Various chemical fixation regimes were studied using leaves of the grass Lolium multiflorum Lam. as a test system. To evaluate the quality of fixation for electron microscopy two parameters were chosen; the extent of cell damage to mesophyll cells and damage and change in size of chloroplasts. Reducing the concentration of glutaraldehyde from 3% to 1% resulted in extensive cell damage. The addition of paraformaldehyde to 1% glutaraldehyde did not totally eliminate cell damage and resulted in dilation of thylakoids within the chloroplasts. In tissue fixed and washed in solutions matched to the total osmolality of the primary fixative solution there was little cell damage but the chloroplasts were larger, diameters 4.5times2.9 μm, than when only the buffer vehicle osmolality was maintained during subsequent washings, diameters 3.5times1.6 μm. Malachite green added to the primary fixative solution abolished cell damage regardless of buffer concentration and resulted in the appearance of osmiophilic droplets on the surface of and within the chloroplasts which were not present in the absence of malachite green. It is suggested that malachite green stabilizes cell membranes during primary fixation.
The microstructure of high‐rank coals at lattice resolution
Authors: Little J. A. , Gronsky R.
Published: August 2011

The microstructures of both anthracite and meta‐anthracite have been examined using high resolution electron microscopy. The microstructure of the native, untreated meta‐anthracite shows carbon fibrils consisting of as many as thirty parallel layers and being as long as 30 nm. In comparison with a previous investigation of anthracite microstructures this corresponds to an anthracite heat‐treated to at least 2500°C, or to a soft carbon precursor heated at 1500°C.

The anthracite microstructure shows little long‐range ordering but confirms earlier work, and consists of very small stacks of carbon layers in a preferred orientation. However, the low volatility of the anthracite has been used to resolve the interface between the coal and some minerals commonly found in all coal structures and an attempt has been made to use these observations to generalize on the possible catalytic activity of such minerals in other coals used for coal gasification. The interfaces were found to consist of numerous ledges and microledges all of which would increase the surface area of the mineral particle and give rise to high energy sites suitable for enhanced catalytic ability.
A comprehensive set of stereological formulae for embedded aggregates of not‐necessarily‐convex particles
Authors: Miles R. E.
Published: August 2011

Theoretical stereological approaches to particle aggregates characteristically suppose that each particle is convex, so that upon sectioning, each particle gives rise to either 0 or 1 convex planar profile. Actually, the essential requirement in such approaches is that each planar section profile be connected; such particles need not be convex.

In this paper, aggregates of quite general non‐convex particles, whose individual planar section profiles may thus comprise a number of disjoint connected regions, are considered. For this, it is necessary to anticipate the ultimate technical development of ultra‐sensitive devices (or other methods) capable of identifying in a planar section those disjoint connected regions stemming from the same particle. With this assumption, it is shown how, using the ‘4‐linc’—a new line section characteristic—to estimate the aggregate volume moment ratio , the standard isotropic section theory fully extends. To complete the picture, the recent development by Jensen & Gundersen of estimators for is extended to the ‘restricted’ case considered here. Allowing the particle volume distribution to belong to a two‐parameter family, these two parameters, Nv and the aggregate mean values may all be estimated.
Stereological estimation of the volume‐weighted mean volume of arbitrary particles observed on random sections*
Authors: Gundersen H. J. G. , Jensen E. B.
Published: August 2011

A stereological estimator of the weighted mean volume of particles of arbitrary shape is described. This unbiased estimator is based on simple point‐sampling of linear intercept lengths. The complete absence of shape assumptions effectively breaks the long‐standing ‘convexity‐barrier’: the only requirement here is that individual particles can be unambiguously identified by their profiles on random sections. Practical details of the simple estimation procedure and an example with very irregular particles are reported. Finally, an estimator of the variance of the weighted distribution of particle volume is discussed. This estimator is also valid for particles of arbitrary shape. For any mixture of ellipsoids (spheres, oblates, prolates and triaxial ellipsoids) the estimator is reduced to a simple function of measurements of diameters in the section plane.
Signed line intercept count
Authors: DeHoff R. T.
Published: August 2011

Elements of surfaces that bound a phase (β) in a two phase mixture (α+β) may be classified as: (a) convex (++) if both principal curvatures are positive; (b) concave (− −) if both are negative; and (c) saddle (+ −) if one is positive and the other negative. This classification excludes the limiting cases for which one or both of the principal curvatures is zero. The traces of these surfaces that form the boundaries of the β areas on a representative two dimensional section may also be: (a) convex (+) if the local curvature is positive; or (b) concave (−) if it is negative. Line intercepts may be tabulated separately for intersections with convex (+) and concave (−) segments of boundary. This paper presents a derivation of fundamental stereological formulae that relate these counting measurements to three‐dimensional geometric properties of the structure they sample.