bluuebunny/crossref_metadata_2025 huggingface dataset:
Cathodoluminescence of biological molecules, macromolecules and cells
Authors: Barnett W. A. , Wise M. L. H. , Jones E. C.
Published: August 2011

Cathodoluminescence observations on biological compounds are compared with previously established data from ultra violet and visible light excitation studies. The comparison demonstrates that the same molecules are responsible for the luminescence properties of macromolecules independent of the type of exciting radiation. Natural cathodoluminescence was also observed from cells. Moreover, advantages gained by the absorption of strongly cathodoluminiscent dyes into cells are demonstrated.
Distilled glutaraldehyde: its use in an improved fixation regime for cell suspensions
Authors: Gillett R. , Jones G. E. , Partridge T.
Published: August 2011

A method is described for the sequential fixation of cell suspensions, suitable for use at room or culture temperatures. Though an adequate method for fixing cell suspensions does exist in the literature (Hirsch & Fedorko, 1968), it involves the use of a mixed glutaraldehyde–osmium tetroxide fixative. Since these two components inter‐react, this method has many drawbacks.

Previously described weaknesses of a sequential fixation regime (Hirsch & Fedorko, 1968; Jones, Yeh & Hirsch, 1972) with glutaraldehyde and osmium tetroxide have been overcome by the use of vacuum distilled glutaraldehyde as the primary fixative. The results, using a mixed glutaraldehyde–osmium tetroxide fixative and using the two components sequentially on a variety of cell types, are compared. The advantages of a sequential fixation made possible by the use of vacuum distilled glutaraldehyde rather than commercial glutaraldehyde are discussed.
The observation of crystalline materials in the scanning electron microscope (SEM)
Authors: Joy David C.
Published: August 2011

Contrast effects from electron channelling in crystalline solids can be observed in the scanning electron microscope by the correct choice of operating conditions. The crystallographic symmetry and orientation of specimen areas as small as 1 μm in diameter can be determined, and detailed quantitative information obtained about the density, nature and position of defects contained within the crystal structure. The backscattered, absorbed current and transmitted electron signals may all be used. With anticipated improvements in instrumentation the technique will be capable of providing all the information currently supplied by the more complex procedures of transmission electron microscopy.
Preparing sections of skeletal muscle for transmission electron analytical microscopy (TEAM) of diffusible elements
Authors: Sjöström Michael , Thornell Lars‐Eric
Published: August 2011

Comparative morphological examination and elemental analysis were carried out in structural compartments of sections of skeletal muscles. These had been prepared either by conventional plastic embedding technique or by various methods of cryo‐ultramicrotomy. The analyses were performed in a Philips EM 301 with an Edax energy‐dispersive X‐ray spectrometer.

Spectra obtained from sections of plastic‐embedded muscle depended on the reagents used for fixation and staining and were absent if these were omitted. Brief fixation with glutaraldehyde resulted in gross ionic changes, and sectioning of frozen material with trough liquid led to extraction of elements. Sections cut from unfixed and frozen muscle without trough liquid showed numerous peaks. (Mg, P, S, Cl, K, Ca). In the superficial parts of the fibres of freeze‐dried sections reproducible spectral differences were found between different structures. Thus, rapid freezing of unfixed tissue, dry cutting in the frozen state, and freeze‐drying should be the procedure of choice if data on diffusible ions are desired.
Tungsten coating—a method of improving glass microtome knives for cutting ultrathin sections
Authors: Roberts I. M.
Published: August 2011

Glass knives for ultramicrotomy were considerably improved by coating the cutting edge with a film of evaporated tungsten metal. Knives treated by this method gave up to ten times as many acceptable sections as uncoated glass knives. They could also cut thinner sections and harder tissues than ordinary glass knives, and eliminated some of the cutting artefacts produced by them. No explanation for this improvement was found although several possibilities were examined.
Detection of ultra‐soft X‐rays with a variable geometry proportional counter fitted to a transmission electron microscope (TEM)
Authors: Armigliato A. , Bentini G. G. , Ruffini G.
Published: August 2011

The ability of an end‐window variable geometry flow proportional counter to detect the ultrasoft BeK and SiL X‐ray lines induced by the electron beam of a transmission electron microscope (TEM) has been demonstrated. This counter has been used in the current work in the energy dispersive mode, and has been equipped with a Formvar window about 400 nm thick, aluminized on both sides with a layer of 65 nm total thickness. X‐ray peaks generated by specimens of other light elements such as boron, carbon, oxygen, fluorine and aluminium, and detected with this technique, are also reported. In the case of the BK line, the counting rate obtained with this detector has been evaluated and compared with that of a crystal spectrometer equipped with a conventional side‐window flow proportional counter. Finally, a linear relationship between X‐ray energy and detected pulse height was found for the lines examined, i.e. from the SiL up to the SiK X‐rays.
Lipid losses during processing of cardiac muscle for electron microscopy
Authors: Ward Barbara J. , Gloster Josephine A.
Published: August 2011

The lipid retaining properties of several methods of processing tissue for electron microscopy (EM) have been assessed quantitatively.

