Showing papers in "Journal of Microscopy in 1986"

Stereology of arbitrary particles. A review of unbiased number and size estimators and the presentation of some new ones, in memory of William R. Thompson

Abstract: This paper deals with isolated, countable items, often termed particles, in three-dimensional space. Its substance is the unbiased stereological estimation of the number, height, surface and volume of such particles without any assumptions about their shape. The full range of estimators is described, some of them for the first time, some in an improved form, several in more than one version, and all of them under the single, absolute requirement that one can in fact identify what one is quantifying on sections. In terms of the minimal number of sections for the analysis, the estimators may be classified as follows: On a single section it is possible to estimate vV, the mean volume of particles in the volume-weighted or 'sieving'-distribution. On two parallel sections, separated by a known distance, estimators exist of particle number and of all mean sizes (height, surface and volume) in the ordinary number distribution, as well as of SDN(v), the standard deviation in the number distribution of particle volumes. If the containing space is relatively transparent the sections may be two optical sections within one thick physical section. On a stack of parallel sections, at least as high as the largest particle, and separated by known distances, one can get twelve mean sizes and twelve distributions of individual sizes: all combinations of three sizes: height, surface and volume in four different types of distributions: number, height, surface and volume. Fulfilling the sampling requirements of the above two estimation principles it has been shown very recently that by combining them one may even estimate mean sizes and number of arbitrary particles in a stack of sections with constant but unknown separation. Finally, a unique, unbiased estimator of the total number of items in a specimen is described for the use of which one need not measure the distance between sections, nor their thickness, nor the volume of the specimen, nor assume anything about shrinkage/swelling, sectioning compression or lost caps. It is the fractionator.

Estimation of surface area from vertical sections.

Abstract: SUMMARY ‘Vertical’ sections are plane sections longitudinal to a fixed (but arbitrary) axial direction. Examples are sections of a cylinder parallel to the central axis; and sections of a flat slab normal to the plane of the slab. Vertical sections of any object can be generated by placing the object on a table and taking sections perpendicular to the plane of the table. The standard methods of stereology assume isotropic random sections, and are not applicable to this kind of biased sampling. However, by using specially designed test systems, one can obtain an unbiased estimate of surface area. General principles of stereology for vertical sections are outlined. No assumptions are necessary about the shape or orientation distribution of the structure. Vertical section stereology is valid on the same terms as standard stereological methods for isotropic random sections. The range of structural quantities that can be estimated from vertical sections includes Vv, Nv, Sv and the volume-weighted mean particle volume vv, but not Lv. There is complete freedom to choose the vertical axis direction, which makes the sampling procedure simple and ‘natural’. Practical sampling procedures for implementation of the ideas are described, and illustrated by examples.

Application of cryoultramicrotomy to immunocytochemistry.

Abstract: This paper reviews the most recent status of immuno-cryoultramicrotomy. The technical aspects of each step of the method are also analysed in detail with the intention of providing a useful source of information for investigators using this method.

A new method for the two-dimensional analysis of bone structure in human iliac crest biopsies

Abstract: A new method is described for the analysis of the two-dimensional structural pattern of trabecular bone in human iliac crest biopsies. 8 microns thick undecalcified sections stained with the von Kossa technique were examined at a magnification of X 9. Using an Ibas II image analyser, the ratio of nodes to free ends and the length of different strut types (cortex to free end, node or cortex, free end to free end and node to node, loop or free end) expressed as a percentage of total strut length were assessed. The reproducibility of the method was good for most of the measured indices but inter-observer and inter-section variation were greater. Comparison of biopsy sections obtained from eleven young healthy control subjects and eleven patients with hepatic osteoporosis revealed a significantly higher node to free end ratio, node to loop and node to node strut length and significantly lower cortex to free end and free end to free end strut length in the controls. No significant differences were seen in node to free end, cortex to cortex or cortex to node strut length. This approach to trabecular bone structure analysis should prove useful in determining patterns of bone loss in health and disease and in examining the effects of treatment on bone structure in osteoporosis.

