Showing papers in "Journal of Microscopy in 1975"

The quantitative analysis of thin specimens

Abstract: SUMMARY Results are reported concerning the use of an energy dispersive X-ray detector to carry out the analysis of thin foils in the electron microscope. The combination of a thin specimen and the extreme stability of the energy dispersive X-ray detector enables the experimental determination of a calibration curve of X-ray production—detection efficiency vs characteristic X-ray energy. Quantitative analysis can be carried out using the calibration curve without reference to standards at the time of analysis.

Electron microscopy of critical point dried whole cultured cells.

Abstract: SUMMARY To determine the overall fine structure of whole, unsectioned cells, cells from rat embryos were cultured on Formvar, glutaraldehyde/osmium-fixed, transferred to grids, dehydrated, critical point dried, then examined by transmission electron microscopy at either 80 or 1000 kV. In contrast to air-dried material, critical point dried cells revealed each component clearly and with excellent contrast. All normal cytoplasmic structures (including coated vesicles, polyribosomes, microtubules and other fine components) were readily identifiable. Extensive structures such as microtubules and the endoplasmic reticulum (which appear fragmented in sections) were well displayed. At 1000 kV the beam readily penetrated even the thick nuclear and perinuclear cell regions and produced exceptionally crisp images. The methods described provide a simplified approach to the study of overall cell fine structure.

Timber—a review of the structure-mechanical property relationship

Abstract: SUMMARY Following a brief introduction concerned with present consumption and future prospects of timber and timber products the principal structure-property relationships of this low density, cellular, polymeric composite are reviewed and discussed. Structure is examined at four levels of magnitude—macroscopic, microscopic, sub-microscopic and chemical—and the various models used to interpret its composite nature are described. The dimensional instability of timber and loss of strength on wetting are discussed in terms of its fine structure. At low levels of stressing, and for short periods of time, timber can be treated as an elastic material, but at higher stresses and prolonged periods, especially with alternating humidity, timber behaves as a linear orthotropic viscoelastic material; the various factors influencing the elastic constants and the relationship of creep to fine structure are discussed. The anisotropy of wood is related to cell arrangement and microfibrillar orientation: strength and its variability are discussed in terms of structure at all four levels. Comparison of the strength of timber with that of other constructional materials especially on a weight basis shows timber in a very good light; the combination of high stiffness and high toughness is unique. Recent models using three-dimensional anisotropic elastic analysis to understand strength and deformation of timber are described. The morphology of fracture under different forms of stressing is illustrated.

The preparation, examination and analysis of frozen hydrated tissue sections by scanning transmission electron microscopy and x-ray microanalysis.

Abstract: A method is reported for preparing, examining and analysing frozen hydrated tissue sections using transmission electron microscopy and X-ray microanalysis. Use of this method permits localization and measurement of water soluble or diffusible elements within the hydrated cell matrix. Since any change in total fresh weight of the specimen will affect the concentration of all components, great care has been taken to demonstrate that the mass neither increases nor decreases and to ensure that the tissue remains frozen-hydrated. Criteria for assessing whether or not the tissue remains frozen-hydrated are reported. After quench freezing, 1-2 mum thick sections of mouse liver were cut at 193 degrees K and picked up on a specially designed annular specimen holder covered with an aluminium coated nylon film. Using a transfer device which prevents contamination of the tissue sections while maintaining them at a low temperature (below 143 degrees K), the sections are transferred either to the vacuum evaporator cold stage or the scanning microscope cold stage. The tissue sections may be coated with an aluminium layer to improve electrical and thermal conductivity. The specimens are examined in the scanning transmission imaging mode and analysed using an energy dispersive X-ray analyser. Concentration of intra-nuclear and intra-cytoplasmic K, P, S and Cl are reported for mouse hepatocytes as ratios of the characteristic radiation to the continuum radiation used as a measure of mass. Ratios for all four elements were higher in the nucleus than the cytoplasm. Examples are given of this method as applied to plant and insect tissue.

Abel type integral equations in stereology. II. Computational methods of solution and the random spheres approximation

Abstract: SUMMARY As mentioned in Part I of this paper, Part II is mainly concerned with the construction of stable computational methods for the solution of integral equations of Abel type which occur in stereology. However, in order to develop a rationale for the types of methods which we shall propose, it is first necessary to review the different types of methods which have been suggested to date in the stereological literature. This is done in section 2. From the review, we conclude that the construction of stable methods can be based on the use of spectral differentiation and product integration to evaluate appropriate inversion formulae, when they are known. Using the Random Spheres Model as exemplification, we examine the nature of such methods in section 3. The potential of the proposed methods is illustrated in section 4 by examining the reliability of the random spheres approximation—an unsolved problem in stereology proposed by Moran (1972). We conclude from this examination that the random spheres approximation is quite reliable for particles which approximate spheres.