Guinea‐pig hearts were perfused in vitro at 37°C with 3H‐oleic acid bound to albumin. The hearts were fixed by perfusion with 4% glutaraldehyde in 0.1 M cacodylate buffer. Pieces of left ventricle and interventricular septum were removed., weighed and processed for EM. The fluids used at each stage of processing were monitored for loss of radioactive lipid by scintillation counting. Lipids were extracted from the processed tissue immediately before the embedding stage using a mixture of chloroform: methanol (2:1 v/v). Counts from processed tissue were compared with counts from tissue extracted directly after perfusion fixation in order to monitor subsequent losses during processing.

A modified version of Epon processing, omitting 100% ethanol, acetone or propylene oxide, gave a lipid retention of only 20.6%. The addition of para‐phenylenediamine to the procedure did not improve the retention although this has been shown to be a useful stain for intracellular lipid. Water soluble Durcupan which does not involve ethanol or acetone dehydration has an average retention of 63% with 100% recovery while the lesser known polymer GACH, a mixture of glutaraldehyde and carbohydrazide used both for dehydration and embedding showed a lipid retention of 82% of the counts recovered although recovery was only 69%.

An attempt was made to determine which classes of lipids were present in the tissue after perfusion fixation using thin layer chromatography. It was found that the presence of any of the processing fluids affected the polarity of the lipids and their rates of migration on thin layer plates.
Contrast enhancement of negatively stained macromolecules and biomembranes by single sideband phase contrast interference
Authors: Harris J. R. , Kerr J.
Published: August 2011

A straightforward procedure is described for the production of contrast enhancement of negatively stained macromolecules and biological membranes by single sideband phase contrast interference (electron optical shadowing). The instrumental adjustment required to produce this type of phase contrast illumination is readily achieved by beam deflection from the strioscopic (dark field) mode. Part of the hollow cone of electrons from the annular condenser aperture that are unscattered by the specimen are permitted to pass through the objective aperture and interfere with the scattered beam. The electron optical shadowing effect is produced because only one side of the unscattered beam is used. Careful adjustment of the beam tilt control, with the ability to tilt in any azimuth, allows optimal illumination conditions to be achieved. The results presented show the increased image contrast obtained using as specimens the purified cylindrical macromolecule from human erythrocyte membranes, purified nuclear envelopes and collagen fibres.
Section diameter method for non‐spherical objects
Authors: Chang K. S. , Dullien F. A. L.
Published: August 2011

A theory of section diameter method has been developed for simply connected convex objects randomly distributed and oriented in three‐dimensional space. All objects have been assumed to be of a similar geometrical shape and they differ only in size. No geometrical symmetry is assumed in the most general case considered, however, considerable simplifications are obtained if the objects have certain symmetry properties. Solution of the integral equations giving the size distribution of two‐dimensional features in the sections presupposes the knowledge of a function which is determined entirely by the geometry of the objects. This function may be found, in principle, by parallel sectioning many identical bodies of random orientations in space.
The influence of the embedding medium when staining for electron microscopy: the penetration of stains into plastic sections
Authors: Horobin Richard W. , Tomlinson Annette
Published: August 2011

Using a sectioned‐section procedure it was found that only a few structures, e.g. the connective tissue elements, were deeply penetrated by phosphotungstic acid (PTA) and uranyl acetate (UA). Reynold's lead citrate appeared to penetrate more generally. Using stains selective for the embedding medium, and also observing the surfaces of shadowed sections, it was concluded that structures readily penetrated by polar stains were only poorly infiltrated by non‐polar embedding media. It is argued that this differential infiltration leading to differential penetration by polar stains largely controls the pattern of staining of ultrathin sections by PTA and UA.
X‐ray analysis without the need for standards
Authors: Nasir M. J.
Published: August 2011

A technique has been developed for X‐ray analysis using energy dispersive solid state detectors commonly used with electron microprobes or scanning electron microscopes (SEM). This technique does not require the use of analysed standard alloys, instead the elemental peaks are directly compared to each other and simple corrections applied to convert these to weight percentages. A new empirical table for X‐ray fluorescence yields for the L X‐ray spectra is also given in this article.
Scanning electron microscopy of renal vascular casts
Authors: Bielke S. R. , Nagle R. B. , Trump B. F. , Bulger R. E.
Published: August 2011

The use of Cementex to form casts of the renal vascular system is described. These casts are useful in delineating the vascular microarchitecture of the kidney including that of the glomerular capillaries, but do not appear to reproducibly reflect physiological vascular tone. The postglomerular vascular system is often incompletely filled using this technique. The amount of filling can be altered by the techniques used in cast preparation. The effect of perfusion pressure, concentration of Cementex, fixation, and type of colour additive are compared with respect to the cast structure.
A new metachromatic stain technique for paraffin‐embedded neural tissue using thionin
Authors: Eichler Victor B. , Taylor Patricia W.
Published: August 2011

