bluuebunny/crossref_metadata_2025 huggingface dataset:
A specimen carrier, storage disc system for scanning electron microscopy (SEM): evaluation of stainless steel as a substratum for cell culture in vitro
Authors: Yeger Herman , Lea Peter J.
Published: August 2011

A method of sample preparation for scanning electron microscopy (SEM) studies based on the use of stainless steel discs as a cell culture substratum is described in detail. A number of different cell lines were grown on stainless steel, and the growth patterns and biocompatibility of cells cultured on stainless steel were compared to identical cells cultured on aluminium, glass and plastic substrata. Stainless steel provides cells with an excellent growth surface which allows these cells to retain their normal growth characteristics and appearance. The non‐toxic stainless steel discs can be manipulated through any combination of fixatives and organic solvents. The discs have been incorporated into a versatile system of sample preparation for SEM.
A simple electron beam gun for platinum evaporation
Authors: Hagler Herbert K. , Schulz Werner W. , Reynolds Rolland C.
Published: August 2011

A simple electron beam gun for platinum shadowing of freeze‐fractured or freeze‐etched surfaces is described. In the Steere‐Denton freeze‐etch instrument, this inexpensive device consistently yielded high quality replicas. The compact design possibly renders this gun operable in most freeze‐etch instruments.
On the methodology of the thyroid epithelial cell thickness determination
Authors: Kališnik Miroslav , Jakopin Primož , ŠUštaršič Janez
Published: August 2011

The average thickness of the thyroid epithelial cells may be determined either directly or indirectly. By the direct or caliper method, this thickness is overestimated as a result of which its empirical value must be divided by a correction factor Kd. By the indirect method, on the other hand, the thickness of the thyroid gland epithelium is calculated as the ratio of the double volume density of the epithelium to the sum of the inner and the outer surface density of the epithelium; in this case the sought for thickness value is underestimated and must consequently be multiplied by a factor Ki.

Both correction factors are algebraically defined. Their values are calculated and graphically represented as a function of the thyroid activation index (the ratio between the volume density of the epithelium and the colloid) for the range from 0·1 to 100.

The validity of the theoretical interpretation of the discrepancy between the values obtained for the average thickness of the thyroid epithelium by the direct and the corresponding values obtained by the indirect method, is empirically tested. It is shown that by the introduction of appropriate correction factors the difference between the results obtained by each method can be reduced. These improved direct and indirect methods for determining the wall thickness of a hollow sphere seem to lend themselves to being used in the stereological analysis of other biological, perhaps even non‐biological materials of similar structure.
A simple method for holding electron microscope grids during autoradiography of serial sections
Authors: Heywood Peter , Hodge Lon , Davis Frances , Simmons Tobianne
Published: August 2011

A simple, reliable, and inexpensive method is described for holding electron microscope grids during the application of photographic emulsion and during subsequent storage and processing. This method has proved to be especially useful in autoradiographic studies using serial sections.
Visualization of vesicles in semi‐thin sections by the heat removal of epoxy resins
Authors: Uhrík B. , Zacharová D.
Published: August 2011

A partial removal of the embedding medium from sections of tissue with hollow structures results in the formation of a phase object. The removal of epoxy resin by the heat of a gas‐burner flame is a simple method which does not lead to shifts and losses of sections from glass slides.
Polymeric cryoproteetants in the preservation of biological ultrastructure
Authors: Franks Felix , Asquith M. H. , Hammond Catherine C. , Skaer Helen le B. , Echlin Patrick
Published: August 2011

The solid states formed by vitrified and frozen aqueous solutions of some hydrophilic polymers, able to act as biological cryoproteetants, have been studied by differential scanning calorimetry and freeze fracture electron microscopy. Glass transitions, devitrification, recrystallization and melting behaviour of aqueous solutions of polyvinylpyrrolidone, hydroxyethyl starch and dextran have been established. The vitrified polymer solutions exhibit a characteristic microspherical morphology which is not induced by the quench cooling process but is an inherent feature of the solutions themselves.
Polymeric cryoproteetants in the preservation of biological ultrastructure
Authors: Skaer Helen le B. , Franks Felix , Asquith M. H. , Echlin Patrick
Published: August 2011

Two high molecular weight polymers, polyvinylpyrrolidone (PVP) and hydroxyethyl starch (HES), have been used as cryoproteetants for preparing specimens to be freeze fractured. Solutions of 25% (w/w) suppress the formation of intracellular ice in single cells and tissue blocks from both plants and animals to the extent that fine structural details of the cell can be elucidated. The mode of action of these cryoproteetants, together with the structures they reveal and the peculiar advantages attached to their use, is discussed.
Ultrastructure of unfixed plasma membranes in ultrathin frozen sections
Authors: Vogel Frank
Published: August 2011

Ultrathin sections of unfixed kidney tubular cells were obtained by cryo‐ultramicrotomy using the ‘wet’ method. The results showed that extremely thin sections permit high resolution studies on unfixed plasma membranes. Structural units on the inner surface of the plasma membrane could be detected.
Preparation of thin cryo‐sections for electron probe analysis of calcifying cartilage
Authors: Ali S. Y. , Gray J. Craig , Wisby Angela , Phillips M.
Published: August 2011