Abel type integral equations in stereology: I. General discussion

Abstract: SUMMARY In developing numerical methods for the solution of computational problems of an involved and pragmatic nature, as occur for example in stereology, the approach should be first to classify the distinct types of numerical problems which arise. If available, appropriate results from numerical analysis can then be used to develop stable numerical processes for their solution. In fact, many of the results relating to the construction of basic numerical methods, such as spectral methods for the stable differentiation of experimental data and product integration methods for the evaluation of integrals with oscillatory and singular integrands, have been resolved. Thus, we are now in a position to apply the above approach to the more complex computational problems which arise in stereology. Many of these problems belong to one of the following two classes: (i) the solution of integral equations of Abel type; and (ii) the solution of some numerical differentiation problem. In Part I of this paper, we illustrate the generality in stereology of the Abel type integral equation and numerical differentiation formulations. As well, we derive the specific properties required in Part II which is mainly concerned with the construction of stable computational methods for (i). Included among these properties are the explicit inversion formulae which are known for general Abel type integral equations. It is these inversion formulae which we use to construct stable computational methods. Often, when estimates for linear properties of the solutions of (i) and (ii) are required, the numerical solution of (i) and (ii) can be circumvented by estimating the linear properties directly from the given observational data. In deriving such estimates, use of the properties of the Abel type integral equation and differentiation formulations plays an essential role. Because of the close connection between such estimation problems and (i) and (ii), the estimation of linear properties from truncated observational data is also examined in Part I.

Tungsten coating—a method of improving glass microtome knives for cutting ultrathin sections

Abstract: SUMMARY 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.

Preparing sections of skeletal muscle for transmission electron analytical microscopy (TEAM) of diffusible elements.

Abstract: SUMMARY 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.

Analysis of droplets from iso‐atomic solutions as a means of calibrating a transmission electron analytical microscope (TEAM)

Abstract: SUMMARY This paper describes a spraying method of producing iso-atomic droplets (≅3 μm diameter) of uniform size, distribution and chemical composition. These droplets were used to calibrate a TEAM system operated under given conditions, so that the relative sensitivity of the instrumentation for the elements Na, Mg, P, S, (Cl), K, Ca and Co (‘overall sensitivity factors’) could be determined. These factors (15, 40, 83, 90, (43), 104, 109, 100% respectively) were used to convert observed X-ray intensity ratios from standard droplets to relative amounts of elements with a fair degree of accuracy (error generally less than ± 10%). It was proposed that the sampling and quantification methods could be applied for the absolute quantitative bulk-analysis of fluids and tissues (after solubilization) with an introduced reference element, e.g. cobalt, and potentially to the study of relative elemental ratios in fluid microvolumes and in biological thin sections.

A wet stage modification to a scanning electron microscope.

Abstract: SUMMARY A modification to the vacuum system of a JSM2 scanning electron microscope has enabled hydrated specimens to be placed inside the specimen chamber of the instrument and to be surrounded by water vapour at a pressure up to approximately 1.3 kPa (10 Torr). The surface topography was observed by detecting the backscattered electrons using a wide angle backscattered electron detector placed close to the specimen. The microscope was operated in the normal scanning mode which allowed the examination of the surface topography of the specimens, whilst still retaining the depth of focus which is a feature of the SEM. This modification has enabled a resolution of approximately 0.2 μm to be obtained from biological specimens partially immersed in water at temperatures just above 0°C.

Removal of glass coverslips from cultures flat embedded in epoxy resins using hydrofluoric acid.

Abstract: SUMMARY A technique is described by which the glass coverslip of a culture, flat embedded in epoxy resins can be removed easily using hydrofluoric acid. Removal by this method leaves the surface of the resin quite smooth and suitable for subsequent thin or ultrathin sectioning for electron microscopy.

Preparation of vascular endothelium for scanning electron microscopy: a comparison of the effects of perfusion and immersion fixation.

Abstract: A comparison of currently used methods of tissue fixation for scanning electron microscopic study of vascular endothelium revealed that in situ fixation by intravascular perfusion is superior to immersion for the preservation of endothelial surface morphology.

