bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparison of two sampling procedures for stereological analysis of cell pellets
Authors: Mayhew T. M. , Williams M. A.
Published: August 2011

Electron micrographs of macrophages within pellets of rat peritoneal exudate cells were analysed by the morphometric point‐counting technique to compare the volumetric and surface‐to‐volume parameters obtained by two distinct sampling procedures. The distinction between the two sampling procedures was dependent upon the nature of the cell profile recorded on the micrographs. The two types of profile compared were (1) equatorial profiles (each containing a substantial nuclear component to facilitate cell identification) and (2) mixed profiles (a random mixture of equatorial profiles, anucleate profiles and cell profile fragments). Exudate cells were obtained from normal animals and from animals stimulated 5 days previously by a single intraperitoneal injection of Freund's Complete Adjuvant in Hank's solution. Volumetric data for intracyto‐plasmic organelles were found to be in excellent agreement when the two sampling procedures were compared. In addition, the results gave satisfactory consistency for the recorded decrease in the surface‐to‐volume estimate of the stimulated macrophage. It is concluded that equatorial profile analysis, in which cell identification is facilitated by virtue of the presence of the nucleus, is suitable for comparative morphometric cytology in the present study and that the biased sampling procedure may have a wide applicability.
Quantitative autoradiography: an evaluation of visual grain counting, reflectance microscopy, gross absorbance measurements and flying‐spot microdensitometry
Authors: Goldstein D. J. , Williams M. A.
Published: August 2011

Kodak AR.10 and Ilford G5 autoradiographs of a variety of radioactive resin standards and labelled cell preparations were investigated by visual grain counting, reflectance microscopy, gross (non‐scanning) absorbance measurements and integrating microdensitometry (the last using the Vickers M85 flying‐spot instrument).

The reflectivity of silver grains is affected by the type of radiation to which the emulsion was exposed, the conditions of development and other factors. Reflectance results in arbitrary machine units were converted to absolute units by comparing them with results obtained under identical experimental conditions on fully‐blackened standard autoradiographs.

With relatively low grain counts, the exposure is approximately proportional to the visual grain count, the reflectance, the gross absorbance and the integrated absorbance. With a fractional coverage of emulsion by developed grains of more than about 0.4, or absorbance more than about 0.22 (corresponding with Kodak AR.10 to grain densities of more than about 1.5/μm2), the exposure remains directly proportional to the absorbance and the integrated absorbance; experimental observations confirmed the theoretical prediction that the apparent fractional coverage, as measured by reflectance microscopy, is approximately equal to unity minus the antilog of minus the absorbance.

Visual grain counting is reliable in the range 0.01–0.4 grains/μm2 (about 30 grains/10 μm diameter cell), and although tedious is widely applicable. Reflectance measurements are objective and relatively rapid, and are useful with grain counts up to about 150 grains/cell, but results may be affected by staining of the emulsion or by fluorescence of the emulsion or specimen. Both gross and flying‐spot absorbance measurements are directly proportional to the exposure over a large range. Gross absorbance measurements are applicable only to extended, homogeneous autoradiographs, while integrating, flying‐spot measurements may be used also with discrete and non‐uniform specimens. Both absorbance methods are best applied to unstained preparations.
Nomarski differential‐interference contrast microscopy in transillumination: Its use on unstained or stained sections, smears, and wet mounts, or on fluorochromed sections and cell‐culture monolayers
Authors: Geissinger H. D. , Duitschaever C. L.
Published: August 2011

Unstained, lightly stained and conventionally stained microtome tissue sections of two different thicknesses (ca. 4 μm and 1 μm) and also unstained or stained wet mounts of cells were photographed under the microscope using bright‐field, positive phase contrast, and Nomarski differential‐interference contrast (DIC) in transillumination. The photomicrographs were critically compared. It was found that the density of various stains did not adversely affect the better resolution of the DIC image (as compared to the bright‐field image); however, Optical sectioning' of darkly stained objects is not possible.

Unstained or stained smears of blood or of epithelial cells of buccal mucosa were examined with DIC in transillumination, then after certain preparatory techniques, the same preparation was examined in the scanning electron microscope, and finally the same areas of the slide were viewed with DIC in epiillumination. Particular attention was given to structures (nuclei and cytoplasm) which appeared in positive or negative relief in the photomicrographs taken by the various techniques. It was concluded that the optically more dense nucleus which always appeared in positive relief by the various methods of examination, was in fact geometrically raised from the surrounding cytoplasm.

Acridine orange (AO) stained cell‐culture monolayers and H and E stained sections were examined under a fluorescence microscope with DIC optics. By comparing photographs which had been taken with DIC, epi‐fluorescence or fluorescence in transillumination, and DIC‐fluorescence, it was concluded that the DIC image, which had been superimposed on the fluorescence image, contributed a definite gain in information.