Stereology for anisotropic cells: Application to growth cartilage*

Abstract: A number of either new or recently available stereological methods are described for estimating volume, surface area and number of anisotropic cells. The methods are illustrated with direct reference to the epiphyseal growth plate. Different estimates of a given quantity are obtained by applying alternative methods to the same set of sections, in order to compare the relative merits of the methods. For instance, the surface area of the cells is estimated via the Dimroth-Watson model (which gives a measure of the degree of anisotropy in addition to the surface area estimate) and from vertical sections using cycloid test systems. Cell number is estimated by traditional unfolding methods and by the new disector method. Also, volume-weighted mean cell volume is estimated from vertical sections via point-sampled intercepts using two different kinds of rulers to classify intercept lengths. Finally, nested design statistics is applied to a set of data from twelve animals in order to compare the relative impacts of biological and stereological (sampling) variations on the observed coefficient of error of a group mean estimate. The preferred methods are listed in the final section.

Post-embedding cytochemistry with gold-labelled reagents: a review.

Abstract: The detection of antigens and glycoconjugates with the protein A-gold and the lectin-gold techniques, respectively, is reviewed. Special attention is directed to the necessary conditions for fixation and embedding as well as to the staining procedures of tissue sections for light and electron microscopy.

A new 3‐d reconstruction scheme applied to the 50s ribosomal subunit of e. coli

Abstract: We developed a reconstruction scheme which is applicable to all macromolecules having a preferred orientation in the electron microscopical preparation with respect to the specimen grid and random in-plane orientations. A projection of the tilted specimen provides a complete conical tilt series with random azimuthal angles (Frank et al. 1978). In the crown view of the 50s ribosomal subunit the orientation of the main part of the particle is well defined with respect to the specimen plane (Verschoor et al. 1985) A pair of micrographs were taken, one with the specimen tilted by 5 0 \" (Fig.la) and one without tilt. The azimuthal angles were determined from the untilted image. The vertex angle of the conical tilt series was known from the goniometer rea$ing. A new weighted backprojection scheme was developed for random orientation applicable to any geometry. In contrast to published schemes (e.g. Gilbert 1972, Radermacher & Hoppe 1978) the weighting function is calculated numerically using the angles actually present. In the plane ( ~ ~ ' , y ~ $ ' ) of the Fourier transform of the projection at angles 17 y i t h e weighting function is: i'

Elemental mapping with elastically scattered electrons

Abstract: SUMMARY We describe a technique for efficient, quantitative, standardless elemental mapping using a high-angle annular detector in a scanning transmission electron microscope (STEM) to collect elastically scattered electrons. With a single crystal specimen, contrast due to thickness variations, diffraction, and channelling effects can be avoided, so that the resulting image contrast quantitatively reflects variations in impurity concentration. We compare a number of simple analytical approximations to the elastic scattering cross sections and show that a standardless analysis is possible over a wide range of atomic number and inner detector angle to an absolute accuracy of better than 20%.

Imaging intracellular elemental distribution and ion fluxes in cultured cells using ion microscopy: a freeze-fracture methodology.

Abstract: A freeze-fracture methodology was standardized for tissue culture cells to study intracellular distribution of diffusible elements with ion microscopy. Chinese hamster ovary (CHO) and normal rat kidney (NRK) cells grown on a silicon substrate were sandwiched using another smooth surface (silicon, glass, mica) in the presence of spacers and fast frozen in liquid nitrogen slush. The sandwich was fractured by prying the two halves apart under liquid nitrogen. This procedure produced large areas on the silicon substrate containing hundreds of cells grouped together and fractured at the apical cell surface. After freeze-drying, these cells revealed a subcellular distribution of Na, K, Ca, Mg, P, Cl and S with the approximately 0.5 micron lateral resolution of the ion microscope. Between the nuclei and the cytoplasm of cells, no major differences were observed for Na, K, Mg, P, Cl and S intensities. Calcium alone, however, exhibited a remarkable distribution. Calcium accumulated more in the cytoplasm than in the nuclei of cells. Even within the cytoplasm its distribution was heterogeneous, suggesting Ca binding sites. The fractured cells consistently exhibited high K-low Na intensities. The injured or dead cells were easily recognized among the healthy ones due to their abnormal ion composition. This simple freeze-fracture methodology allowed fracturing of cells without removing the cells from the substrate. In addition, it eliminated the need for washing the nutrient media away and cryo-sectioning before ion microanalysis. The methodology was successfully extended to 3T3 mouse fibroblast, PtK2 rat kangaroo and L5 rat myoblast cultures.