A metachromatic staining procedure which differentiates Nissl substance in neuron cell bodies, neuropil area, and axonic fibres of passage with distinct colours with no counterstaining has been developed for paraffin‐embedded tissue utilizing thionin stain. Frozen sections of neural tissue fixed in formalin may also be satisfactorily stained by this procedure, and both paraffin‐embedded and frozen sections have been found to retain the distinct colours for a year with little fading. Since metachromatic staining of paraffin‐embedded neural tissue has not been successfully achieved before, the following procedure will be especially valuable for studying small vertebrate brains, or other central nervous tissue which must be processed by the paraffin method.
An inexpensive spray‐freezing unit for preparing specimens for freeze‐etching
Authors: Lang R. D. A. , Crosby P. , Robards A. W.
Published: August 2011

The construction and method of use of an inexpensive spray‐freezing device are described.
Improved resolution in cathodoluminescent microscopy of biological material
Authors: Haggis G. H. , Bond E. F. , Fulcher R. G.
Published: August 2011

This paper continues work reported in an earlier paper† on modification of a Cambridge Stereoscan Mk IIA to improve the quality of cathodoluminescent micrographs of biological material. In the work presently described the microscope gun has been offset laterally by 2 mm, to prevent light from the filament passing down through the column to the specimen chamber. The electron beam is brought onto the column axis by deflection coils. This modification effectively eliminates background light in the chamber, and a full parabolic mirror is fitted to maximize light collection. Results for yeasts and wheat seed sections are described.
The application of optical microscopy to studies of dust filtration: II. Specimen preparation techniques for analysing the distribution of dusts in filter fabrics
Authors: Rothwell E. , Bickham D. R.
Published: August 2011

Microscopic examination of dust cakes supported on filter fabrics is limited by difficulties in preparing useful sections by impregnating in cold setting resins; cake movement and sedimentation of dust particles result in sections which are not representative of the initial materials. An evaporator‐condenser assembly has been designed to enable sections to be prepared in which the fibres and dust particles are retained in their initial locations. Specimens of dust cakes supported on filter fabrics are placed in a thermal gradient in this assembly and a volatile monomer is condensed from the vapour phase on to dust particles and fibres of the fabrics. In situ polymerization effectively cements individual dust particles to each other and to the supporting fabric. After this treatment, specimens are vacuum impregnated in cold setting resin and sectioned by established techniques.
Simple magnetic holders for critical point drying of microspecimen suspensions
Authors: Cohen William D.
Published: August 2011

Microspecimens can be critical point dried from suspension in holders consisting of three magnets and two filters. These holders are inexpensive, require no construction, and are easily assembled and disassembled.
Molecular image of copper phthalocyanine
Authors: Murata Y. , Fryer J. R. , Baird T.
Published: August 2011

The structure of copper phthalocyanine has been studied by high resolution transmission electron microscopy (TEM). To reduce radiation damage, the technique of minimal beam exposure has been used together with X‐ray film for recording the image. This resulted in very noisy micrographs but optical averaging clearly revealed the molecular shape of the copper phthalocyanine, and also showed that a slight discrepancy exists between the averaged image, and the model structure which had been assumed to be isomorphous with platinum phthalocyanine.
Critical evaluation of histogram densitometry of Feulgen‐stained normal human lymphocyte nuclei
Authors: Boss J. M. N. , Mays R. G. W.
Published: August 2011

By means of a Vickers M85 microdensitometer, combined with a histogram unit, the distribution of the coloured product of the Feulgen reaction in single nuclei of human lymphocytes was examined by taking the fraction of each nucleus occupied by DNA at different concentrations. Tchacarof's index of homogeneity, derived from these fractions, was found to rise with the mean nuclear DNA concentration, and the calculated linear relation between the index and the concentration was found, in a preliminary experiment, to be independent of the degree of flattening of the nuclei. Further analysis, of the contribution of more and less densely packed areas to differences in mean intranuclear DNA concentration, showed that these differences depended on differences in both denser and less dense areas. The effects of hydrolysis on homogeneity were controlled by the use of Tchacarof's index. The procedure for using the Vickers M86 microdensitometer, with histogram unit, for analyses such as those reported here was developed to become established critically.
Some methods for more efficient processing of SEM specimens
Authors: Sweney L. R. , Laible N. J. , Shapiro B. L.
Published: August 2011

Three techniques are described for increasing the quantity of specimens which may be processed for SEM while maintaining the quality of the final product. They are: a simple stainless steel holder for safe manipulation of coverslips during incubation and fixation; a coverslip carrier which permits four or more coverslips to be dehydrated and critically point dried at the same time; and a simple pattern for making baskets to hold relatively large but delicate specimens during processing for SEM.