Some common errors in the interpretation of the DIC image are discussed; methods of avoiding improper use of equipment are given. The conclusion is drawn that the DIC system is superior to positive and negative phase contrast for the examination of a variety of unstained or stained preparations. Therefore, this method can be used to advantage not only for the examination of unstained preparations, but also on some specimens which have been routinely stained or fluorochromed.
Scanning electron microscopy of L‐phase streptococci
Authors: Bibel David J. , Lawson John W.
Published: August 2011

Protoplasts and L‐phase variants derived from Streptococcus fæium strain F24 were studied by scanning electron microscopy. Effects on morphology due to several osmotic stabilizers, the duration and kind of fixation, the type of specimen mounting, and the method of drying were examined. Conditions for optimal results were found which will allow further biological study of the L‐phase. Whereas air‐drying of broth‐grown L‐phase variants was satisfactory, agar‐grown colonies required the critical point method.
Specific labelling of thiol groups in mammalian keratin suitable for electron microscope studies
Authors: Dobb M. G. , Murray R. , Sikorski J.
Published: August 2011

Quantitative Polarographic studies of the reaction between methylmercuric iodide and variously reduced wool keratin indicate that reaction occurs specifically with the thiol groups from cystine. Since loss of protein from fibres is negligible, this organomercurial is preferred as a selective amino acid label in electron microscope studies. Similar reactions of wool with parachloromercuribenzoate (PCMB) result in extensive structural modifications.

The mercury mercaptides were shown to be thermally unstable in the electron beam and cooling of the specimen between — 140°C and — 88°C coupled with low beam intensities was found necessary to study the original distribution of the mercurials in the samples. Since a direct relationship between the electron scattering sites of the mercurial and cystine within the keratin was established, it has been possible to demonstrate unambiguously for the first time that:

(a) There exists a radial distribution of cystine within cuticular cells. Indeed a new sulphur‐rich component at the outer edge of the ‘a’ layer in peripheral cuticular cells has been identified.

(b) Para‐cortical cells of intact keratin contain a higher proportion of cystine than ortho‐type cells.

(c) A dense concentration of cystine labelled by the electron scattering centres (mercurials) corresponds closely to an intermicrofibrillar‐matrix system in the cortex.

Furthermore, a method has been devised for determining the cystine content of the exocuticle using data obtained from electron micrographs.
Identification of structure by the common‐sense approach*
Authors: Elias Hans
Published: August 2011

Mistakes are often made and perpetuated in the three‐dimensional interpretation of single sections. Such errors can be avoided and corrected if the observer considers carefully what kind of three‐dimensional shape has the greatest probability of yielding profiles such as are seen in the microscope; and if he does not take certain published or verbal statements at their face value.
Recent progress in automatic image analysis*
Authors: Moore G. A.
Published: August 2011

A general survey has been made of the present utility of computing equipment and automatic devices for common problems in stereology and morphometry. Three situations have been evaluated:

(1) Commonly available computer facilities permit easy and rapid prediction of two‐dimensional measurement distributions characteristic of any mathematically defined spatial structure. Tabulated predictions can be computed by direct simulation of sampling processes and random perturbations may be introduced if desired. Resort to formal mathematical solutions is not normally required.

(2) For purposes of controlling the quality of useful solid materials, the materials engineer can be satisfied by description of a Gestalt which characterizes the material as a whole and which can reasonably be presumed to control the behaviour of the material in service. Six stereologically valid parameters are sufficient to describe such a Gestalt. These are: volume fraction (Vv), mean free path in the matrix (l̄α), mean intercept width of particles (l̄β) and measures of material variability, general anistropy, and degree of patternness. Practical measurements of the latter parameters have been defined. Current scanner models are intrinsically suitable for these measurements but will benefit from standardized data outputs and improvements to increase measuring accuracy.

(3) While Gestalt measurements may perhaps be applicable to some biological problems, many problems in this field are specifically concerned with the micro‐architecture of individual objects or cells. Micro‐architectural observations such as counting and measuring, or identifying, individual object sections are inefficiently performed in ordinary computers. Large programming effort is required for limited machine intelligence and the operating speeds are unsatisfactory. These machine operations are not yet competitive with human observers. A partnership combining human identification with machine measurement of identified objects is practical and equipment is presently available. There is reason to presume that computer methods of object identification which have been employed are not similar to the methods actually employed by humans. New hardware systems appear to be necessary either to obtain satisfactory operating speeds by known processes or, if possible, to economically simulate human processes of image recognition.
Applications of quantitative microscopy in materials engineering
Authors: Fischmeister H. F.
Published: August 2011

Among the examples reviewed are volume‐fraction measurements in phase diagram and kinetic studies, properties dependent upon internal surface area, the characterization of phase arrangements by contiguity, proximity and dihedral‐angle measurements. The use of grain boundary and particle spacings in studies of mechanical properties is discussed in relation to current theories of strengthening mechanisms. Applications of size‐distribution analysis in kinetic studies and in the assessment of inclusion contents in steels are surveyed.
Stereological characteristics of atomic arrangements in crystals
Authors: Mackay A. L.
Published: August 2011

A major problem in dealing with complex structures is to identify simple parameters which will summarize significant information. For crystal structures the simplest measure is volume per atom. The Voronoi polyhedron (or Dirichlet region) about each atom can be calculated exactly or statistically. This has been done for a number of structures with unexpected results.

