bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron diffraction on frozen hydrated polysaccharides
Authors: Chanzy H. , Guizard C. , Vuong R.
Published: August 2011

Electron diffraction data were obtained on three different frozen hydrated polysaccharide specimens: pustulan single crystals, algal cell wall fragments consisting of β, 1–3 xylan, and fungal microfibrils of β,1–3 glucan. For pustulan, the diffraction pattern yields two of the crystalline lattice parameters of this polymer: a = 2·44 nm and b= 1·77nm. The results with β,1–3 xylan and β,1–3 glucan are identical to already known X‐ray data, but also provide information on the molecular orientation at the micrometre level. In all three cases the hydrated electron diffraction diagrams are compared with the corresponding diagrams of the dried specimen.
Beam heating of a moderately thick cold stage specimen in the SEM/STEM
Authors: Talmon Yeshayahu , Thomas Edwin L.
Published: August 2011

A mathematical model for the electron beam heating problem of thick specimens (thickness less than the electron range) in the cold stage of SEM/STEM is proposed, and an analytic solution for the problem is provided. The use of the model is demonstrated by calculating the maximum temperature rise in a red blood cell mounted on a metallized polymer film, for a large range of operating conditions. With a probe size of 20 nm and probe current of less than 10 nA the temperature rise was found to be on the order of 5 K. This temperature rise increases proportionally to the probe current if all other variables are kept constant. Although the amount of energy dissipated in thick specimens is large relative to thin films, temperature rise can be minimized by mounting the thick sample on a metal stub or on a metallized film thus providing large heat transfer area through a high thermal conductivity substrate. Of course our calculations only have meaning if the specimen is in good thermal contact with the underlying support.
Bright field and hollow cone dark field electron microscopy of palladium catalysts
Authors: Freeman L. A. , Howie A. , Treacy M. M. J.
Published: August 2011

The usefulness of hollow cone dark field imaging for the study of Pd catalyst particles on γ‐alumina substrates is investigated. By varying the depth of the specimen in the objective lens field, different Bragg reflections can be conveniently selected. Typically, about 70% of the particles appear in either the 111 or 200 Bragg reflections. Rather more particles can be visible in bright field because of other Bragg reflections as well as absorption effects. Size distributions in bright field and dark field are compared.
Photoelectron microscopy of cell surfaces
Authors: Dam Rudy J. , Nadakavukaren Karen K. , Griffith O. Hayes
Published: August 2011

Photoelectron micrographs of fixed, unstained, uncoated chicken embryo fibroblasts and the absolute photoelectron quantum yields in the 180–230 nm wavelength band of l‐fucose, d‐galactose, d‐glucose, n‐acetyl‐d‐glucosamine, and the sucrose polymer Ficoll are reported. The quantum yields of the saccharides are low compared to the reference dye, phthalocyanine, and fall in the same range as those previously measured for amino acids and membrane phospholipids. Photoelectron micrographs of the unstained and uncoated cells exhibit considerable surface detail. The photoelectron quantum yield data and the micrographs indicate that surface relief is the dominant source of contrast.
A device for dark field microphotography at low magnifications
Authors: Holzgraefe M. , Wolff J. R.
Published: August 2011

A simple illumination system is described that can be easily adapted to a laboratory stereomicroscope allowing for low power high contrast dark field surveying. The system uses commercially available fibre optic lamps, standard bright field objectives and produces a large, evenly illuminated field suitable for photography.
The analysis of fibre distributions in fibre reinforced materials*
Authors: Stroeven Piet
Published: August 2011

Different spacing factors and various orientation efficiency factors, applied to characterize the spatial distribution of the (steel) fibres in fibre reinforced cementitious materials, cannot constitute a sound basis for a mutual comparison of experiments or for an evaluation of experiments in a fracture mechanical sense.