Developments of new Lowicryl resins for embedding biological specimens at even lower temperatures.

Abstract: SUMMARY Two new Lowicryl resins have been developed for embedding biological materials at temperatures down to 210K (hydrophilic K11M) and to 190K (hydrophobic HM23). They have similar properties to Lowicryl K4M and HM20. The new resins were first tested for low temperature applications by the ‘progressive lowering of temperature’ procedure and this shows that the low viscosity of K11M and HM23 is favourable for the infiltration of biological specimens. Hardening is achieved through photo-polymerization at these lower temperatures. These properties make K11M and HM23 suitable for cryosubstitution of rapidly frozen material and it is speculated that the preservation of antigenicity may be further improved.

Extraction of proteins and membrane lipids during low temperature embedding of biological material for electron microscopy.

Abstract: SUMMARY The extraction of proteins and membrane lipids from biological materials during embedding procedures for electron microscopy carried out at temperatures down to 223 K was studied. Glutaraldehyde-fixed cells of Acholeplasma laidlawii mainly served as test material. More than 99% of the protein and 88% of the lipid of these cells were retained after dehydration with ethanol or acetone between 277 and 223 K and infiltration with methacrylate at 223 K. When methanol was used for dehydration, only 54% of the lipid was retained. The amount of extracted lipid was essentially independent of the ratio between volume of extraction liquid and amount of material subjected to extraction. The cytoplasmic membrane of sectioned Acholeplasma-cclls dehydrated and infiltrated as described above appeared more diffuse than that of cells fixed with glutaraldehyde and osmium tetroxide in epoxy resin at room temperature. Glutaraldehyde-fixed erythrocyte ghosts retained 85% of their phospholipid content when dehydrated with ethanol between 277 and 223 K and infiltrated with methacrylate at 223 K. Spinach chloroplasts and thylakoid vesicles retained 61% and 35%, respectively, of their cholorophyll content.

Reconstitution of molecule images analysed by correspondence analysis: a tool for structural interpretation.

Abstract: Correspondence analysis is gaining increasing importance in the analysis of electron micrographs of macromolecules. Partial or complete reconstitution of images from their factorial representations is introduced as a useful tool in interpreting variational patterns and tracing their physical origin. The value of image reconstitution is demonstrated with two examples, one using a set of model images and the other a set of images of a haemocyanin molecule assembly product.

Cryo-electron microscopy and three-dimensional reconstruction of actin filaments.

Abstract: Actin filaments have been examined by electron microscopy whilst in a frozen-hydrated state. Filaments embedded in a vitreous water layer are basically similar to those prepared by negative staining and show characteristic helical substructure, where the pitches of the helices are about 70 nm and 6 nm. Variability in spacing between long pitch helix cross-over points has been observed, which is consistent with intrinsic angular disorder between successive filament subunits. Fourier transforms of the most ordered filaments show four strong layer lines that index as the first, fifth, sixth and seventh orders of a 35 nm repeat. A three-dimensional helical reconstruction, calculated to a resolution of about 4 nm, shows the individual subunits to be orientated with their long axes roughly perpendicular to the filament axis. Each subunit is somewhat curved and is resolved into two domains. Most connections between successive subunits appear to be made close to the filament axis. We also report on the performance of the specimen holder (Philips PW 5699) used in this work.

Electron beam radiation damage to organic inclusions in vitreous, cubic, and hexagonal ice

Abstract: SUMMARY Frozen hydrated specimens of various latex spheres were used as well-defined systems for the study of electron beam radiation damage to organic inclusions in vitreous, cubic and hexagonal ice. We found that radiolysis of organic material is modified by the presence of ice and that radiolysis in vitreous ice is different from that in crystalline ice. The pattern of damage depends also on the nature of the irradiated polymer, e.g., damage to poly(vinylchloride) is quite different from damage to polyacrylates, although in both polymers the main radiolytic process is chain scission. Some polymers such as polyacrylates were found to be much more stable in vitreous ice than in crystalline ice. The experimental results indicate that free radicals formed at the ice–organic matter interface play an important role in the radiolysis process which affects both the ice and embedded organic particles. Ice may play also a physical role in the process by limiting the diffusion of free radicals away from the interface. Although net mass loss is not much affected by ice, massive structural changes including repolymerization take place in its presence.