Conventional methods of fixation, dehydration, embedding and wet‐sectioning can produce artefacts in the chemical composition of mineralizing tissues. Cryoultramicrotomy was adopted for a more reliable approach to electron probe analysis of initial apatite formation in calcifying cartilage. Fresh rabbit epiphyseal cartilage was mounted on silver pins, frozen by immersion in liquid nitrogen, and sectioned with the specimen temperature at 133 K and the knife temperature at 273 K. Dry cryo‐sections (30–70 nm in thickness) were manipulated on to coated grids and examined the same day. These cryo‐sections showed good morphological and cytoplasmic detail, with large areas relatively free of ice‐crystal damage. Sections stained either with osmium vapour or negatively stained with silicotungstic acid showed areas with well‐preserved mitochondria with granules and endoplasmic reticulum. Unstained sections also showed dense granules (50–120 nm in diameter) in the mitochondria of chondrocytes and preliminary electron probe analysis of these has indicated a Ca/P mass ratio of approximately 1·14. In the longitudinal septa, about 2 μm away from the chondrocytes, matrix‐vesicle‐like particles could be seen with crystal needles inside them. Micro‐analysis of two of these gave a Ca/P mass ratio of 1·73 and 2·68. Cryo‐ultramicrotomy appears to confirm a number of conclusions derived from conventional ultrastructural study of growth cartilage and suggests for the first time how amorphous calcium phosphate and crystalline apatite can be shown to exist in different organelles in the same cryo‐section of the tissue.
Freeze‐fracturing: a review of methods and results
Authors: Sleytr U. B. , Robards A. W.
Published: August 2011

Freeze‐fracturing may be accomplished either under vacuum, or at atmospheric pressure. The devices available for freeze‐cleaving are discussed in relation to the vacuum and temperature conditions prevailing during cleaving, etching and replication. It is concluded that most specimens can be satisfactorily cleaved (even at 4 K), and processed using simple cleavage devices and systems that provide ample cold‐trapping protection for the specimen. Only for special purposes are microtome assemblies or ultra‐high vacuum units essential.

The fracturing process may produce artefacts by plastic deformation. Such artefacts have been noted in both non‐biological and biological polymers cleaved at temperatures as low as 4 K. Contamination of the frozen surface need not be a problem provided that the specimen is transferred under ‘safe’ conditions, and is protected by well‐designed cold traps.

Freeze‐cleavage and freeze‐sectioning are compared. It is considered that there will be a temperature range for most heterogeneous specimens within which both cleavage and fracturing may occur, depending upon the nature of the molecules in the cleavage/sectioning plane. Local heating during freeze‐sectioning will produce deformation artifacts as in freeze‐cleavage, and may also lead to more general surface ‘flow’. The relationships between sectioning and fracturing biological specimens at low temperature require further clarification.
Evaluation of membrane structure facts and artefacts produced during freeze‐fracturing
Authors: Bullivant S.
Published: August 2011

The freeze‐fracture technique is now widely used in the study of membranes, but it should be stressed that it shows internal hydrophobic planes of membranes, prepared under physical conditions far removed from those prevailingin vivo.Hence there is considerable potential for artefact. Work on the membrane lipid component, the intramembrane particles, and their aggregation under certain conditions, is reviewed in the context of fact versus artefact. Particular attention is paid to the results of complementary replica experiments, performed in the author's laboratory and elsewhere, which indicate that lipid collapse and protein particle deformation contribute to the appearance of the membrane fracture face seen in the final replica. A model, showing the effects of freeze‐fracturing on membranes, is presented.
Interpreting the results of freeze‐etching
Authors: Branton Daniel , Kirchanski Stefan
Published: August 2011

Morphological data obtained by freeze‐fracturing and other low temperature techniques must be interpreted in terms of molecular organization and function. Interpretation is aided by physical and biochemical approaches. Physical approaches such as rotary replication and ultralow temperature fracturing can improve resolution and preserve molecular arrangements which are difficult or impossible to observe with standard freeze‐etching techniques. Biochemical approaches such as dissociation‐reconstitution experiments can establish the molecular parameters underlying electron‐microscopically visible forms. Both approaches are illustrated by investigations of the human erythrocyte membrane.
Freeze‐drying process of biological specimens observed with a scanning electron microscope
Authors: Nei Tokio , Fujikawa Seizo
Published: August 2011

A pit membrane was observed with a cryo‐SEM during the course of dehydration at low temperatures. The freeze‐drying process of sea‐urchin eggs and parenchyma cells of higher plants was also examined with this microscope. Conditions for observation of frozen specimens in the native state were discussed on the basis of morphological studies of alterations such as shrinkage or deformation which appeared during the freeze‐drying process.
Contrast and quantitation in uniform regions of thin sections using transmission electron microscopy
Authors: Edie John W. , Karlsson Ulf L.
Published: August 2011