This paper therefore presents a complete framework, based on geometrical probability theory, providing the investigator with methods to obtain three‐dimensional information from two‐dimensional images produced by X‐ray radiography. In this approach the fibres are assumed to be disposed in such a way that on the average a partially planar‐oriented structure is obtained. In addition, because of their high aspect ratio, the fibres are considered to be lineal features.

Two methods are elaborated, i.e. the feature counting technique and the method of directed secants. These operations have to be performed in the projection plane. Both methods yield data on the degree of inhomogeneity (segregation) and the degree of orientation (anisometry), as well as on spacing and orientation efficiency. By substituting design data in the presented formulae, this framework can also serve to characterize the spatial properties of the fibre structure in the designed mix.

The application of the theory is illustrated with the help of data that confirm the occurrence of segregation and preferred orientation effects due to vibration of the specimens.
Fifty‐nine tetrakaidecahedra as grain models
Authors: Hucher Monique , Grolier Jacques
Published: August 2011

A catalogue of the Schlegel graphs of all the three‐valent tetrakaidecahedra with faces having at least four edges and at most six edges is presented. These fifty‐nine polyhedra can be used for all purposes in stereology, either as morphological models of grains in microscopic studies of all kinds of materials where the unit cells are supposed in equilibrium, for the experimental and theoretical study of three‐dimensional packing and non‐packing problems or as a conceptual basis for the theoretical frequency computation of occurrences of the different types of polygons in materials observed in slides or polished sections.
A simple low‐cost critical point dryer with continuous flow dehydration attachment
Authors: Brown J. N.
Published: August 2011

A simple low‐cost critical point dryer with continuous flow dehydration attachment is described. By using the critical point pressure chamber for all fluid exchanges, loss of material and air‐drying artefacts due to specimen handling are reduced. An operating schedule that allows the optimum path around the critical point process to be followed is proposed.
Application of fluorescence microscopy in coal petrology and oil exploration
Authors: Teichmuller M. , Wolf M.
Published: August 2011

Fluorescence microscopy of polished surfaces allows more satisfactory study and identification of the hydrogen‐rich constituents (liptinites) that are the precursors of petroleum and which, in soft brown coals, fine‐grained clastic and carbonate sediments can hardly be detected using normal reflected light. Diagnostic properties of liptinites and lipoid substances are their colour, intensity and variation of fluorescence (both colour and intensity) during irradiation which may allow distinction of organic and inorganic constituents in coals and bituminous shales. Fluorescence properties also change with increasing degree of diagenesis (‘rank’) of organic matter, although organic constituents cease to fluoresce beyond the medium‐volatile, bituminous‐rank stage. In this paper fluorescence microscopy as a means of studying the rank of coaly and lipoid substances in sedimentary rocks is discussed after a brief description of the method. The influence of facies on fluorescence properties is considered and the relative merits of fluorescence and reflectance measurements in different parts of the rank range are discussed. The importance of fluorescence microscopy in petroleum exploration is also emphasized. Finally, consideration is given to the future of fluorescence microscopy in relation to the ‘bituminization range’ of coalification which has important implications to both petroleum generation and to the coking and hydrogenation of coals.
The variation of the microhardness and reflectance of coal under conditions of oxidation simulating weathering
Authors: Nandi B. N. , Ciavaglia L. A. , Montgomery D. S.
Published: August 2011

This investigation indicated that different types of microhardness impressions were obtained on different ranks of coal by the Vickers microhardness test and that oxidation transformed the plastic state of fresh vitrinite into an elastic state. The transformation occurred rapidly in high volatile coals, but more severe oxidation was required to cause this change in low volatile coals. Reflectance also increased with oxidation. As oxygen penetrated a coal particle, it produced changes in the microhardness impressions and in the reflectance which were rather abrupt at some point between the outer surface and the centre. The microhardness impressions combined with the reflectance data were capable of defining the boundary where rapid transition from the plastic to elastic state occurred. Movement of this boundary made it possible to follow the oxygen penetration into coal particles.
‘Large’ and ‘small’ nuclear pore complexes: The influence of glutaraldehyde
Authors: Willison J. H. M. , Rajaraman R.
Published: August 2011