The interferometric methods applied to the studies of fibrous materials

Abstract: This paper describes the use of interferometric methods in fibre science. Different techniques of double-beam and multiple-beam microinterferometry are given, together with their application in the investigation of fibre properties. The use of the polarizing microscope in this respect is also given. Double-beam and multiple-beam interferometric methods were applied to determine the mean refractive indices and birefringence of polyester fibres. The results are illustrated by microinterferograms. Accurate results are obtained when considering the area under the interference fringe shift representing the path difference integrated across the fibre. The double-beam interference microscope is a quick method for the determination of the optical properties of fibres. The interference fringes produced from the multiple-beam interferometric methods are extremely sharp and the amount of information in this case is considerable. The fringe displacement in this case is proportional to twice the phase difference introduced by the fibre. Therefore, multiple-beam interferometric methods are much more sensitive than the double-beam methods.

On crystal size and cooling rate

Abstract: A theoretical model is proposed which is used to derive a quantitative relationship between the critical cooling rate and average crystal size at any location within a biological specimen of given shape subject to rapid freezing. The model is applicable to the slamming, plunging or spraying methods of cryofixation provided the ice crystal size is at least 5 times greater than the size of the critical nucleus. Complete vitrification of pure water or aqueous solutions is shown to take place at cooling rates in excess of about 3 X 10(6) K/s.

Quantitative image analysis of finite porous media: I. Development of genus and pore map software

Abstract: SUMMARY Software for the reconstruction of branch-node charts from serial sections is tested with a simple cubic lattice, and is applied to determine the genus per unit volume (specific genus) of a Berea sandstone sample from seventy-eight serial sections. The genus is found analytically for the cubic lattice as a function of depth, cross-section and volume. These results enable us to draw conclusions about the relative importance of depth and cross-section and provide useful information on how to treat finite sample data to obtain the specific genus of an infinite homogeneous porous medium. It is shown that it is more important to have a deep sample (many serial sections) than a sample of large cross-section. For samples which are too small in the direction of sectioning, the genus and the rate of change of genus with volume are too low because some of the large loops are not completed within the sample boundaries. For the Berea sample, this ‘shallow-sample depth’ is about 2·2 grain diameters (forty-five serial sections). Only data points at depths in excess of this value are used in determining the genus per unit volume. The slope of the genus versus volume curve is the better predictor of the genus per unit volume of the unbounded medium, of which the sample is a small part, than the value of genus/volume of the sample. For samples of finite cross-section, the Gmin and Gmax versus volume curves are divergent and they only become essentially parallel when the dimensions in the plane of sectioning are large multiples of the grain size. It is shown that it is reasonable to assume that the arithmetic mean slope found for a small cross-sectional area is a good estimate of the common slope that would be attained at high cross-sectional areas. Therefore, samples of small cross-section can be used, resulting in a reduction in the amount of data to be processed and an enhancement of the resolution of small features. The Berea sample data, beyond the ‘shallow-sample’ region, show the same qualitative features as predicted analytically for the cubic lattice. The ratio of the slopes of the Gmax and Gmin curves is 1·22 and the genus per unit volume from the mean slope is 52·6 × 10−8 μm−3.

The preparation and X-ray microanalysis of bulk frozen hydrated vacuolate plant tissue

Abstract: SUMMARY A series of modifications have been devised which allow the peak to background ratio X-ray analytical method to be used more effectively to measure elemental concentrations in large vacuolate plant cells. Planar, frozen-hydrated fracture faces of bulk plant tissue are coated with a thin film of evaporated chromium, which prevents surface charging. Provided the film is sufficiently thin, c. 5–10 nm, there is no attenuation of the electron beam and only a small absorption of soft X-rays. The chromium makes a small but measurable contribution to the spectral background and suitable corrections may be made to the quantitative results. An improved back-scattered imaging system is described, which helps to overcome the problem of spurious X-ray signals from rough surfaces. The microscope column has been modified to permit a continuous readout of beam current, sensu stricta, during X-ray microanalysis and to allow rapid exchange of the electron gun assembly during low temperature operation. Calculations are given relating the size of the X-ray interactive volume to electron penetration and X-ray emission in both frozen hydrated and frozen dried cells. The problems of X-ray microanalysis are discussed in relation to the highly vacuolate cells found in most mature plant tissues and an example given of the distribution of four major cations in tobacco leaves.