A direct approach to quantitative measurements of uniform regions in thin sections is described. Accelerating voltages around 80 kV and objective aperture angles of about 9·3 mrad will provide conditions where contrast is directly proportional to specimen mass thickness. An extensive treatment of electron scattering in Formvar films for wide ranges of electron microscopic operating conditions is summarized in a simple, empirical equation. The extent to which Formvar results may be generalized to other materials, both embedding media and structures within the thin section, is treated. Using these results, precise measurements of local section thickness and of specimen density and/or dry mass of regions which penetrate the entire section thickness are possible, with the accuracy dependent upon irradiation effects and specimen makeup.
Preparation of specimens for microscopic examination of edge profiles
Authors: Allen D. M. , Horne D. F. , Stevens G. W. W.
Published: August 2011

A method of mounting small fragile components in an optically clear medium has been developed which results in good edge retention on an undistorted specimen.
Cyanide removal of gold from SEM specimens
Authors: Sela J. , Boyde A.
Published: August 2011

The cyanide method is highly recommended for the purposes of the removal of gold coatings from SEM specimens prior to (a) further treatment before further SEM, and (b) staining and mounting for LM.
Accurate stigmating of a high voltage electron microscope
Authors: Krivanek O. L. , Isoda S. , Kobayashi K.
Published: August 2011

To reach the 0·2 nm point‐to‐point resolution possible with some high voltage electron microscopes, the astigmatism of the objective lens must be compensated to within 5 nm. Due to a number of factors the resolution of the image seen on the viewing screen of the high voltage microscope is, however, quite poor and does not permit compensation of such accuracy.

We describe a technique for evaluating and correcting the astigmatism that starts from a recorded micrograph of a thin amorphous specimen. The astigmatism is determined from the optical diffraction pattern using a variation of the Thon method. This variation avoids any direct measurement of the radii of the contrast transfer zones, and is extremely rapid and convenient. Adjusting the stigmator coil currents, calibrated in terms of their stigmating power, for zero astigmatism completes the correction in less than 10 min after the recording of the micrograph.
Preservation of shock‐frozen myocardial tissue as shown by cryo‐ultramicrotomy and freeze‐fracture studies
Authors: Sætersdal T. S. , Røli J. , Myklebust R. , Engedal H.
Published: August 2011

Myocardial tissue was quickly quench‐frozen by letting the specimen fall either into liquid nitrogen (LN2) or into LN2‐slush, or, by drill‐propelling the specimen into LN2 at a speed of 1·5 m/s. The preservation of the tissues was studied in ultra‐thin, dry‐cut and freeze‐dried cryo‐sections. Shock‐freezing by propelling into LN2 yields extensive areas of well preserved tissue without hole damage. Quenching into LN2‐slush shows variable results, while a maximum of hole damage is obtained by letting the specimen fall into LN2. Replicas of freeze‐fractured tissue which had been quench‐frozen by the same methods confirmed these observations. By the drill‐propelling, areas 2000–5000 μm2 wide reveal good preservation without visible ice crystals. Such areas occur within a superficial band of tissue to a depth of c. 20 μm. Even at 35 μm and 50 μm depth good preservation may be registered at the periphery of the replicas.

Description is given of modifications in accessories and methods in order to make the LKB CryoKit more suitable as a routine instrument. These modifications cover replacement of the knife coolant container by a coolant brass container in open connection with three copper tubes, replacement of one of the LKB Dewar flasks by a 25 litre Dewar equipped with a Balzers filling device, and replacement of the LKB plastic sleeve fixed to the back of the specimen coolant container, by two perspex‐glass discs mounted in the slit between the Ultrotome base and the cryo‐chamber. In addition, modified constructions are presented of the grid‐carrier, the press‐assembly as well as of the container for freeze‐drying and warming up of the frozen sections.
Freeze‐etching of unglycerinated tissue dispersions by application of the oil emulsion technique
Authors: Buchheim W. , Welsch U.
Published: August 2011

The oil emulsion technique has been applied to rat brain and liver tissue for freeze‐etching preparation without antifreeze pretreatment. It has been demonstrated that the dispersed tissue exhibits a high degree of vitrification throughout the mostly globular fragments up to at least 30 μm in diameter. Signs of insignificant ice crystal formation of 50 nm and less could only rarely be detected. Although the dispersion process seriously affects the tissue organization as a whole, most cellular organelles and the plasma membrane show satisfactory preservation of structure. In larger tissue drops, a local overall structural preservation of the original cellular organization is recognizable. The results obtained demonstrate the applicability of this method to various biological objects for realizing sufficient cryofixation in the absence of cryoprotectants.
Developments in automated reflectance microscopy of coal
Authors: Davis Alan , Vastola Francis J.
Published: August 2011

Automated reflectance microscopy (ARM) systems employ a microscope photometer in conjunction with a computer which controls a motor‐driven stage drive and records and categorizes the photomultiplier output. In a system being developed at The Pennsylvania State University, the object stage moves continuously under the microscope objective at a constant speed and in a predetermined raster. With a scan speed of 1600 μm/s and the computer recording the photo‐multiplier output every 1.25 ms, each recording represents a scan of 2 μm. An aperture below the photomultiplier is set to give an effective area of measurement of 2 μm square, so that a continuous series of readings is obtained on adjacent fields of this size. The difference in reflectance between coal and pyrite particles in polished coal pellets enables pyrite chord‐size distributions to be measured; consecutive recordings on pyrite grains are summed to give a measure of chord length. Size distributions determined by ARM compare well with values measured by visual microscopy. In the course of analysis of a single coal pellet, approximately 400,000 individual readings are recorded, the entire operation of scanning and printout of data taking approximately 18 min. Coal reflectograms obtained by ARM may be used for rank measurements and to indicate petrographic compositions.
The standardization of petrographic analysis of coal
Authors: Juckes L. M. , Pitt G. J.
Published: August 2011