Processing soft tissues for scanning electron microscopy (SEM) presents many problems depending on the size, shape and type of specimen. Cohen (1974) presented an overall view of this problem. Methods for handling cells in suspension have been suggested by Baker & Princen (1975), Newell & Roath (1975) and Rostgaard & Christensen (1975). Tissue culture specimens are also difficult to process for SEM. These may be long‐term cultures such as fibroblasts or kidney cells, or short‐term preparations such as the collecting of white blood cells or bacteria on coverslips. Nemanic (1972) described a method using Tygon tubing with slits to hold the coverslips after incubation. Perecko et al. (1973) gave details of a holder for processing six small (6 mm) coverslips after incubation. Rice et al. (1976) designed several complex multipurpose carriers for 9 × 22 mm coverslips and for cells on silver or cellulose membrane filters. Broers et al. (1975) used a mesh receptacle for bacteria attached to silicon dioxide slivers. Many investigators simply process pieces of plastic cut from the tissue culture dish itself. Using the plastic directly avoids the problems of incubating cells on glass coverslips. Coverslips are fragile and difficult to remove from the culture dish or flask. Each coverslip must be processed individually, increasing the risk of breakage and limiting the number which can be done at one time. For processing larger tissues there are commercial baskets available. Cohen (1974) illustrated mesh baskets for odd sizes and shapes of soft tissues.

We have devised a simple holder which facilitates the use of 12 mm 0 grade coverslips in several types of culture dishes. We have developed a multipurpose carrier which increases the number of coverslips which can be processed for SEM and reduces the number of manipulations needed for each individual coverslip. This carrier can be used for histological and histochemical studies as well as SEM processing. We have modified Cohen's technique (1974) for mesh baskets for larger soft tissue specimens.
The application of optical microscopy to studies of dust filtration
Authors: Rothwell E. , Bickham D. R.
Published: August 2011

Needle‐felted fabrics which are used in modern continuously operated dust filter units comprise a loose assembly of synthetic fibres interlocked in the manufacturing process by the action of needle barbs, which pull tufts of fibres through the depth of the fabric; the high porosity of the fabrics and their lack of rigidity prevents their sectioning by conventional microtome techniques. Specimens for incident and transmitted light microscopy are prepared after vacuum impregnating with an embedding resin using modified metallographic preparation techniques. The advantages and limitations of these techniques in revealing details of the structure of needle‐felted fabrics are discussed and illustrated with typical photomicrographs.
The anti‐static pistol as an aid to ultrathin sectioning
Authors: Fraser T. W.
Published: August 2011

The use of a piezo‐electric anti‐static device to eliminate a block charging defect during ultramicrotomy is described.
A method of embedding tissue culture preparations in situ for transmission electron microscopy
Authors: Codling B. W. , Mitchell J. C.
Published: August 2011

A method for embedding tissue culture preparations, grown on Millipore filters, in Araldite is described. Accurate light microscopic localization of areas suitable for ultrathin sectioning is possible. The plane of sectioning relative to the Millipore filter can be varied to suit individual experimental needs.
Suppression of background on stripping‐film autoradiographs: A review
Authors: Stevens G. W. W.
Published: August 2011

Suppression of background in autoradiographic materials can be influenced by control in manufacture, transport and storage, and user handling. Fogging by radiation during transport and storage is a definite risk, but for some materials can be combated by latent‐image fading during storage before transfer to the specimen. Leaching out of emulsion constituents during transfer enhances the risk of background growth, and can be counteracted by transferring from a suitable solution. Background growth is positively promoted by chemographic agents derived from wood and other materials. The transfer solution helps to block chemographic action, as does storage of the transferred emulsion at a low temperature or in a non‐porous and photographically inert container.
Analysis of electron microscope autoradiographs using the hypothetical grain analysis method
Authors: Parry D. M. , Blackett N. M.
Published: August 2011

Cross‐fire between adjacent labelled sources can often introduce problems in the analysis of electron microscope autoradiographs. A general method is described which makes allowance for this cross‐fire and is then applied to the analysis of autoradiographs of 3H‐proline in the accessory olfactory bulb of the mouse.

It is shown how the method allows estimates to be made of the radioactivity of the various cell types and the accuracy of these estimates and the errors in obtaining them are discussed.
High‐resolution autoradiography of 3H‐ouabain binding in salt transporting epithelia
Authors: Stirling C. E.
Published: August 2011

The distribution of salt transporting sites in epithelia is a problem of special interest to electrolyte metabolism. Autoradiography of freeze‐dried plastic embedded tissue was used to localize 3H‐ouabain in rabbit small intestine and renal medulla and gills of killifish. Resolution approaching theoretical limits for tritium at the light microscope level was achieved. The results of several tests are presented which demonstrate the resolution of the method and the specificity of the observed ouabain binding patterns. A large uptake of ouabain was found along the basolateral membranes of the enterocyte, thick ascending tubules and chloride cells (fish gill).
Autoradiographic studies of DNA synthesis in lymphoid tissues
Authors: Harris G.
Published: August 2011

Using long exposures of stripping film autoradiographs before processing, mixtures of weakly and strongly labelled nuclei were seen in different areas of the mouse spleen. Previous results (Harris et al., 1973) led to the conclusion that many cells, not in division cycle, were labelling with (3H) thymidine and that this process was important for the development of specific antibody‐producing cells following stimulation with an antigen such as sheep red cells (SRC). The present data are an analysis of the (3H) thymidine labelling kinetics in the spleens of mice reared in conventional or germ‐free conditions.