Pulsed laser atom probe analysis of semiconductor materials

Abstract: SUMMARY As the size of semiconductor devices is reduced the active volumes of material in each device is also decreased. Under these circumstances it becomes more important to understand the microchemistry of semiconducting materials, as small fluctuations in composition can dramatically affect both the operation of the devices, and of the contacts to semiconductors. Atom probe microanalysis has been shown to be able to analyse the microchemistry of metallic materials with plane-by-plane resolution, and by using a pulsed laser to replace the more conventional voltage pulses the analysis of semiconducting and insulating materials becomes possible. The pulsed laser atom probe has been shown to give very accurate chemical analysis of the stoichiometry of extremely small volumes of III-V compound semiconductors, and the composition of the interfacial layer between silicon dioxide and silicon has been identified as SiO of thickness about 0.3 nm. It has been shown to be possible to prepare specimens for analysis from thin films of semiconductors, thus allowing the microanalysis of a wide range of materials that are deposited in thin film form.

Crystallization and vitrification in aqueous systems

Abstract: SUMMARY Some key ideas and experimental findings concerning the probability that crystallization of a liquid or its binary solutions will occur at moderate cooling rates are discussed. The use of cryoprotectants and of pressure to favourably influence these probabilities is rationalized, and some of the newer findings on small sample supercooling phenomena are reviewed.

Fast cryofixation technique for X-ray microanalysis

Abstract: SUMMARY The proposed cryofixation technique uses a tubule-shaped needle chilled in liquid propane for simultaneous excision and freezing of a tissue specimen. Due to this simultaneity, ionic shifts created by traumatic influences are avoided even in the outermost cells of the specimen. Moreover, it is shown here that stopping the blood flow for more than about 10 s results in notable ionic shifts between cells and extracellular space in rat heart and liver. Such preparative ischaemic injury is minimized by the Fast Cryofixation Technique because it can be easily performed on organs within the circulatory system, whilst the heart of the animal is still beating. Intracellular concentrations of the monovalent ions in rat heart and liver, obtained by this method, tally well with recent results from different independent techniques reported in the literature. As demonstrated by cross-sectioning and freeze-fracturing, the structural preservation of the freezing technique is sufficient for X-ray microanalytical work.

Light microscopical detection of single 5 and 20 nm gold particles used for immunolabelling of plasma membrane antigens with silver enhancement and reflection contrast.

Abstract: To detect plasma membrane antigens, cytocentrifuge preparations of the macrophage-like cell line P388D1 were incubated with monoclonal antibodies and labelled with 5 and 20 nm gold particles conjugated to immunoglobulins or protein A. The 20 nm, but not the 5 nm, particles could be observed by reflection-contrast light microscopy. Single 5 nm particles, however, were clearly demonstrable with silver contrast enhancement, both in absorption contrast and in reflection contrast. The method proved to be compatible with cytochemical methods such as acid phosphatase.

HREM image contrast from supported small metal particles

Abstract: SUMMARY HREM, image calculations and small probe diffraction/AEM have been used to characterize structure and contrast of supported small metal particles of ≤5 nm diameter. Such small particles are thought to be active species in industrial applications such as in heterogeneous catalysis where, in general, the particles employed as catalysts are supported. Image calculations (HREM and diffraction contrast) carried out at both 200 keV and 400 keV at various defoci and support thicknesses have shown that in HREM, particle images are obscured by the support contrast with the loss of edge definition and particles appear to be smaller than they actually are. The particle visibility is better at 400 keV. The calculations have also indicated that particle shape varies as a function of support thickness and defocus. The results have clear implications for identification and interpretation of surface structure of the supported small particles accurately by HREM if not performed under controlled conditions and for determining their size and shape.

Locating water‐soluble vital stains in plant tissues by freeze‐substitution and resin‐embedding

Abstract: SUMMARY Vital stains, moving in the transpiration stream in leaf apoplast, may be kept in place through freezing, freeze-substitution, embedding and sectioning, to reveal their position in the living plant. This technique has been used to study the details of movement of water out of the veins of leaves, and has wide application in histochemistry with water-labile dyes, and for following dye movements in protoplasm. Patterns of water movement in the leaf of Zea mays L. are presented as an example.