Petrographic analysis is used to characterize coals in terms of the maceral group composition and the reflectance of the vitrinite. Interchangeability of results and applicability of prediction methods have been hampered up to now through differences between the methods used by different petrographers. Some aspects of a proposed standard method being developed in this country are described.
The rank and anisotropy of anthracites: the indicating surface of reflectivity in uniaxial and biaxial substances
Authors: Hevia V. , Virgos J. M.
Published: August 2011

Theoretical and empirical formulae are given which relate the principal reflectivities of uniaxial and biaxial substances to the mean values of random, maximum and minimum reflectivities measured on particulate samples. Instead of using values of R̄a and R̄a − R̄c determined along the principal directions, a method is described for determining respectively: (a) coal rank through the mean random vitrinite reflectivity, and (b) the bireflectance using the difference between the mean random maximum and minimum reflectivities measured on particulate samples, irrespective of uniaxial or biaxial character.
The accuracy of refractive and absorptive indices derived from reflectance measurements on low‐reflecting materials
Authors: Cook A. C. , Murchison D. G.
Published: August 2011

Errors in the refractive and absorptive indices derived from reflectance measurements on relatively low‐absorbing organic substances in two media are discussed, with particular reference to coal macerals and cokes. Variation of errors in refractive and absorptive indices at different levels of reflectance, and the effect of different combinations of sign and magnitude of reflectance error on the two parameters, are presented in both tabulated form and as diagrams. The errors in the refractive and absorptive indices vary in relation to reflectance level, the reflectance difference between standard and unknown, the difference between the refractive indices of the unknown and the immersion oil and the wavelength of reflectance measurement within the visible spectrum. Despite the variation, it is possible to obtain useful values for the parameters within the low‐absorbing range. If the absorption is negligible, it may not be possible to obtain realistic values for absorptive index, but even where this is the case, satisfactory values for refractive index can still be obtained. Suggestions are made on how to minimize reflectance errors and to increase the accuracy of dispersion curves for derived refractive and absorptive indices within the visible spectrum.
Microscopical investigation of the transformation (diagenesis) from peat to lignite
Authors: Ting F.T.C.
Published: August 2011

A silicified Palaeocene peat layer discovered in a lignite bed in western North Dakota, U.S.A., has proved to be excellent material for studying the transformation from peat to lignite. Detailed anatomical structures are well preserved in the peat and similar to the level of preservation of Carboniferous coal balls. A compaction of 4: 1 results when the peat is converted to lignite. Loss in volume is primarily caused by the loss of cellular cavities and intergranular spaces and consequent loss or redistribution of moisture. Huminite formed from secondary xylem and phloem sclereids differs in appearance under the microscope from huminite derived from periderms and young cortical tissues. Resinites are formed from fillings of resin canals, secretory cells and sclereids of secondary phloem and may also originate from resinous materials occurring in the palisade and spongy mesophyll of leaves. Cutinite can form from either cuticles or from compressed cortical tissues of young roots. Charred leaves and charred secondary xylem and phloem comprise an important portion of the fusinitic and semifusinitic fractions of the coal. Pyrite grains tend to occupy the cell cavities of early wood and ray cells. Differences between collinite and telinite developed during the peat stage, while progressive coalification tends to accentuate the lack of structure in the collinite.
Prediction methods in coal and coke microscopy
Authors: Mackowsky M.‐Th.
Published: August 2011

Prediction of coke strength based on measurements of vitrinite reflectance and data from group‐maceral analyses requires information about the coking power of the different coal macerals in relation to rank. A range of bases are considered by different individuals to be important in prediction studies, e.g. the optimum ratio of reactive to inert constituents for each ‘V‐step’ (vitrinite oil reflectance step × 10); the proportion of vitrinite and clarite and rank; the amount of inertinite with no consideration given to an optimum ratio of reactives to inerts; the rank with little regard given to varying maceral composition. Entirely satisfactory results can be obtained by all investigators for coke‐strength predictions from certain coals under given coking conditions. For other coals, some of the determined values lie well outside the permissible limits of error. Reasons for these deviations may lie in an erroneous, or at least in an inadequate evaluation, of the different microscopical parameters. With this introduction, different predictive methods are discussed and tested. Limits of applicability are demonstrated. Possible improvements are suggested and questions, which have so far not been answered entirely satisfactorily, are posed. Above all, the underlying importance of light microscopy to a major industrial field is stressed.
High resolution electron microscopy of carbon structure
Authors: Crawford D. , Marsh H.
Published: August 2011