Algorithms for examining the local atomic configurations by the calculation of N‐dimensional volumes are described. Other proposals for the statistical examination of known crystal structures are put forward. Crystals are significant in themselves and in that they provide model structures which can be handled exactly.

Formulæ, convenient for handling the packing of spheres and polyhedra in N dimensions, are listed.
The texture analyser
Authors: Klein J. C. , Serra J.
Published: August 2011

The texture analyser is an apparatus for making morphological measurement which starts from a TV scan and specific hardware. We have conceived and realized this machine at the Centre de Morphologie Mathématique of the School of Mines of Paris. It inaugurates a new generation of apparatus in this field. It is based upon a new conceptual background, using the random set theory of G. Matheron, and the hit‐or‐miss transformation. Its sophisticated hardware is accessible but with the help of specific software. Thus, besides traditional measurements (e.g. lengths of intercepts) it allows the determination of new and useful parameters: radius of curvature, bi‐dimensional size distribution, covariance matrix, bi‐dimensional clumping measurements, and numerous other new parameters.
Current capabilities and limitations of available stereological techniques
Authors: Weibel(Editor) Ewald R. , Fisher Colin , Gahm Joseph , Schaefer Andreas
Published: August 2011

The various methods available for stereological analysis of sectioned material are reviewed with special emphasis on a critical evaluation of their capabilities, advantages and limitations. The review deals with four main topics: (I) Comparison with structural standards; (II) Point counting methods; (III) Image analysis with the scanning microphotometer; (IV) Automatic image analysis based on television systems. It is concluded that each of these approaches has its place, and that the choice essentially depends on the properties of the material and on the nature of the problem.
The use of a low‐viscosity epoxy resin in the preparation of undecalcified bone sections for light microscopy
Authors: Xipell J. M. , Gladwin R. C.
Published: August 2011

A method using the Spurr low‐viscosity epoxy resin medium for the preparation of Von Kossa impregnated undecalcified bone sections for light microscopy is described. The method gives high quality thin sections (0·6–1·0 μm) of cancellous bone and overcomes some of the difficulties experienced with other plastics. The procedure is relatively simple and is well suited for use in a routine diagnostic laboratory.
Synthesis and turnover of DNA in hepatocytes of neonatal rats
Authors: Viola‐Magni M. P.
Published: August 2011

The incorporation of 3H‐thymidine has been studied in the hepatocytes nuclei of rats during the first 3 days of life. The synthesis of DNA which occurs in 18% of nuclei after a single injection of the labelled precursors 2 h after birth, cannot be considered as premitotic since:

The hepatic cell number is constant during the first 3 days of life.

The mitotic index is less than 0·1%, 20 times lower than that expected on the basis of the labelling index and of the length of cellular cycle.

The percentage of labelled nuclei does not increase in the following 3 days.

The average number of grains per nucleus is unchanged for 18 h after the injection, a period longer than an entire cellular cycle (13 h).

This synthesis is better explained as due to an increase of DNA content per nucleus, previously described, in the hepatocytes during the first 3 days of life.

After 20 h of life (18 h after label injection) a decrease of label of about 30% takes place without variation of DNA‐Feulgen dye content per nucleus. It is concluded that synthesis and turnover of DNA are present in the hepatocytes nuclei and that these processes are probably related to the functional maturation of the cells.
Studies on cell proliferation of liver epithelia in 3‐week‐old rats
Authors: Schultze Brigitte , Maurer Werner
Published: August 2011

Cell proliferation of liver epithelia in 3‐week‐old Wistar rats was re‐investigated. Up to a time interval of 54 h the percent labelled mitoses curve shows only one peak of labelled mitoses. From this peak a S‐phase of 8 h was derived. The results are compatible with the model of an exponentially growing population of liver cells with a growth fraction of unity and an' instantaneous' generation time of 7–1 days. The difficulties in describing the special mode of growth of liver cells in 3‐week‐old rats are discussed.

The results are in complete contradiction to those from experiments of Post, Huang & Hoffman> (1963) and of Grisham (1969).
Performance and applications of the Toulouse 3 million volt electron microscope
Authors: Dupouy Gaston
Published: August 2011

This paper describes the electron microscope and gives details of its performance at an operating voltage of 3 MV.