The application of optical densitometry in the study of wood structure and Properties

Abstract: The importance of density as an indicator of wood quality, and the need to measure density rapidly, accurately and on a large scale, have led to the development of the techniques described in this paper.

Wood anatomy of Crypteroniaceae sensu lato

Abstract: SUMMARY The wood anatomy of Alzatea from South America, Axinandra, Crypteronia and Dactylocladus from South East Asia, and Rhynchocalyx from South Africa is described in detail. Special attention has been given to the ultrastructure of vestured pits in the five genera. The grouping of these genera into one family as suggested recently is not supported by wood anatomy. Alzatea andRhynchocalyx differ widely from the three other genera and appear to share many characters with both Lythraceae and Melastomataceae; the latter family shows a wide wood anatomical range. The wood anatomical affinities of Axinandra, Crypteronia and Dactylocladus are with Melastomataceae. Comparisons with some other Myrtalean families, notably Oliniaceae and Sonneratiaceae, indicate intimate relationships between Melastomataceae, Lythraceae, Oliniaceae, Sonneratiaceae and Crypteroniaceae s.l. because of overlapping of the wood anatomical range among these families.

Preparation of nematodes for scanning electron microscopy.

Abstract: Nematodes from the orders Tlyenchida and Rhabditida were fixed and processed in several different ways for examination with the scanning electron microscope (SEM). Four processes produced good preparations of fixed nematodes. Drying from acetone was the simplest of these techniques and most useful for regions of the tylenchid nematodes supported by skeletal tissue. Critical point drying, a more complicated procedure, gave good preparations, but they required special care in processing. Nematodes infiltrated with glycerol and a conducting agent were the most life-like but were difficult to examine. Specimens infiltrated with an epoxy resin looked natural and this was the most promising process tried.

Radiation damage in electron microscopy of organic materials: effect of low temperatures.

Abstract: SUMMARY As measured by the life-time of their electron diffraction patterns, the radiation sensitivity of anthracene and coronene at 500 kV is reduced by a factor of three to four at liquid helium temperature in comparison to room temperature. For l-valine the ratio is about 1·8 but there is a wide variation in the results, possibly due to differences in crystal thickness. The end-dose at 20°K for valine is equivalent to 13 electrons/A2; for anthracene and coronene it is about 600 electrons/A2 at room temperature. The variation of end-dose with temperature shows that at least two mechanisms must be involved in damage to such compounds, possibly concerning the breaking of intermolecular and intramolecular bonds, respectively.

Quantitative analysis of micrographs by computer graphics.

Abstract: SUMMARY A computer-aided graphical analysis system is presented which can be used to quantify several types of information from micrographs of cross-sectioned peripheral nerves. A simple input consisting of eight points, corresponding to two diameters of each axon and two for each axon plus myelin, is used to compute statistical and numerical estimates of these diameters, the myelin thickness being expressed as a function of axon diameter, and both the spatial position and distribution of nerve fibres within the bundle. The computer programs presented and summarized here describe the procedure for digitizing the input data and the subsequent computations for editing and analysis. The general applicability of this approach for automated analyses of other anatomical areas is also presented.

Changes of particle frequency in freeze‐etched erythrocyte membranes after fixation

Abstract: The frequency of particles on the membrane fracture faces of freeze-etched human erythrocytes was measured, and the effect of fixation procedures on the particle frequencies was studied. Fresh blood, buffer washed cells and cells fixed in one of the following ways were examined: glutaraldehyde, glutaraldehyde followed by osmium tetroxide, osmium tetroxide alone. Quantitative analyses showed that some treatments produced a significant reduction in the number of particles on the fracture faces as compared with the fresh cells. After both osmium tetroxide fixations, the loss of particles was greater from the outer fracture face (OFF) than the inner fracture face (IFF), whilst after the other treatments approximately the same number of particles were lost from both fracture faces. The results are discussed with respect to some current concepts of the molecular architecture of the erythrocyte membrane and the action of fixatives. The reduction of particle frequencies is thought to be due to both leaching of membrane proteins, and deviations of the usual fracture plane within the membrane. Glutaraldehyde alone was shown to have less effect on particle frequency than the other fixatives and it is therefore a suitable fixative for the preparation of freeze-etch specimens.

High resolution electron microscope study of lattice images in biological apatites.