An investigation is made of the inherent performance of a high‐resolution transmission electron microscope applied to the study of developing graphitic‐sheet structures in heat‐treated, coal‐tar pitch carbons. Image detail is shown to be highly dependant on instrumental defocus. It is not obvious which form will be assumed by artefacts in these images; consequently, anomalous features are illustrated by reference to a specific electron‐optical case and a corresponding light‐optical analogy. Despite the difficulties associated with locating an optimum level of focus, optical diffractometry confirms that adequate conditions of microscopy are attainable on a routine basis. In a quantitative analysis of morphology in coal‐tar pitch carbons, the technique reveals the cause of incipient, thermally‐induced, molecular distortions which evidently result in a regression in the improvement of order otherwise developing with progressive heat‐treatment.
Elateritic hydrocarbon from South Crofty Mine, Cornwall: a preliminary note
Authors: Harrison R. K. , Beer K. E.
Published: August 2011

Black, viscous, subelastic elateritic hydrocarbon associated with ‘peach’ (tour‐maline‐quartz‐chlorite‐rock), forms a seepage in a movement zone in granite near its contact with killas at South Crofty Mine, near Redruth, Cornwall. The hydrocarbon is paragenetically associated with granular pyrite, but infiltrates the ‘peach’. Readily soluble in aromatic and halogenated alkane solvents, the hydrocarbon is insoluble in paraffinic solvents suggesting a condensed structure. Infrared spectroscopy and N.M.R. spectroscopy indicate a mainly aliphatic content with some aromatic component. Both carbonyl (>C=0) and hydroxyl (−OH) functions are present. Strong methyl absorption bands show a considerable amount of branching or methyl side groups. Gel permeation chromatography and visual spectrometry show no detectable porphyrins. The origin of the hydrocarbon is uncertain; it may represent a distillate of carbonaceous killas (Mylor Series) entrapped in granite at depth, but an abiogenic origin cannot be ruled out.
Determination of the mass thickness of biological sections from electron micrographs
Authors: Misell D. L. , Burdett I. D. J.
Published: August 2011

Theoretical results are given for the evaluation of the mass thickness of unstained and stained biological sections based on the scattering contrast model for in‐focus electron microscope images. Densitometer measurements on photographic images enable the scattered electron intensity (I) and the incident electron intensity (I0) to be determined, and it is shown that the ratio I/I0 can be related to the mass thickness ρt of a particular region of the specimen by I/I0 = exp (−Spρt). The parameter Sp is given for a range of incident electron energies (E0 = 20–100 keV) and objective aperture sizes (semi‐angle α subtended at the specimen 5–15 mrad). Experimental results on thin stained sections of Bacillus subtilis for E0 = 40–100 keV and α = 3.8–17.5 mrad demonstrate the internal consistency of the theoretical curves for the calculation of mass thickness of both the embedding medium and the biological material. The sensitivity of this method for the determination of mass thickness depends on E0 and α; variations in mass thickness of ±2 mg m−2 can be determined for E0 = 40 keV and α = 5 mrad, becoming less sensitive as E0 and α are increased, so that for E = 80 keV and α = 10 mrad the sensitivity is reduced to ±5 mg m−2. This means that density variations of ±40 and 100 kg m−3, respectively, can be determined in sectioned biological material. Because of the requirement of a linear relation between log (I/I0) and ρt, the theoretical calculations are limited to a maximum mass thickness of 200 mg m−2 or a section thickness of about 200 nm for material of density 1000 kg m−3.

These calculations are relevant to applications in microbiology, stereology and autoradiography.
Cryomicrotomy applied to the preparation of frozen hydrated muscle tissue for transmission electron microscopy
Authors: Varriano‐Marston E. , Gordon J. , Davis E. A. , Hutchinson T. E.
Published: August 2011

A method is described by which frozen hydrated bovine muscle is prepared for the transmission electron microscope. The importance of maintaining specimen temperatures at −110°C (163°K), or below, during the preparation of frozen thin sections and during specimen transfer and microscopical observations is emphasized and documented.

Electron micrographs of a section of muscle were followed through three levels of dehydration. Electron micrographs of frozen hydrated muscle were compared with micrographs of fixed and embedded muscle. In general, the micromorphology of muscle prepared by the two methods was found to be quite similar.
Autoradiographic studies on the cellular uptake of Technetium‐99m and Chromium‐51
Authors: Barth Rolf F. , Jr John Clancy , Pugh Jill M.
Published: August 2011

The present study was designed to investigate the cellular uptake of technetium‐99m (99mTc) and chromium‐51 (51Cr) by autoradiographic methods and to compare these data to those obtained by gamma scintillation counting. HeLa cells, a fibrosarcoma of murine origin (Sarcoma I) and human peripheral blood lymphocytes were labelled with either 99mTc‐sodium pertechnetate or 51Cr‐sodium chromate. Cells were non‐uniformly labelled with both radionuclides. Nevertheless, uptake as determined by both autoradiography and gamma scintillation counting revealed that each cell type had a characteristic labelling pattern which appeared to have a relationship to cell surface area. This was determined to be linear for 99mTc when counts per minute were plotted as a function of area (μm2). Grains were randomly scattered over the entire cell surface and occasionally tracks could be seen as a trail radiating out from the periphery. HeLa cells had the largest surface area, the highest grain counts and the greatest uptake of both 99mTc and 51Cr as determined by gamma counting. Lymphocytes had the smallest surface area, the lowest grain counts and the least uptake. Both radionuclides are non‐specific cell labels which are taken up independently of mitosis and protein synthesis. The favourable chemical properties of 99mTc‐sodium pertechnetate have led to its use as a label for a wide variety of inorganic and organic compounds which are employed as radiopharmaceuticals. Autoradiographic methods could be useful in determining the cellular localization of these compounds, as well as any protein molecule with which pertechnetate forms chelates. Although the 6 h half life of 99mTc limits its use to short term experiments, it does permit brief exposure times prior to development.
A combined top and side entry specimen stage for a Hitachi H500 transmission electron microscope
Authors: Chapman S. K.
Published: August 2011