Some initial results are reported concerning: penetrating power; resolving power; studies of radiation damage, and other interactions between electrons and crystalline materials. Several observations are also described concerning biological materials (bacteria, frog skeletal muscle, filaments from mushroom spores …).

Because at high voltage, radiation damage is considerably reduced, the results obtained indicate a new range of applications of high voltage electron microscopy in biological ultrastructure research.
The application of stereo‐micrography in the high voltage electron microscope to studies of cell‐wall structure and deposition
Authors: Cox Guy , Juniper Barrie
Published: August 2011

Stereo‐pairs taken from thick sections have proved useful in solving two problems concerned with the unusual cell walls of collenchyma. These are the three‐dimensional architecture of the cellulose microfibril skeleton of the wall and the organization of the plasmalemma and its derivatives, the paramural bodies.

The basic structure of the wall is lamellate, as has been known for some decades, but the fine details of the lamellation have been the subject of controversy. A model has already been deduced on the basis of thin‐section work, but stereo‐micrography presents the opportunity of direct observational confirmation, as well as more detailed information.

Wall deposition involves both the normal plasmalemma and the elaborate paramural bodies derived from it. The high voltage microscope presents the opportunity of looking at membranes in diagonal and face views, impossible in conventional ultrathin sections. In this way an increased knowledge of the structure of the membrane, as revealed in positively stained sections, has been obtained, as well as a more detailed picture of its configuration in the cell. The paramural bodies are elaborate tubular, lamellar or multivesicular bodies, continuous with the plasmalemma. Stereo‐micrography provides a much clearer view of their organization than has been possible heretofore.
The preparation and observation of thick biological sections in the high voltage electron microscope
Authors: Favard Pierre , Carasso Nina
Published: August 2011

Techniques for the preparation and investigation of thick biological sections in the high voltage electron microscope (HVEM) have been studied using the Toulouse 1.5 and 3 MV microscopes.

Sections several micrometres thick can be made using conventional ultra‐microtomes, but, in order to know their actual thickness, it is necessary to resection them perpendicularly to their surface after embedding them in Araldite.

When successive thick sections are made with glass knives, damage may be observed on their upper surface. This can be avoided by alternating several thin sections between each thick section. Such damage is further reduced if thick sections are made using diamond knives.

The maximum thickness that can be observed at a voltage of 2.5 MV is not limited by the penetrating power of the beam but by the damage suffered by irradiated Araldite. The limit is approximately 10 μm. Below 10 μm a general uniform loss in thickness is observed in irradiated areas; above 10 μm, that loss in thickness is irregular and craters are formed under the beam.

Contrast can be obtained by staining tissues before or after embedding them in Araldite. The cellular structures can be uniformly stained using uranium or lead salts, or selected parts of them can be stained using colloidal tracers, Gomori's lead method, osmium or silver impregnations. The latter techniques are the more convenient as far as sections several micrometres thick are concerned.

The resolving power depends on section thickness and accelerating voltage. At 2.8 MV, by using selective staining techniques, sections several micrometres thick can be investigated with a sufficient resolving power, especially with regard to understanding the spatial organization.

Stereoscopic viewing allows a better interpretation by removing the inconveniences resulting from overlapping structures, but the depth of field must then be considered. Its value, for a given resolving power, taking into account the focal length of the objective lens, depends on the objective aperture: by using a 5 μm aperture one can get a depth of field of 4 μm at a resolving power of 2.0 nm and 12 μm at a resolving power of 6.0 nm, the focal length of the Toulouse 3 MV electron microscope being 10 mm. At this voltage aberrations due to diffracting phenomena are not very disturbing.
Current developments in the high voltage electron microscopy of metals and other materials
Authors: Goringe M. J.
Published: August 2011

The principal advantages of the high voltage electron microscope are first described. Examples are then quoted of various topics in the study of materials in which these advantages are being applied at present. The most obvious improvement, that of greater penetration is utilized in the study of thicker specimens, useful particularly for observation of multiphase solids, experiments in the microscope and environmental cells. The reduction in ionization damage with increasing energy allows observation of many susceptible materials, while the onset of displacement damage interferes with certain other observations. However, the study of displacement damage in nuclear materials using the very fine flux conditions in the HVEM has proved to be of great technological importance. Finally it is shown how a particular high voltage effect, the critical voltage, may be utilized.
Observation of bubble domains in the high voltage electron microscope
Authors: Jones G. A. , Grundy P. J. , Goringe M. J.
Published: August 2011

A variable height holder has been constructed for the EM7 which allows the specimen to be displaced along the optic axis of the microscope. In this way a thin foil can be subjected to a large range of vertical magnetic field associated with the objective lens. So far the device has been used for the observation of magnetizing processes in thin single crystals of cobalt with the c‐axis normal to the foil. As the field on the specimen is increased by lowering it further into the lens, the usual plate domain structure is progressively converted to arrays of bubble domains. On reducing the field from saturation, bubble domains are seen to renucleate.
Combined transmission electron microscopy and X‐ray micro‐analysis of ultrathin frozen dried sections—an investigation to determine the normal elemental composition of mammalian tissue
Authors: Weavers Barry A.
Published: August 2011