Aliquots of lymphocyte cell suspensions were pretreated according to the following three schedules before freeze fracturing: (a) prefixed with 2% glutaraldehyde before infiltration with 25% glyercol in medium RPMI‐1640; (b) frozen in medium RPMI‐1640 without additional pretreatment; and (c) frozen after pretreatment with 25% glycerol in medium RPMI‐1640. The diameters of the fractured nuclear pore complexes of cells prefixed with glutaraldehyde were normally distributed within the range 70–120 nm (median 90 nm). The nuclear envelopes of 66–75% of cells processed through schedules b and c, which omitted glutaraldehyde fixation, had 70–120 nm diameter pores, while the remainder had pores with diameters in the range 120–175 nm. The large pores were structurally similar to the smaller pores except for their dimensions. These results indicate that glutaraldehyde gives rise to shrinkage of the larger pores to the minimum, smaller, diameter. Apparent orifices of at least 30 nm diameter were sometimes observed at the centres of these large pore complexes. We propose that the variation in pore diameters may indicate opening and closure of this orifice, and that the widely reported ‘central granule’ of the nuclear pore complex corresponds with the orifice in a closed configuration.
Detection of Concanavalin A receptors by affinity to peroxidase and iron dextran by scanning and transmission electron microscopy and X‐ray microanalysis
Authors: Baccetti Baccio , Burrini Anna G.
Published: August 2011

A method of cell surface mapping has been developed on spermatozoa. Concanavalin A binding sites have been simultaneously revealed both by peroxidase DAB reaction and by iron dextran coupling. The areas are examined in scanning and transmission electron microscopes and submitted to electron probe X‐ray microanalysis.
Staining reticulin in beef dorsal lymph nodes for electron microscopy
Authors: Voyle C. A.
Published: August 2011

Reticulin has been classically identified by virtue of its argyrophilia, as observed by light microscopy. The similarity between reticulin and collagen has been a matter for considerable discussion. This paper describes the application of a silver‐staining procedure which demonstrates the fine structure of these fibres when studied by electron microscopy, giving a direct indication of the morphological identity of the argyrophilic structures, i.e. reticulin, with collagen. Beef lymph node tissue was used for this study.
Signal to noise enhancement in dark field electron micrographs of vasopressin: filtering of arrays of images in reciprocal space
Authors: Ottensmeyer F. P. , Andrew J. W. , Bazett‐Jones D. P. , Chan A. S. K. , Hewitt J.
Published: August 2011

A technique for filtering arrays of images of dispersed molecules is presented which takes advantage of the crystallographic properties of regular arrays and the rapidity of optical treatment. A filter in reciprocal space consisting of perforations in a square or rectangular lattice, as determined by the image arrangement, reduces noise and averages the images simultaneously by transmitting only regularly recurring image structure. The filter is universal for all image contents and introduces no additional biases. The signal to noise improvement approaches √N for N independent images of the same molecule in the same configuration. The procedure, which is simple, rapid and inexpensive, is demonstrated with the aid of dark field electron micrographs of the protein vasopressin and combined with iodination of that molecule to elucidate its structure.
Optical properties of graphite
Authors: Kwiecinska B. , Murchison D. G. , Scott E.
Published: August 2011

Reflectances of natural surfaces of fresh graphite peels parallel to the (0001) plane have been measured throughout the visible spectrum using only slightly modified microphotometric equipment and measuring procedures. There is satisfactory agreement between the level and trends of reflectances of graphites from different metamorphic environments throughout the spectrum, the maximum relative difference in the most difficult conditions in the blue region being approximately 4%. There is less good agreement between the dispersion curves of derived refractive and absorptive indices for which the maximum relative difference is as high as 12% in the red region. At 546 nm, the wavelength at which optical data in organic petrology are principally reported, relative errors below 1% for all the optical parameters were attained between samples. Values of Rair = 31.40%, Roil = 17.85%, n = 2.49 and k = 0.61 compare well with other recent estimates and although there is still not complete agreement between laboratories, the true constants for graphite at this wavelength must lie close to these values. The use of semi‐graphites as metamorphic indicators in a scale with graphite as the end‐point is considered, as are the implications on such a scale of the current use of natural surfaces for reflectance measurement, because of an inability of many laboratories to produce specularly polished surfaces of semi‐graphites and graphites of high uniform quality.
The effect of six fixatives on the areas of rabbit neurons and rabbit and rat cerebral slices
Authors: Deutsch K. , Hillman H.
Published: August 2011