The Hitachi H500 transmission electron microscope has been modified in order that both the top and side entry specimen stages may be fitted simultaneously. This made possible top entry multi‐specimen operation up to a maximum magnification of × 100,000 and a resolution of 1.8 nm, combined with the normal side entry stage facilities of ±60° tilt, 0.45 nm resolution and × 400,000 magnification.
A simple technique for the phase contrast and acridine orange fluorescence microscopy of the same living cells
Authors: Söderström Karl‐Ove
Published: August 2011

A simple technique for the detection and localization of changes in the DNA structure of living cells is described.
Use of an automated image analyser to quantitate cellular hyperplasia in urinary bladder epithelium
Authors: Stinson S. F. , Sporn M. B.
Published: August 2011

The feasibility of using an automated image analyser to evaluate quantitatively hyperplasia caused by N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine in bladder epithelium was studied. The number of cells per unit length of epithelium was counted manually, and compared with automated measurements of: (1) the number of nuclei, (2) the number of positive tangents to the lower edge of nuclei, (3) the nuclear cross‐sectional area, and (4) the epithelial cross‐sectional area per unit length respectively. Regression of each of the automated measurements on the manual counts all revealed close linear relationships with correlation coefficients in excess of 0.9. Coefficients of variation for repetitive automated measurements were less than or equal to 0.06 in each of the four modes. The automated system resulted in a great saving in time over manual counting. It is concluded that the automated image analyser provides an accurate, precise, and efficient tool for estimatimg epithelial cell numbers in normal and hyperplastic bladder epithelia.
Preparation of Biological Material For X‐Ray Microanalysis of Diffusible Elements
Authors: Sevéus Lahja
Published: August 2011

Any method used for the analysis of diffusible elements within a cell should involve conditions which eliminate the redistribution of those elements. Redistribution of diffusible substances is caused by (a) ice damage during freezing, (b) thawing during sectioning, and (c) thawing during handling of sections. To eliminate this sections were cut (a) in the 5–8 μm ice crystal free surface layer obtained by freezing in melting nitrogen, (b) at a specimen temperature of 133 K and a knife temperature of 173 K to 153 K, and the temperature of the chamber was kept low by evaporating nitrogen from an extra reservoir inside the chamber, by regulating the output of a foil heater. A grid carrier was designed to simplify the section collection and the sections were affixed on to the grid in a press assembly. The sections were hard and translucent, and showed faint interference colours.
Preparation of Biological Material For X‐Ray Microanalysis of Diffusible Elements
Authors: Barnard Tudor , Sevéus Lahja
Published: August 2011

The appearance of ultrathin, dry‐cut cryosections of brown adipose tissue and liver was found to be strongly dependent upon adequate freeze‐drying. If freeze‐drying was inadequate, diffusion of substances could be demonstrated and freezing damage was not apparent. Diffusion was manifested as an electron dense film over structural features such as triglyceride droplet profiles and the edges of the section; when this film was thick, X‐ray signals for P, S, Cl, K and Ca could be detected from it, in different proportions to those found in the section. The frequency and intensity of diffusion were lowered by decreasing the temperature in the cryochamber from about 200 K ± 5 K to 163 K ±5 K by forced evaporation of liquid N2 using an extra heater. The lowest incidence of diffusion was obtained in conjunction with this device, by leaving the sections in the cryochamber for at least 2 h during drying, either over a drying agent or under moderate vacuum. Such sections showed a narrow zone (A) at the edge that lacked obvious ice‐crystal damage, a thicker zone (B) of moderate ice‐crystal damage and the bulk of the interior (zone C) severely damaged by freezing. Qualitatively different, reproducible X‐ray spectra could be obtained from ultrastructural features even in areas showing some signs of diffusion.
Holographic Microscopy of Glycerination of Amoeba Proteus
Authors: Opas Michal
Published: August 2011

The process of glycerination of Amoeba proteus was observed under the holographic microscope with coherent noise elimination. It was found that during glycerination redistribution of cell material occurs, and is accompanied by reversible deformation of the cell cortex. The role of the cortex in shape maintenance in glycerinated models was demonstrated.
Direct Deposition Autoradiography: Can it Really Be Detecting Radioactivity?
Authors: Williams M. A.
Published: August 2011