A variety of mammalian tissues have been prepared for electron microscopical examination by cryo‐ultramicrotomy. In conjunction with the ultrastructural investigation of the cellular and subcellular morphology of frozen sections, X‐ray micro‐analysis has been performed on various organelles in an attempt to determine their elemental composition and to monitor any variations in elemental concentrations.
Processing small delicate biological specimens for scanning electron microscopy
Authors: Marchant Harvey J.
Published: August 2011

To overcome the problems of collecting and handling delicate cells while dehydrating them for scanning electron microscopy, a method is suggested whereby cells are collected on solvent resistant Millipore filters. The schedule used for successfully processing a wide range of algae, protozoa and some other organisms is outlined as is the construction of a simple carrier to hold the filter in the critical point drying apparatus.
A method for staining incompletely dehydrated Araldite embedded biological material for light microscopy
Authors: Ansell P. L. , Marshall J.
Published: August 2011

A method is described for high contrast staining of Araldite embedded biological material that has a high water content due to erroneous dehydration.
A design for a simple electronic exposure meter for use with an electron microscope
Authors: Hills G. J. , Garner R. T.
Published: August 2011

A circuit has been developed to enable a simple yet sensitive exposure meter to be made for any electron microscope. The circuit is based around an inexpensive operational amplifier and will indicate a current range 10 −4−10 −11 A which corresponds from maximum illumination to a level of fluorescent screen illumination which is not detectable by eye. The exposure meter measures the charge on the fluorescent screen which is correlated to exposure time for photography.
Calculations and observations of the weak‐beam contrast of small lattice defects
Authors: Häussermann F. , Katerbau K. H. , Rühle M. , WILKENS M.
Published: August 2011

Dynamical many‐beam calculations were performed for the investigation of the diffraction contrast of small lattice defects observed in the electron microscope under weak‐beam conditions. A new computational method was developed with which the depth position zo of the defect and the thickness t of the foil were eliminated from the integration procedure of the differential equations. The contrast figures for dislocation loops which were inclined to the foil plane and for spherical inclusions were calculated for various excitation errors of the weakly excited beam and for various combinations of t and z0. The dependence of the contrast figures on z0 (‘depth oscillations’) were studied in detail. The results of the calculations are compared with experimental observations.
Evidence of dislocation dissociation in nearly stoichiometric tantalum carbide using the weak‐beam technique
Authors: Martin J. L.
Published: August 2011

The dissociation of dislocations in nearly stoichiometric tantalum carbide has been revealed by the weak‐beam technique; this is in agreement with the assumptions of a model of dislocation glide which had been derived for this compound. An order of magnitude of the partial spacing for the screw and the edge dislocation is 2·7 and 5.2 nm respectively, which leads to a stacking‐fault energy of about 170 mJ m−2 (erg cm−2).
Instrumental aspects of image analysis in the electron microscope
Authors: Mulvey T.
Published: August 2011

Methods of analysing electron micrographs are reviewed, with special reference to the needs of the practising electron microscopist. It is concluded that a simplified optical diffractometer and a projection ‘Periodograph’ can be particularly helpful, especially in biological investigations, to which extensive reference is made in the bibliography.
Defocusing phase contrast effects in electron microscopy
Authors: Johnson D. J. , Crawford D.
Published: August 2011

The theory of defocusing phase contrast is presented in terms of Scherzer's (1949) ‘integrated‐ray’ and Heidenreich's (1964) ‘single‐ray’ approach to spherical aberration; contrast transfer theory in terms of the Scherzer expression is included. Fine structure in transverse sections, negative stain and carbon film is shown to be the result of defocusing phase contrast. Examples of interpretative confusion are given for various macromolecular systems and quantitative support is found for a simplified ‘rule‐of‐thumb’ approach to phase‐contrast enhancement. High‐resolution studies of carbon‐fibre lattices demonstrate contrast reversal, hence it is possible to show that a practical expression for the phase change due to spherical aberration falls between the Scherzer and the Heidenreich terms; the defocus term is also found to require some adjustment.
The application of stereo‐techniques to electron micrographs
Authors: Hudson B.
Published: August 2011

It is demonstrated that the only requisite for a true three‐dimensional image is the presence of parallax in the stereo‐pair. This parallax is introduced into electron micrographs by tilting the specimen between exposures. After deriving the usual parallax equation several examples of the use of stereo‐measurements in electron microscopy illustrate the extra information retrieved by its use.
Resolution limits in the surface scanning electron microscope
Authors: Catto C. J. D. , Smith K. C. A.
Published: August 2011

An approximate theory is developed for estimating the limiting topographical resolution of the scanning electron microscope operating under certain idealized conditions. Limitations imposed by electron beam shot noise and electron diffusion effects within the specimen are considered for the case of an instrument incorporating a field emission source and in which there is ideal collection of the secondary electron signal. The specimen is assumed to be an homogeneous, isotropic solid with the beam incident normally to its surface.