Rationale for the application of thin, continuous metal films in high magnification electron microscopy.

Abstract: SUMMARY Various metal films of different thicknesses were deposited on to a particle test specimen and their effects on topographic contrast generation and specimen preservation were determined. Tobacco mosaic virus adsorbed on to thin carbon supports or silicon chips was imaged in TEM or high resolution SE-I SEM at a magnification of 350,000×. Tantalum films of 1–2 nm (average mass) thickness produced best contrasts and prevented volume loss of the particles from electron beam damage. Excessively thick films of 5–10 nm thickness blanketed fine structures and caused severe volume losses. Discontinuous 2 nm thick films of gold or platinum decorated the surfaces, caused a loss in topographic contrasts and induced very high volume losses. Thin continuous metal films were necessary to generate high topographic contrast and to prevent volume loss from beam damage by providing sufficient mechanical stability for small topographic features and increased thermal conductivity of the specimen surface.

Specimen preparation technique for high resolution transmission electron microscopy studies on model supported metal catalysts

Abstract: SUMMARY A new technique is described which can be used for preparing transmission electron microscopy (TEM) specimens suitable for high resolution studies on supported metal catalysts. By conventional silicon processing techniques 200 × 200 μm2 Si3N4 membranes on Si wafers are produced. These membranes are extremely flat and have a uniform thickness of 13 nm. They can be used as a support in various kinds of thin film deposition. A TiO2 film, optimally structured with respect to the requirements for high resolution TEM work in TiO2–metal cluster systems, is deposited on the Si3N4 layer. It consists of one monolayer of 10–25 nm TiO2 crystallites. TiO2 lattice images show that a line resolution down to 0.19 nm is possible. Examples of TiO2–Pd and TiO2–Rh are given using respectively photodeposition and impregnation reduction to produce l.5–4 nm metal clusters.

An extrapolation method for the determination of Cliff‐Lorimer kAB factors at zero foil thickness

Abstract: SUMMARY An extrapolation method is proposed to be useful for the determination of the Cliff-Lorimer kAB factor at zero foil thickness. The method consists of measuring kAB factors as a function of the measured foil thickness, tM, and extrapolating the relationship toward tM=0. The intersection between the extrapolated line and the ordinate of tM=0 gives (kAB)0 which is free from the effect of absorption. The straight line extrapolation that can be achieved by a linear-least squares method is particularly developed to eliminate arbitrariness introduced in the extrapolation process. The extrapolation method is applied to data available in the literature. It is shown that the method yields the (kAB)0 factors compatible with those predicted from the theoretical calculation. It is also shown that this method can circumvent several problems which make it complex and difficult to determine accurate values of the absorption-free kAB factors. Using the straight line extrapolatioin, it is possible to estimate the degree of the thickness overestimation which arises when the foil thickness is measured by the contamination spot separation (CSS) method. Validity of the straight line extrapolation is further discussed.

Preparation of serial sections in dry snow specimens

Abstract: SUMMARY Pore space in a dry snow specimen is filled with a water-insoluble liquid which is frozen solid. The specimen is microtomed, polished with carbon dust, and micrographed through a photomicroscope mounted directly over the microtome. Serial sections are prepared and micrographed at the rate of about ten per hour. The micrographs are automatically digitized, and converted to binary images for microstructural analysis.

Beam induced mass loss in high resolution biological microanalysis.

Abstract: SUMMARY Electron beam induced loss of mass from the organic matrix and from higher Z constituents of biological samples was measured by monitoring bremsstrahlung and peak changes in EDS spectra. When any effects of contamination, extraneous X-rays, beam current drift, specimen drift, and specimen shrinkage were monitored and corrected for, the three types of samples gave consistent and similar results at 296 K. Bremsstrahlung losses averaged 45%, 46% and 50% respectively for muscle homogenate, salivary gland sections and albumin. Sulphur losses average 74%, 72% and 86% for the same three sample types. No other elements suffered significant losses. Dl/e for bremsstrahlung averaged 0·14 C/cm2. Bremsstrahlung loss at 93 K began approximately one order of magnitude higher in dose, and the extent of loss varied. Sulphur losses, however, were greatly reduced at low temperatures.