The literature concerning the ‘direct deposition method’ of electron microscope autoradiography has been examined and certain calculations made. Studies have been made on the interaction of gold chloride with protein films, tissue sections and ultrathin sections. It is concluded that in its present form the direct deposition method does not offer an autoradiographic approach of practical value.
A Method For The Study of Internal Spaces in Hard Tissue Matrices By SEM, With Special Reference to Dentine
Authors: Martin D. M. , Hallsworth A. S. , Buckley T.
Published: August 2011

Infiltration of a porous hard tissue with methyl methacrylate, followed by dissolution of the tissue provides a three‐dimensional methacrylate cast of spaces within the tissue. Examination of such a cast by scanning electron microscopy provides information on the nature and extent of the pore system to a degree that cannot readily be visualized by the direct examination of fractured surfaces. The findings described concern human dentine but the technique may have a wider application in studies on the internal structure of other hard tissues.
Low Cost Internegatives From Electron Micrographs of Metal Shadowed Objects
Authors: Boye Hans , Rønne Mogens
Published: August 2011

Some observers prefer a contrast reversal of electron micrographs of metal shadowed objects, so that the metal‐free shadows appear dark on the print. There are several ways of obtaining contrast reversal, and the reasons for using 35 mm internegatives are given and a setup for re‐photography of electron microscopy films is described together with a modified enlarger. Both are equipped with near‐point illumination, which can be diffused in a simple manner with minimal loss of intensity. Other features are a particularly rigid camera stand combined with a numbering device, and a glass‐ and scratch‐free negative holder for the enlarger. The choice of lens for the re‐photography is discussed.
Optical Anisotropy of Poly (P‐Phenylene Terephthalamide) Fibres
Authors: Hamza A. A. , Sikorski J.
Published: August 2011

The principal polarizabilities of poly(p‐phenylene terephthalamide)—PPT—fibres were calculated using the molecular model of Northolt, together with the values of bond polarizabilities given by Denbigh and also by Bunn & Daubeny. Following generally accepted practice, the Lorentz‐Lorenz expression was utilized to derive the principal refractive indices (and the birefringence) of the PPT fibres.

The experimental values of the refractive indices, for four different PPT samples, were obtained with the aid of the Pluta polarizing interference microscope; some comments on the relevant procedures being included.

Finally, discussion on the discrepancy between the calculated and observed values of refractive indices is attempted.
Stereology of the Dermal System of Fruit
Authors: Considine J. A.
Published: August 2011

Methods for determining cell numbers and cell size in the skin or dermal system of fruit have been examined, using as an example the grape, Vitis vinifera. Though the cells are commonly six‐sided, projected cell area is more accurately determined if the cells are assumed to be circular. Sources of error and assumptions are considered.
A Method For the Removal of Epoxy Resins From Tissue in Preparation For Scanning Electron Microscopy
Authors: Steffens W. L.
Published: August 2011

A method for unmounting entire resin‐embedded samples for SEM observation is described. This technique is particularly useful when correlation of TEM and SEM images is desired for material that is no longer available for conventional SEM preparative procedures. Samples embedded in a variety of epoxy‐type resins were trimmed of excess resin and placed in a concentrated solution of sodium methoxide. After complete dissolution of the resin, the tissue was washed in a graded series of sodium methoxide in methanol‐benzene, transferred to acetone, critical point dried, mounted on stubs, and coated with gold‐palladium. Upon viewing in the SEM, the tissue samples showed remarkable preservation of detail at relatively high magnifications.
The Sampling, By Quadrats, of Planar Aggregates
Authors: Miles R. E.
Published: August 2011

The estimation, for planar particle aggregates, of their number or density per unit area and of their distributions (e.g. of area or perimeter), by means of randomly placed quadrats, is complicated by the question of deciding whether or not to include particles truncated by the quadrat boundaries. Previous unsatisfactory solutions to this problem have led to unnecessary bias in the estimates. A general ‘associated point’ solution is presented, and its theory is developed in some detail.
X‐ray microanalysis of epon sections after oxygen plasma microincineration
Authors: Barnard Tudor , Thomas Richard S.
Published: August 2011

Dark gold sections of osmium tetroxide‐fixed, Epon‐embedded brown adipose tissue before and after low‐temperature oxygen plasma microincineration were examined using a high‐resolution scanning transmission electron microscope and an energy‐dispersive X‐ray spectrometer. Microincineration produced ash patterns which were free of organic matrix, chlorine (from the Epon) and probably osmium (from the fixative). X‐ray sensitivity was improved by a factor of 2–4 owing to decreased background, and sulphur, calcium and probably phosphorus were detected in the ash. Fidelity of the ash patterns permitted microanalytical spatial resolution of 0.1 μm or better. Oxygen plasma microincineration is thus shown to offer advantages for high resolution X‐ray microanalysis of conventionally sectioned biological material. Its future application to shock‐frozen, frozen‐dried, unstained sections is indicated.
Ring counting in field‐ion micrographs
Authors: Webber R. D. , Walls J. M. , Smith R.
Published: August 2011