It is estimated that, under these ideal conditions, the limiting resolution for a metallic specimen lies in the region of 1 nm. The possibilities of realizing a resolution of this order in a practical instrument are discussed.
Reconsideration of resolution in EM autoradiography using a biological line source
Authors: Gupta Brij L. , Moreton Roger B. , Cooper N. C.
Published: August 2011

Pieces of integument containing cuticle and hypodermal cells from the desert locust Schistocerca gregaria were pulse‐labelled to produce very narrow bands of radioactive chitin or resilin in the cuticle. Araldite sections of such cuticle were used as a radioactive line source to test the resolution and the possible effect of self‐absorption due to heavy metal staining of tissue‐sections in EM autoradiographs.

The approximate position of the source and the half‐distance of resolution (HD) in EM autoradiographs were determined by replotting the grain‐distribution in integrated form, producing a sigmoid‐shaped curve. The values of HD, the position of the source and the total number of grains were then used to superimpose a theoretical grain‐density distribution function on the experimental grain‐distribution histograms. The results confirm the line or very narrow band nature of the radioactive chitin or resilin in the tissue‐sections used.

When the source was resilin situated in the cuticle at about 1 μm from the cell‐surface, the values for HD were 130–180 nm in Ilford L4 and about 100 nm in Agfa‐Gevaert NUC 307. When the source was chitin situated just outside the cell surface, the corresponding value for HD in Ilford L4 was 85–125 nm. Possible adverse effects of electron beam damage on resolution in resilin‐containing samples are discussed.

In sections with radioactive chitin at the cell surface, the total number of grains in the cell‐side of the histogram was always significantly lower than that in the cuticle‐side. This marked asymmetry in grain distribution is considered to be indicative of much greater self absorption of beta‐particles from the tritium source, in heavily stained areas of the sections.
Plastic embedding and surface erosion techniques in soft tissue scanning electron microscopy
Authors: Alexander I. G. S. , Capicchiano P. M. , Ritchie B. C. , Maloney J. E.
Published: August 2011

The maintenance and true pictorial representation of the complex cyto‐architecture of tissues is the aim of the preparatory methods for scanning electron microscopy. The procedure employed eliminates complications associated with and due to the vacuum technology necessarily employed in electron optic instruments using a scanning beam. Tissues are fixed by vascular perfusion after the administration of heparin eliminating clot formation. The infusion of Araldite in the tissues prevents the collapse and disorganization of cells due to the combined effects of dehydration, the vacuum of the scanning electron microscope and the scanning beam. The specimens are scoured with acetone and the blocks cured, this latter process ensures their permanence; the standard procedures of gold coating, stub mounting and painting the bases of specimens with a colloidal silver solution are then carried out. The method allows magnifications of the order of 10,000 diameters and an acceptable resolution of some 50 nm is obtained. Cytoarchitectural features demonstrated by the method described are borne out by transmission microscopy.
About quantitative studies on the cerebral cortex*
Authors: Bonin Gerhardt Von
Published: August 2011

Among the well established facts we can count that the cortex increases in volume against the thalamus as well as against the basal ganglia as we go from smaller to larger forms, and that the thickness of the cortex does not increase uniformly with the size of the brain. The details of the size of the thalamus and of the basal ganglia are not satisfactorily established. The values for the brains of whales and of man, which were measured by several individuals, but by the same technique, agree very well with each other.

The number of cells in the cortex is not known with any degree of accuracy nor is the theoretical value of the grey cell coefficient well established. We advocate abandoning it, and replacing it with some measure of the number of synapses and/or the number of nerve fibres entering and leaving the cortex which can be established with greater accuracy.
Morphometric model, tissue sampling and test of stereologic procedures
Authors: Schroeder H. E. , Münzel‐Pedrazzoli S.
Published: August 2011