The labelling seen in the 24 h following an injection of (3H) thymidine could best be interpreted on the basis of synthesis of unstable DNA. The changes in the pattern, and distribution of labelled nuclei as well as the intensity of their labelling was not compatible with cell division only, but was also the result of movement of labelled material between the lymphoid cells of the organ.

Germ‐free mice were followed for 24 days following a single injection of (3H) thymidine. The rate of uptake of label into the spleen was much slower than has been found previously in mice reared in conventional conditions. When SRC were injected 2 h after giving (3H) thymidine the labelling of lymphoid cells in the spleen and blood was quite different to controls given (3H) thymidine alone. Detailed analysis indicated that turnover of labelled material, presumably DNA, as well as cells was involved. This turnover of DNA could be considered to be metabolic in the sense that renewal, increase in amount, loss, and transfer to other cells were involved. These, and other studies, in vivo (Harris & Olsen, 1973) and in vitro (Harris et al. 1975) indicate that such processes, involving DNA, are highly relevant to the development of antibody‐producing capacity by cells responding to antigenic challenge.
The possibility of electron microscopic autoradiography of steroids after freeze drying of unfixed testes
Authors: Frederik P. M. , Klepper D.
Published: August 2011

A technique has been developed for the autoradiographic localization of steroids in testes at the light and possibly electron microscope level. During processing of the tissue there is no contact of tissue with water up to the moment of photographic development of the autoradiographs.

Tritium labelled steroids have been introduced into the testis through perfusion of the isolated organ. Small tissue samples were rapidly frozen, freeze dried and fixed with osmium vapour. The fixed tissue was embedded in Epon and thin sections could be cut from this material on an ultra‐cryotome without the aid of floatation liquids. Results with light microscopic autoradiography indicate the potential of this technique. This procedure which avoids any contact of the tissue with water offers good prospects for autoradiography of steroids and other soluble compounds at the electron microscope level. A comparison is made with other proposed techniques for steroid autoradiography.
A copper block method for freezing non‐cryoprotected tissue to produce ice‐crystal‐free regions for electron microscopy
Authors: Dempsey G. P. , Bullivant S.
Published: August 2011

Two methods are described for fracturing unfixed, uncryoprotected mouse liver that had been frozen by contact with a copper block at liquid nitrogen temperature. The best method involved precise fracturing of a few microns of surface tissue with a cooled glass knife, using the Cryokit attachment of the LKB III ultramicrotome. The resulting replicas were free from electron microscopically‐visible ice crystals. A noticeable feature of replicas from unfixed, uncryoprotected tissue was the extent of plastic deformation of certain cellular structures. Cytoplasmic macromolecules, and to a lesser extent intramembranous particles, often appeared to be ‘stretched’ to form fibrils.
Electron microscope image contrast with double beam illumination from crystal supports
Authors: Hines R. L.
Published: August 2011

When a cluster of atoms on the bottom side of a crystal is viewed in the vicinity of a bend contour, a double image of the cluster is formed. The crystal acts as a beam splitter so that an object on the bottom of the crystal is illuminated by two coherent but non‐parallel electron waves. The possibility of using double beam illumination to enhance the contrast of amorphous phase objects is discussed. Experimental results are presented for the image contrast of clusters formed by evaporating gold and aluminium on to the bottom side of graphite and silicon crystals.
The application of X‐ray analysis in the transmission electron analytical microscope (T.E.A.M.) to the quantitative bulk analysis of biological microsamples
Authors: Davies T. W. , Morgan A. J.
Published: August 2011

This paper describes: (a) methods for the high and low temperature ashing of biological tissue samples about 10−4–10−5 g in weight; (b) a method of spraying the solubilized ash on to specimen grids which produces droplets of a size convenient for their quantitative analysis in a transmission electron analytical microscope (T.E.A.M.), but without the need to know the actual volume of individual droplets; (c) the construction of standard curves for determining the absolute concentration of Na, Mg, P, S, K, and Ca simultaneously in a given sample. For most of the elements the error in analysis was about 5% or less. It was proposed that the technique is useful for detecting local element‐compartments in complex tissues, and thus may be an important aid in the interpretation of the results of point analyses performed on sections taken from adjacent micro‐volumes.
X‐ray microanalysis of ultrathin frozen and freeze‐dried sections of human sperm cells
Authors: Chandler J. A. , Battersby S.
Published: August 2011

X‐ray microanalysis of air dried and ultrathin frozen and frozen dried sections of human sperm cells has indicated a large deviation of elemental composition between cells in a single sample and between samples. Analysis of air dried cells indicates a similar subcellular elemental distribution to that found in sectioned material. Sodium to potassium ratios are found to be similar in both preparations. Air drying is considered a valid method for the preparation of sperm cells for analysis.
An improved culture flask for photographic and visual observation of living cell cultures
Authors: Fox Cecil H. , Chaconas Peter G. , Sanford Katherine K.
Published: August 2011

A new type of culture vessel is described, identical in shape to the classic T15 vessel but with the advantage of optical surfaces that allow photography at medium resolution (with numerical apertures of 0.60–1.0). Details of flask manufacture are given.
Characterization of particulate material in very pure waters
Authors: Biddle P. , Miles J. H.
Published: August 2011

Techniques are described which enable exceptionally pure waters to be handled without contamination. The solids content which may be less than 1 part/108, can be obtained on the electron microscope grid and transmission electron micrographs and diffraction patterns obtained. X‐ray spectra obtained using a Li drifted Si detector attached to a scanning electron microscope (SEM), are enhanced by use of an auxiliary circuit, which causes the electron beam to dwell on the particle surface much longer than over blank film. The problems involved in converting X‐ray count rates into an analysis are discussed. The analyses gave valuable information on the origin of the particulate material.