Abstract: SUMMARY Lattice (Fourier) images of crystallites in human bone and teeth, and calcified atherosclerotic plaque were studied using high resolution transmission electron microscope techniques. The lattice images observed in the normal and diseased calcified tissue were compared with the images of synthetic hydroxyapatite crystallites.

Ice crystal growth in skeletal muscle fibres

Abstract: SUMMARY Ice crystal growth was studied in rapidly frozen skeletal muscle fibres which were treated with cryo-protective additives (glycerol, DMSO, sucrose) or which were untreated. Freeze cleaving and etching was the basic method, with conventional plastic embedding and cryo-ultramicrotomy as complementary techniques. Extensive crystal growth occurred during freezing in all unprotected fibres. Just below the fibre surface the crystals were numerous but small, while deeper in the fibre they were fewer but larger. The deeper within the specimen a fibre was located, the larger, in general, was the crystal size. The crystal volume density was about 55%, irrespective of crystal size. Ice recrystallization was practically absent at the temperature normally used in cryo-sectioning (–70°C). Anti-freeze treatment eliminated crystal growth. If the anti-freeze agents were used in non-toxic concentrations, however, their effect on crystal growth was very limited. ‘Dry’-cut, freeze-dried ultra-thin cryosections of protected and unprotected fibres confirmed these observations, while sections obtained by ‘wet’ cryo-cutting showed no apparent signs of crystal growth. In plastic sections of frozen and thawed fibres a previous occurrence of crystals was only slightly indicated. In interpreting the ultrastructure in ‘wet’-cut cryo-sections of unprotected frozen mucle fibres, the distorting effects of ice crystals through mechanical compression and alterations in sectioning conditions, must be taken into consideration. Crystal growth also strongly limits the possibilities of using ‘dry’-cut sections of untreated frozen tissue for analytical electron microscopy; only the most superficial parts of the fibres seem to be suitable for microanalysis.

The pore arrangement in the emu (Dromaius novæhollandiæ) eggshell as shown by plastic models

Abstract: SUMMARY Pores arising in the cone layer merge with a plexus of tubules running just under and parallel to the outer surface of the emu eggshell. The outer surface of the shell is irregular due to dark green crystalline material arranged as flat domes. The plexus of tubules vent to the ambient environment via short pores opening in the valleys between these domes.

The precise measurement of the thickness of ultrathin sections by a ‘re‐sectioned’ section technique

Abstract: SUMMARY The thickness of ultrathin tissue sections embedded in Epon-Araldite and cut with a diamond knife was measured by re-sectioning and electron microscopic examination of the section profiles. A secondary section mounted on a Formvar-coated slot grid provided enough normally cut segments (seven to seventeen) for measurements giving a precise estimate of mean thickness, comparable to that obtainable by interference microscopy (±2.3% or less for grey to dark gold sections). The standard deviation of section thickness within sections was never more than 5 nm, corresponding to a coefficient of variation of 6.5% or less for sections more than 48 nm thick. This suggests that variation in section thickness, within sections, may be less than has been supposed, so that quantitative work may be based on thickness measurements made over a limited representative area. A silver interference colour was associated with sections 49–60 nm thick.

A multipurpose specimen-carrier for handling small biological objects through critical point drying.

Abstract: SUMMARY A specimen-carrier for safely handling, protecting and drying minute biological specimens down to 1·0 μm in size during the various steps of the critical point drying procedure is described. Free, unattached cells or subcellular fragments can be processed without loss and without risk of unintentional air-drying.

Radiation damage in stained catalase at low temperature.

Abstract: SUMMARY Radiation damage, which occurs in the beam of the electron microscope, has been studied in uranyl acetate stained catalase crystals. Damage is observed in terms of the disappearance of higher orders of the electron diffraction pattern. In this study it has been found that the damage at very low specimen temperatures proceeds both more rapidly and to a greater final degree than is the case at room temperature.

Handling and staining epoxy resin sections for light microscopy

Abstract: SUMMARY Epoxy resin sections 0.1-1.0 μm thick of specimens embedded for electron microscopy, were collected from the ultramicrotome using strips of cover slip, and, after drying on a hot plate, were bulk stained in specially made troughs. After treatment with periodic acid, the sections were stained first in basic fuchsin at 70°C and then in alkaline methylene blue at room temperature. The handling technique allows accurate collection of serial sections without interrupting the sectioning process, and the stain combination is suitable for different specimens in different epoxy resins and is unaffected by storage for more than 1 year.

A freeze-fracture replication apparatus for biological specimens.