Stereologic point counting procedures were applied to estimate volumetric and numerical densities of tissue components residing in and forming a defined, simplified model of human marginal gingiva. This included the quantitative characterization of inflammatory cell infiltrates occurring in the epithelial as well as the connective tissue fractions of this model. The gingival tissue was harvested from defined intraoral sites of 9–15 year‐old children and processed under standardized conditions for light and electron microscopic observation. Random cross‐sections of this tissue were subjected to morphometric analysis based on a multistage sampling technique. At each sampling level, tests were performed to study (1) the suitability of different test systems, (2) the parameter variation within one and between different biopsies and (3) the comparability and reliability of the data obtained. Based on these tests, a morphometric technique was designed which resulted in an accurate estimation of the various parameters characterizing the model tissue. These parameters were the volumetric densities of the following constituents: oral epithelium, junctional epithelium and its leucocyte content, infiltrated and non‐infiltrated connective tissue, and its respective collagen fibre content. Infiltrated and non‐infiltrated connective tissue fractions were analysed for their respective volumetric and numerical densities of fibroblasts, neutrophilic granulocytes, monocytes, macrophages, small and medium‐sized lymphocytes, immunoblasts, plasma cells and mast cells. Sampling technique, test procedures and the design and results of the morphometric system are discussed.
Three‐dimensional histology; a 35 mm photographic method
Authors: Buchanan James W. , Chmielewski Stanley
Published: August 2011

Commercially available photographic equipment can be used to study specimens histologically in three dimensions. Thirty‐five millimetre colour transparencies are made of serial sections at desired intervals using a photomicroscope with a rotatable stage. The series of transparencies is projected in alternate fashion using two identical projectors linked by a dissolve control. The method has proved useful in anatomic and embryologie studies and presentations as well as in evaluation of pathologic material in which initial histology revealed an abnormality that could be clarified by three‐dimensional study.
Industrial applications of analytical electron microscopy
Authors: Jacobs M. H.
Published: August 2011

Some metallurgical studies carried out using a combined electron microscope — microanalyser (EMMA‐3) are described. A correction procedure is outlined for the quantitative X‐ray microanalysis of thin foils and small extracted particles. The applications discussed include (a) the examination of small crystals of carbides and spinel‐type oxides, (b) the analysis of small, single‐phase grains in splat‐quenched Al‐8%Fe, and (c) the identification of small ferrite grains within an austenite matrix by means of the convergent‐beam diffraction technique.
The use of thin specimens for X‐ray microanalysis in biology
Authors: Hall T. A. , Anderson H. Clarke , Appleton T.
Published: August 2011

The quantitative X‐ray microanalysis of ultrathin biological sections is exemplified by a recent study of the distribution of calcium in mineralizing cartilage and bone. The determination of calcium mass‐fraction in one microarea of a vesicle within a section of rabbit epiphyseal plate cartilage is presented in detail in order to display all steps of the processing of the data. Mass fractions are obtained from an equation which is approximate but which is adequately accurate for most cases of interest, specifically for ultrathin biological sections where the analysed area consists predominantly of organic matrix. We shall examine the physical assumptions behind the computation, and the extent to which these assumptions have been verified in the analytical microscope.

The purpose of this paper is to describe in detail the scheme of data collection and processing, which has now been in use for some years in the Cavendish Laboratory, for the measurement of local elemental mass fractions in thin biological specimens in instruments like the EMMA. After an outline of the physical theory we shall describe the handling of the data by means of working through an example of an actual measurement, and finally we shall add some comments, mainly precautions and reservations.
The application of analytical electron microscopy to the study of diseased biological tissue
Authors: Henderson W. J. , Chandler J. A. , Blundell G. , Griffiths C. , Davies J.
Published: August 2011

A routine method is described for X‐ray microanalysis of thin specimens of biological tissue containing mineral particles in cancerous growths. Such a method allows information to be obtained that relates pathological history to histology, electron microscopy and X‐ray microanalysis.

Classification of minerals is possible in a way that is not provided by bulk analysis.

The technique provides baselines of elemental data of minerals from various sources that may be used to classify particles found present in certain tumour growths.
Cooling bath for rapid freezing in electron microscopy
Authors: Umrath Walter
Published: August 2011

A cryogenic device, freely accessible from the top, is described which allows rapid cooling of specimens for electron microscopy by immersion into melting nitrogen.
The quantitative morphology of anisotropic trabecular bone
Authors: Whitehouse W. J.
Published: August 2011

Dimensional measurements have been made on a small number of tracings of low magnification scanning electron micrographs of trabecular bone sections, using the Quantimet Image Analysing Computer, with a view to expressing the properties of the local bone pattern in a quantitative manner.

Measurements of the orthogonally projected length of the boundary profile of the interface between bone and marrow were made as a function of the direction of projection, and these showed that the majority of the patterns were distinctly anisotropic.

A formula is proposed for calculating the mean widths of the trabecular bands as they appear on the surfaces of the sections, from the mean area of bone and the boundary profile length. As the validity of this formula is not theoretically evident, the results have been compared with direct experimental measurements, and found to agree very well.

The mean intercept lengths in the bone and in the marrow spaces have been calculated as a function of direction across the section. The polar diagrams of these lengths are found to have the shapes of ellipses, to an unexpected degree of accuracy. This makes it possible to express the departure from isotropy of the pattern by a single number, for instance by the ratio of the axes of the ellipse.