Cell bodies of cerebral neurons from rabbits were isolated by hand, transferred to a microscope slide in a ‘199’ medium, and the projected areas of their cytoplasm and nuclei were measured. In sixty‐four cells there was a strong correlation between the projected areas of the cytoplasm and the nuclei (r=0.66, P < 0001), and the ratio of the projected areas was 11.6. The medium was then replaced by the following fixatives: formalin (10% v/v), Bouin's, Carnoy's, Susas, glutaraldehyde (5% v/v), and osmium tetroxide (1% w/v). Cerebral slices were obtained from the grey and white matter of rabbit and rat, and were also measured before and after treatment with similar fixatives. Relative to the unfixed areas, glutaraldehyde and osmium tetroxide had no significant effect on the projected areas of isolated cells, Carnoy's fixative shrunk the areas of the cytoplasm by means of 16.26%, Bouin's by a mean of 49%, and Susas by a mean of 65%. The shrinkage of the cytoplasm and the nuclei was not significantly different from that of the nuclei for each of these three fixatives individually, but with formalin the mean shrinkage of the cytoplasm was 46% while the nuclei did not shrink significantly.

Using the same fixatives the effect on the areas of the cerebral slides from rabbit and rat were as follows: glutaraldehyde and osmium tetroxide caused no change in area; Carnoy's, formalin and Bouin's fixative diminished the areas by a mean of 10–20%, and Susa's by a mean of 35%.

It was concluded that a particular fixative often caused a different degree of shrinkage to the cytoplasm, nuclei and cerebral slice. In general, the lower the osmotic pressure of the fixative, the less shrinkage it induced.
A method for handling free cells through critical point drying
Authors: Barrett L. A. , Pendergrass R. E.
Published: August 2011

A method is described in which free cells were dehydrated in suspension, put on double‐coated tape attached to a specimen stub and dried from CO2. The method provides a simple, fast means of handling free cells when using the critical point method of drying.
Effect of Fixation on Cell Membrane of Early Embryonic Material as Observed On Freeze‐Fracture Replicas
Authors: Stolinski C. , Breathnach A. S. , Bellairs R.
Published: August 2011

Some cell membranes of chemically fixed, cryoprotected and freeze‐fractured chick embryonic material are characterized by 150–250 nm diameter circular smooth blebs. No such blebs are observed, however, on cryoprotected, rapidly frozen but not chemically fixed samples. It is concluded, therefore, that fixation with buffered glutaraldehyde and not cryoprotection with glycerol is mainly responsible for the appearance of the blebs. The suggestion that blebs represent a budding virus is not tenable, since blebs are present in material obtained from virus free embryos.
Localization of Protein in the Rabbit Yolk Sac Splanchnopleur By Two Methods of Autoradiography
Authors: Williams E. W.
Published: August 2011

The distribution of protein labelled with radioactive iodine or tritium within cells of the yolk sac, after it was injected into the uterine lumen of pregnant rabbits, was studied by means of two autoradiographic techniques, one using the classical loop method of Caro & vanTubergen (1962), and the other using Normandin's direct deposition method (Normandin, 1973). The resolution of the latter method was found to be approximately 10 times more sensitive than that of the former (Hemmings & Williams, 1976); thus rendering the site of labelled protein easier to locate. Both techniques are reproducible, but the direct deposition method is easier to handle. Location of protein was detected within vacuoles, and sometimes free in the cytoplasm.
A Method For Photographic Densitometry of Scanning Electron Micrographs
Authors: Hatami‐Monazah Houshang , Boss Jeffrey
Published: August 2011