Paper presented to the symposium on the ‘Use of the microscope in studies on air and water pollution’. Royal Microscopical Society, University of York, 17 December 1974.
The effect of the electron beam on various mounting and coating media in the SEM. II
Authors: Rampley D. N.
Published: August 2011

The results given in this paper supplement those reported in Muir & Rampley (1969). The effect of the electron beam on eight uncoated and eight differently coated specimen adhesives is investigated. The results are reported in tabular form. The investigation was carried out to determine the most beam damage‐resistant combination of adhesive and coating. It must be kept in mind that for certain investigations the choice of adhesive is limited by one or more factors, which may be unrelated to beam damage resistance.
The electron‐beam induced monomerization of di‐p‐anthracene
Authors: Jones William
Published: August 2011

The electron beam stability of di‐para‐anthracene has been investigated with regard to the study, in situ, of the mechanism of photodimerization of anthracene and related materials. It is found that the electron beam converts di‐para‐anthracene to anthracene, the process taking place in a single crystal to single crystal manner. Electron doses greater than 6.0 × 10−5 C mm−2 result in a diffraction pattern corresponding to the anthracene lattice.
Microspectrofluorometry in the study of biogenic amines: Automatic correction of excitation spectra
Authors: Klig Victor , Demirjian Charles , Pungaliya Prem
Published: August 2011

An on‐line digital system for correcting fluorescence excitation spectra for the response of the exciting light system is described.
Errors in area measurement in thick sections, with special reference to trabecular bone
Authors: Whitehouse W. J.
Published: August 2011

A formula is derived for the errors caused by section thickness in the measurement of the mean cross‐sectional areas of opaque material embedded in a transparent medium. There are some simplifying assumptions which can only be justified by limiting the maximum thickness of the section, but no restriction whatever is placed upon the shape of the opaque material.

For isotropic materials a very simple formula is found, relating the relative error in cross‐sectional area to measurements of area and boundary profile length on the section surface. The effect of anisotropy in the material may be very large. To illustrate this, numerical calculations are made for two particular classes of simple anisotropic material, those which might be made from isotropic materials by linear distortions.
The use of freeze‐substitution in the preparation of plant tissue for ion localization studies
Authors: Harvey Diana M. R. , Hall J. L. , Flowers T. J.
Published: August 2011

A method, utilizing freeze substitution, is described for the preparation of plant tissue for analytical electron microscopy. The fine structure of the cytoplasm was adequately preserved after freezing leaf tissue in 2‐methylbutane at −170°C. Furthermore, following substitution in ether, losses of sodium and potassium from the tissue were less than 4% of the original ion content and the loss of chloride was less than 1%. The merits of the procedure as a means of tissue preparation for ion localization studies are discussed.
Stereological method for estimating relative membrane surface area in freeze‐fracture preparations of subcellular fractions*
Authors: Weibel Ewald R. , Losa G. , Bolender R. P.
Published: August 2011

Microsomal fractions from liver cells are a mixture of vesicles derived from a number of different cellular membranes. These can be differentiated on freeze‐fracture preparations by their characteristic density of intramembranous particles. A stereological method is developed which allows the estimation of the relative membrane surface of the various membrane types by using freeze‐fracture preparations. The sample is restricted to concave profiles without cast shadow. The numerical frequency distribution of vesicles with respect to particle density is determined. The estimation of relative surface area must consider the effects on sampling of (a) variable size distributions of vesicles and (b) loss of small profiles. Correction coefficients are derived which allow a differential vesicle count to be transformed into an estimate of relative membrane surface.
Application of various stereological methods to the study of the grain and the crack structure of concrete
Authors: Stroeven Piet
Published: August 2011

Various statistico‐geometrical as well as deterministic stereological methods have been applied to study the structure and the structural defects in plain concrete, subjected to a compressive loading. Up till now use has been made of an artificial spherical single‐sized ceramic aggregate instead of coarse gravel for most of the investigations. Crack patterns which have been visualized with the help of the filtered particle method (based on fluorescence), have been analysed by means of the method of directed secants. Apart from structural information and results for engineering applications, this approach facilitated a mutual comparison of various stereological techniques in this field of materials science.
Estimation of linear functionals by maximum likelihood*
Authors: Nicholson W. L.
Published: August 2011

A classical problem of stereology is estimation of linear functionals of particle size distributions from intersections on planar probes. The maximum likelihood method of estimation is outlined and compared with classical linear methods and with numerical interpolation methods.