The essential limitations of the methods used in these experiments are briefly discussed, in particular the impossibility of finding the exact symmetry properties of the surface patterns by experiments of this type.
A stereological method for calculating internal surface areas in structures which have become anisotropic as the result of linear expansions or contractions
Authors: Whitehouse W. J.
Published: August 2011

It is postulated that there may be classes of microstructures, having anisotropic internal features, which are related to internally isotropic structures by a coordinate transformation. Such structures are known to occur, for instance, in metallography when the bulk material has been subjected to deformation and there are many biological analogies.

The simplest case of coordinate transformation is investigated theoretically in this paper; that of a linear transformation in one dimension. Formulae have been found for the numerical constant which allows the density of the internal surface which separates two components of the material to be calculated from the border profile density on a plane section through the material.

Measurements on trabecular bone have suggested that it may be a structure of this type, and, on this assumption, the errors due to incorrectly assuming isotropy, when calculating the area of the surface which separates the bone from the marrow, are discussed.
A simple fracturing device for obtaining complementary replicas of freeze‐fractured and freeze‐etched suspensions and tissue fragments
Authors: Sleytr Uwe B. , Umrath Walter
Published: August 2011

Techniques for obtaining complementary replicas have already been shown to be valuable in aiding the interpretation of electron micrographs of replicas of specimens prepared by freeze‐etching and freeze‐fracturing techniques, and in the recognition of artefacts. This paper describes a simple and efficient method of obtaining complementary pairs of replicas of all types of specimen. Ordinary hollow brass rivets are used as specimen holders and are frozen in an end‐to‐end position using a special pair of forceps. Up to six rivets are placed in a device consisting of a hinged plate held together with clamps against the force of small springs. When the clamps are released, the pair of rivets are separated, fracturing the specimens. The device is easily adapted for use in any type of freeze‐etching or high vacuum apparatus. On the example of Saccharomyces cerevisiae the application of the technique to the detection of artefacts in complementary replicas are described. It was shown that the fibrils observed on cross‐fractured cell walls are produced by plastic deformation of cell wall components.
A simple device for obtaining complementary fracture planes at liquid helium temperature in the freeze‐etching technique
Authors: Sleytr Uwe B. , Umrath Walter
Published: August 2011

This paper describes a simple technique for obtaining complementary replicas of specimens fractured under liquid helium. Ordinary hollow brass rivets are used as specimen holders and are frozen in an end‐to‐end position. The pairs of rivets are placed in a device consisting basically of two concentric steel tubes which can be twisted and moved relative to each other guided by a peg‐in‐slot mechanism. The rivets are inserted while the device is under liquid nitrogen, and the device is then transferred to liquid helium and the tubes are moved so that the upper rivet is knocked away and falls into a collector. The device is then transferred back to liquid nitrogen, the fracture faces being protected from contamination throughout the process. Replicas are then obtained by any of the standard methods for specimens fractured under liquid nitrogen. The device has been tested using yeast cells as a model system, and some typical results are presented and discussed.
The interpretation of freeze‐etch replicas from plant cell walls
Authors: Parish G. R.
Published: August 2011

If a freeze‐etch replica is not completely clean, some problems may arise concerned with interpretation. These can be particularly misleading in the case of freeze‐etched cellulose microfibrils. The interpretation of such replicas is discussed, and the method of image formation from a clean replica is demonstrated.
An illumination system for specimen block knife alignment with the LKB ultrotome ultramicrotome
Authors: Wyatt John H.
Published: August 2011

The principle of using reflected light to align knife to specimen block proposed by Gorycki (1965) is applied to the LKB ultrotome. A small mirror mounted on the knife holder base at an angle of 5° reflects light from the fluorescent lamp on to the front of the knife from where it is reflected on to the block face. The width of this band of reflected light at the bottom edge of the block is reduced as the knife approaches the block. At the point at which it is no longer visible the block is 2–3 μm from the knife. When the knife is parallel with the block in the cutting plane the line of reflection is parallel with the knife edge. Using reflected light facilitates alignment between block and knife and makes closing the gap prior to the initial cut a simple and certain operation.
A simple method of preparing 1–2 μm sections of large tissue blocks using glycol methacrylate
Authors: Sims B.
Published: August 2011

The method described allows the production of 1–2 μm sections of large tissue blocks. Sections can be obtained from conventional routine histological microtomes. The hydroxyethyl methacrylate is compatible with a wide range of fixatives and a variety of standard histological staining techniques may be applied. Small biopsies, e.g. renal, liver, jejunal etc., can be processed cut and stained within 24 h of receipt. Blocks and stained sections show no apparent deterioration over the 4 years in which the technique has been employed in this laboratory.
Perfusion fixation of the kidney of the domestic fowl
Authors: Rothwell B.
Published: August 2011

A technique is described for the fixation of the fowl kidney using vascular perfusion through the renal portal system. This technique should be generally applicable to avian species.