Abstract: SUMMARY A freeze-fracture apparatus of original design has been constructed which can be fitted onto a standard vacuum evaporator unit. In it, cell suspensions and organized tissue may be processed by inserting a sample into a cylindrical holder. By leaving a small part of the tissue protruding from the holder, pre-selected and aligned portions of the specimen can subsequently be revealed by fracture under vacuum. After rapid freezing, the specimen remains firmly attached to the inner wall of the sample holder, preventing its possible loss during fracturing. A mechanism, in the form of a double-sided converging wedge, which is operated from outside the vacuum chamber, is used to produce a fracture in the specimen. The device gently induces a fracture in the desired part of the tissue and lifts the protruding part of the specimen out of the way. In this way, reasonably flat fracture faces are produced for subsequent replication. As the fracturing mechanism comes into contact only with the outer edges of the specimen, damage and contamination liable to occur when the entire specimen is traversed by a blade, is avoided. In addition the specimen stage is surrounded by a cold metal shroud which acts as an efficient trap for contaminants. In this way, favourable vacuum conditions are produced in the vicinity of the specimen. Such effective enclosing of the specimen also facilitates controlled sublimation of the sample.

Structural components influencing the permeability of ponded and unponded Sitka spruce

Abstract: SUMMARY Study of the gaseous permeability of wood stored in ponds to promote bacterial decomposition of the bordered pit membranes has shown that the pit aperture/pit cavity contributes to the total resistance to flow of liquids through both ponded and unponded wood. In ponded wood the contributions of the pit apertures/pit cavities ranged between 5% and 46% of the total resistance, the tracheid lumina making up the remaining 96–54%. This large variation is attributed to the presence of different proportions of earlywood and latewood in specimens tested. In air-dried unponded wood, however, the pit membrane pores contribute an average of 81% of the total resistance to liquid flow, the tracheid lumina an average of 16%, and the pit aperture an average of only 3%. An implication of these results is that previously used analytical techniques for the determination of the radius and number of conducting flow paths in wood, based on the assumption that only two structural components offer resistance to flow, are not wholly satisfactory.

Adsorption of DNA molecules to different support films.

Abstract: SUMMARY Protein-free adsorption of the DNA of the Escherichia coli bacteriophage T7 to carbon, collodion, aluminium-beryllium and aluminium films was studied. It was found that the appearance of DNA strands depended greatly upon the kind of support film used. Direct adsorption of DNA to aluminium-beryllium or aluminium films yielded specimens with ‘thin and long’ and ‘thick and short’ regions along the strand. Well extended, uncoiled and unaggregated DNA molecules were obtained only when DNA was adsorbed to carbon, collodion or mica in the presence of intercalating dyes such as ethidium bromide. Adsorption properties of the different films are well correlated with their surface charge. Aluminium-beryllium films carry a strong positive surface charge, aluminium films a weak positive charge and carbon films a weak negative charge. It is suggested that for the preparation of specimens by spontaneous adsorption of well extended and unaggregated strands it is necessary that the DNA molecule is stiffened by a ligand such as an intercalating dye, and that the charge on the surface of the support film is opposite to the charge of the macromolecule.

Field ion microscopy of biomolecules

Abstract: A new type of image, existing only at field strengths below the denaturation field strengths of molecules, has been discorvered. This type of image has structure, is not symmetrical and thus differs from previously reported low field strength images. The possibility that macromolecules adsorbed on tip surfaces produce such structured images has been exhaustively investigated. The result is that no observation has been found which disproves this hypothesis and many tests conducted in such attempts yielded correlations consistent with this hypothesis. It is therefore concluded that it is highly probable that biomolecules produce structured images and that it is highly improbable that these correlations represent a chance event. On the other hand, these correlations may be due to some cause unknown to the authors; but we consider this possibility to be unlikely. Some micrographs have been obtained which provide a reasonable basis for the hope that tertiary structure information may be defrived from low field strength imaging of partially embedded biomolecules or of biomolecules that are resistant to field denaturation during imaging. Information may presently be obtained from analysis of low field strength ion micrographs about the size and shape of some biomolecules.

A container for processing small volumes of cell suspensions for critical point drying.

Abstract: SUMMARY The attachment of lymphocytes to glass or filters, in order to facilitate handling for processing prior to scanning electron microscopy, may introduce artefacts in surface topography. A container has therefore been adapted, from an embedding capsule, for the preparation of small volumes of cell suspensions.