A population of spherical particles is distributed in an infinite matrix with position defined by a Poisson field. Diameters of circular intersections on a planar probe constitute a generalized Poisson process. Maximum likelihood theory fits a parametric family of planar density functions to observed diameter frequency data. The integral of an appropriate kernel with respect to the fitted density function is the linear functional estimate.

The approach can handle composite data involving observation at several magnifications. Estimation of complementary cumulative density of pores in uranium oxide fuel pellets illustrates the maximum likelihood method of data collected with two microscopes at four magnifications.
The preparation of thin samples of III‐V semiconductors for TEM studies
Authors: Weiss B. L.
Published: August 2011

An improved anodization technique for the production of thin samples of GaAs and other III‐V semiconductors which are suitable for TEM studies is described.
Cell and organelle shrinkage during preparation for scanning electron microscopy: effects of fixation, dehydration and critical point drying
Authors: Gusnard Debra , Kirschner Robert H.
Published: August 2011

The critical point drying method of preparing samples for scanning electron microscopy is associated with a variable amount of specimen shrinkage. We studied the causes of this phenomenon in isolated mouse hepatocyte nuclei and in human erythrocytes and found that the critical point drying process itself caused most of the shrinkage that we observed (a 25–30% reduction in diameter in both specimens). Glutaraldehyde fixation and ethanol dehydration caused only minimal size reduction, prior to critical point drying. Substitution of an inert (ethylene glycol‐ethylene glycol monethyl ether) dehydration technique did not alter the final result. Previous studies in our laboratory using high resolution SEM and correlative transmission microscopy of isolated nuclei have demonstrated that the shrinkage represents a miniaturization of the organelles in which all structural components retain their usual relationships.
Some proposed improvements in the scanning ion microprobe
Authors: Krohn V. E. , Ringo G. R.
Published: August 2011

Three improvements, which we have investigated, promise to lead to a scanning ion microprobe with a space resolution of about 15 nm. The improvements are: (1) use of a field‐evaporation (EHD) ion source with liquid gallium to give a brightness exceeding 1010 A m−2 sr1 at 21 keV, (2) a high efficiency (10%) collecting system for secondary ions, and (3) enhancement of the secondary‐ion yield by cesium deposition. With this instrument, tracing with stable isotopes would offer a number of advantages over autoradiography.
Thiocarbohydrazide binding for botanical specimens for scanning electron microscopy: a modification
Authors: Postek Michael T. , Tucker Shirley C.
Published: August 2011

The ligand osmium binding technique utilizing thiocarbohydrazide has been a successful alternative to evaporation or sputter coating of botanical specimens. A modification to this technique is presented which we have found successful in our laboratory for the preparation of a variety of specimens. This technique permits an overall reduction in the quantity of osmium necessary to provide conductivity. Further, dehydration time and specimen handling is reduced by the complete chemical dehydration of the tissue utilizing 2‐2‐dimethoxypropane.
Silhouette photography of small active subjects
Authors: Edgerton Harold E.
Published: August 2011

The silhouette system, although requiring a darkroom, gives the experimenter a powerful method of recording the biological populations in large samples of water with great depth‐of‐field. The short exposure also effectively stops the motions of the subjects. No optics are required for the initial exposure but are required for enlargements.
Limitations in the X‐ray microanalysis of thin foils in a scanning transmission electron microscope
Authors: Doig P. , Flewitt P. E. J.
Published: August 2011

The X‐ray microanalysis of thin foils has been investigated using a scanning transmission electron microscope fitted with an energy dispersive spectrometer. Thin foils prepared from an iron‐nickel and an aluminium‐zinc‐magnesium‐copper alloy have been observed and analysed. For foil thicknesses between 200 and 300 nm the X‐ray intenstiy ratios are consistent with X‐ray absorption characteristics. For regions less than 200 nm thickness the measured intensity ratios increase by up to a factor of 5. These results have been explained in terms of a solute enriched or depleted surface layer developed during the preparation procedure.
Freeze‐fracturing of monolayers (capillary layers) of cells, membranes and viruses: some technical considerations
Authors: Nermut M. V. , Williams Lynn D.
Published: August 2011

A novel hinged device for freeze‐fracturing of cell monolayers in the Balzers freeze‐etch unit is described. It is economical on biological material and enables oriented adsorption of sheet‐like membrane fragments.