Ring counting is an important technique in the quantitative analysis of field‐ion micrographs since the information derived is independent of magnification and projection both of which vary in a complex way over the image. However, although the technique is important, its direct use has previously been restricted to the region within which the particular ring system is discernible. In this paper, techniques and procedures are described which usefully extend the range over which the ring count can be performed. The methods are based on determining the numerical interrelationship of the various ring structures. This not only allows ring counting to be effected over large angular distances, but also enables counts to be performed in any direction so that the number of rings of a high‐index plane can be determined through more prominent low‐index regions.
A perspicuous technique for directly visualizing radiation‐damage artefacts in biological electron microscopy
Authors: King Murray Vernon , Parsons Donald F.
Published: August 2011

Levels of impairment of electron‐microscopic images of biological specimens stemming from radiation damage are assessed in a rapid visual procedure that involves taking a pair of low‐fluence micrographs of a specimen area before and after a fraction of the picture area has been more seriously damaged by applying a measured electron fluence. The pair of micrographs is treated as a mock‐stereo pair and is given contrasting colours. Lateral displacements of specimen details appear as false relief and changes in electron lucency as false colour.
Area changes in slices of rat brain during preparation for histology or electron microscopy
Authors: Hillman H. , Deutsch K.
Published: August 2011

Cerebral slices cut from rat brain, either 2–3 mm or 0·27 mm thick, were used to study the effect of embedding and freezing. Paraffin wax sections 6 μm thick were mounted and stained with haematoxylin and eosin or Marsland et al.'s (1954) silver stain, and their areas were examined at each step.

Embedding in paraffin wax of slices 2–3 mm thick, or in Epon of slices 0·27 mm thick, caused a diminution of their areas by 20–30%. Staining of paraffin wax sections did not alter their areas. Glycerol alone at 15% concentration had no effect on the areas, but at 30% concentration they were diminished by approximately 20%. Diminution of the areas of glycerol treated slices 0·27 mm thick also occurred when they were transferred to liquid N2 or to isopentane, but the areas increased after glycerol was replaced by Freon 12.

It was concluded that embedding or freezing cerebral slices caused changes in their areas, but that staining of sections after they had been embedded, sectioned and mounted did not.
Ponceau 2R staining of proteins and periodic acid bleaching of osmicated subcellular structures on semi‐thin sections of tissues processed for electron microscopy: a simplified procedure
Authors: Gori Paolo
Published: August 2011

An aqueous solution (pH 1·5) of 0·5% Ponceau 2R (C.I. 16150) and 2% periodic acid was used at 318 K to stain proteins (brilliant red) and simultaneously to bleach the osmicated non‐proteinaceous cell structures on 1–2 μm thick sections of tissues fixed in glutaraldehyde‐osmium tetroxide and embedded in epoxy resins. This H5IO6 bleaching‐Ponceau 2R staining procedure only stains the proteins so that black‐and‐white films can be used.
An iterative approach to the analysis of EM autoradiographs
Authors: Downs Angela , Williams M. A.
Published: August 2011

An iterative scheme is described for determining the most appropriate ‘model’ for dividing micrographs of EM autoradiographs into identified items of known relative specific activities in such a way that the dispersion of silver grains over the autoradiographs is fully and satisfactorily explained.

A trial model is first set up for which, using a square matrix of estimates of notional cross‐fire between the chosen items, a ‘solution’ is obtained. The quality of the model is then tested using extended cross‐fire data contained in a rectangular matrix. If the model is shown to be unsatisfactory, an improved model (of tissue subdivision) is postulated and the whole process repeated until the model is deemed to be satisfactory. The computations are such that the data can be processed rapidly on the laboratory bench using a small machine. The logical estimation of errors is facilitated by this approach.
The theory of scanning microscopes with Gaussian pupil functions
Authors: Sheppard C. J. R. , Wilson T.
Published: August 2011

The theory of imaging in scanning microscopes with lenses, source and detector all having Gaussian pupil function is developed. This assumption is useful as the expressions may be evaluated analytically. It is shown that Type 2 microscopes exhibit superior performance to those of Type 1. Effects of defocus are considered. It is found that defocus can be used in a Type 2 microscope to observe phase information without the limitation in resolution associated with stopping down the collector of a conventional microscope. It is also found that a Type 2 microscope discriminates against light scattered by parts of the object outside of the focal plane, allowing observation of detail within a thick object.
Refractive index and absorption coefficient as measures of structural organization in carbonized bitumen
Authors: Khavari‐Khorasani G. , Blayden H. E. , Murchison D. G.
Published: August 2011

Microscope photometry has been used to determine reflectances, measured in air and in stable immersion oil, of the carbonized products of the asphaltite bitumen, gilsonite. Comparison of values for parameters (n' and k'), analogous to refractive index and absorption coefficient which are derived from the reflectances, with several X‐ray parameters of the carbonized samples suggests that microscope photometry offers a sensitive alternative method for following changes that take place in the molecular‐structural organization of organic materials during coalification and carbonization processes. Although direct measurement of lattice dimensions is not possible, the optical method is more rapid than X‐ray procedures, is less expensive in terms of capital equipment, while the reflectance measurements can be made on extremely small, discrete and categorically identified field areas, not on the finely ground bulk samples required for X‐ray procedures. Wider application of the microscope photometric method to other carbonized materials and the possibility of absolute correlations between optical and X‐ray data must await more detailed study. The preliminary data are sufficiently encouraging to warrant further extended investigations.