For freeze‐fracturing ‘by hand’ a monolayer is formed on a positively charged piece of mica (with polylysine) and this is covered with another piece of mica, thin brass plate or filter paper. Such a sandwich is frozen in liquid nitrogen and fractured by means of forceps. Several modifications of this technique as well as practical examples are described. Among possible applications are: negative staining of intramembranous protein particles; chemical or physical analyses of single membrane leaflets; identification of protein complexes by immunoelectron microscopy, etc.
Semiconductor material assessment by scanning electron microscopy*
Authors: Davidson S. M.
Published: August 2011

Considerable strides have been made in the application of the cathodoluminescent and beam‐induced current modes of operation of the scanning electron microscope to the high resolution characterization of semiconductor materials. These techniques can be used to measure parameters such as resistivity, carrier concentration, mobility, composition, diffusion length, minority carrier lifetime, luminescence efficiency, dislocation density and temperature from regions of semiconductors as small as one micron across. The theoretical and experimental factors which limit the range of values over which measurements are possible and the accuracy attainable are discussed in detail. Each technique is illustrated by recent experimental results.
The modulation contrast microscope: principles and performance
Authors: Hoffman Robert
Published: August 2011

The modulation contrast microscope produces an image of high contrast and resolution. The image has a three‐dimensional appearance wherein a rounded object appears dark on one side, bright on the other with grey in between against a grey background. The performance features are optical sectioning, directionality, high resolution and control of contrast and coherence. A bright field microscope is converted to the modulation contrast microscope by adding the modulator, a special amplitude filter, in the objective. A slit aperture part of which is polarized is placed before the condenser. Below this is a rotatable polarizer. The modulator processes light from opposite gradients oppositely, that is brighter for one and darker for the other; thereby preserving the sign. The diffraction theory has been extended to show that gradient image intensity is the intensity of the zero order and when modified by the modulator creates a high contrast image.

The modulation contrast microscope is simple and easy to adjust. It is useful in reflected and transmitted light systems, with plastic and glass vessels as well as in combination with fluorescence systems and polarization techniques. There is virtually no limit to the type of specimen that can be studied.
Scanning electron microscopy of freeze‐dried aortic elastin
Authors: Grut W. , Edwards J. , Evans E. J.
Published: August 2011

As part of a larger project aimed at clarifying some of the uncertainties that exist concerning the structural basis for an explanation of the mechanical behaviour of elastin, an examination was made of purified porcine aortic elastin under the scanning electron microscope. In order to avoid structural changes caused by impregnation with organic solvents, specimens were prepared directly from their water‐saturated state by either air‐drying or freeze‐drying processes. Comparison of the results obtained by these techniques demonstrates that freeze‐drying of elastin has the advantage of enhancing fine detail, minimizing distortion and reducing specimen shrinkage.
The restoration of damaged Philips electron microscope specimen holders used in the high‐resolution stage
Authors: Maxwell M. H. , McFarlane J. B.
Published: August 2011

This note describes how damaged Philips electron microscope specimen holders may be repaired simply and cheaply. The damaged part is replaced by a length of 18‐gauge syringe needle which is silver‐soldered into position. This simple and rapid repair caused no subsequent electrical distortion.
Fast freezing device
Authors: Sitte H. , Fell H. , Hölbl W. , Kleber H. , Neumann K.
Published: August 2011

A fast freezing device for cryofixation of biological material, without cryoprotection, has been developed based on metal freezing (Van Harreveld et al., 1964, 1974) combined with preceding sectioning (Bernard & Krigman, 1974; Van Harreveld & Fifkova, 1975) as well as freeze sectioning and freeze‐etch replication (Dempsey & Bullivant, 1976).
Cutting work in thick section cryomicrotomy
Authors: Saubermann Albert J. , Riley William D. , Beeuwkes Reinier
Published: August 2011

The forces during cryosectioning were measured using miniature strain gauges attached to a load cell fitted to the drive arm of the Porter‐Blum MT‐2 cryomicrotome. Work was calculated and the data normalized to a standard (1 mm × 1 mm × 0·5 μm) section. Thermal energy generated was also calculated. Five parameters were studied: cutting angle, thickness, temperature, hardness, and block shape. Force patterns could be divided into three major groups thought to represent cutting (Type I), large fracture planes > 10 μm in length (Type II), and small fracture planes < 10 μm in length (Type III). Type I and Type II produced satisfactory sections. Work in cutting ranged from an average of 78·4 μJ to 568·8 μJ. Cutting angle and temperature had the greatest effect on sectioning. Heat generated would be sufficient to cause through‐section melting for 0·5 μm thick sections assuming the worst possible case, namely that all heat went into the section without loss. Presence of a Type II pattern (large fracture pattern) is thought to be presumptive evidence against thawing.
Frozen‐surface replicas of rat bladder luminal membrane
Authors: Severs N. J. , Hicks R. M.
Published: August 2011

A method for preparing replicas of the luminal surface of frozen, unfractured but deep‐etched whole bladder tissue using a Bullivant type II device is described. A small piece of glutaraldehyde‐fixed (uncryoprotected) rat bladder is rinsed in distilled water, mounted luminal side uppermost on a specimen holder and rapidly frozen by immersion in liquid nitrogen (cooled below its boiling point in a vacuum) or by contact with a copper block at liquid nitrogen temperature. The specimen is processed in the type II device without fracturing and ‘deep‐etched’ by allowing a longer period than usual to elapse before shadowing. The results are assessed with reference to the appearance of the luminal membrane in standard freeze‐fracture replicas, and some preliminary observations on the structure of the normal luminal membrane and its counterpart in bladder tumours are presented.