A method is described for the study of the surface structure of mammalian chromosomes. Densitometric analysis of scanning electron micrographs permits the detection of regularities where overlying material obscures these in directly inspected micrographs. The method has been applied to the study of mammalian chromosomes.
A Simple Method For The Determination of Thickness and Grain Size of Deposited Films as Used On Non‐Conductive Specimens For Scanning Electron Microscopy
Authors: Røli Jakob , Flood Per R.
Published: August 2011

Latex spheres in linear aggregates allow coating materials to accumulate only along their free surface. Transverse to the axis of the aggregate the diameter of a sphere will increase corresponding to the uncoated diameter plus twice the film thickness. Parallel to the axis of the aggregate the diameter of terminal spheres will increase with one film thickness, whereas the diameter of ‘enclosed’ spheres remain constant. By mounting uniform latex spheres on standard electron microscope‐grids and comparing the transverse to the parallel diameter of linearly aggregated spheres in the transmission electron microscope, the film thickness produced by various coating procedures has been determined. The grain size and crack formations in the films may be examined simultaneously.
Metallic Deposition in Specimens Presenting Cavities Using the Sputter Coater
Authors: de Slobodrian Maria‐L. Ligatto , de Estable‐Puig Rosita F. , Estable‐Puig Juan F.
Published: August 2011

Local charging effects in small biological cavities during SEM observation are apparent, even after sputter coating. Using an experimental model, the thickness of metallic coating at the bottom of cylindrical cavities has been measured and found to be less than 50% the thickness on the surface, depending on the relation of diameter to depth of the cylinder.
Electron Beam Heating Temperature Profiles in Moderately Thick Cold Stage STEM/SEM Specimens
Authors: Talmon Yeshayahu , Thomas Edwin L.
Published: August 2011

The analytic solution for the model of electron beam heating a moderately thick STEM/SEM specimen was used to calculate the steady state temperature profiles developed in the bulk of the specimen. For thin specimens one maximum in the temperature profile was found near the surface. The location of this maximum shifted away from the surface with increasing sample thickness. For specimens of thickness approaching the electron range two maxima were found: one close to the surface (as in thin samples) due to a ‘self‐insulating’ effect, and another maximum near the sample‐substrate interface—a result of very rapid increase in the energy loss by the electrons near their penetration range. These results are of particular interest for X‐ray microanalysis where high beam currents are used, resulting in potentially large temperature rises in the bulk of the specimen.
A New Freeze‐Etch Unit For Freeze‐Etch Rotary Shadowing, Low Temperature Freeze‐Fracturing and Conventional Freeze‐Etching
Authors: Elgsaeter Arnljot
Published: August 2011

A new freeze‐etch unit is described in which one can make replicas where hydrated, frozen, fractured specimens rotate during specimen heavy metal shadowing. The freeze‐etch unit can also be used to do low temperature freeze‐fracturing and conventional freeze‐etching.
Freeze‐Fracturing of Biological Material With A New Specimen Table
Authors: Nickel Elvira , Oebel Gertrud , Pscheid Peter
Published: August 2011

The freeze‐fracturing of various biological material is described with the aid of a modified Balzers specimen table. The advantages of this table are easier and faster handling of the specimens and a better thermal contact between the specimen and the cold stage.
Application of Statistical Methods in Quantitative Microscopy*
Authors: Nicholson W. L.
Published: August 2011

The purpose of the paper is twofold: first, to describe the aspects of quantitative microscopy where statistical ideas are being applied today; and second, to describe some ways in which more complete use of statistics could improve quantitative microscopy. The typical estimation problem in quantitative microscopy is described with emphasis on the modelling of variability in estimation. A Poisson field model for imbedded particulates is used to illustrate the value of theoretical treatments of variability. Sampling, estimation and multivariate analysis are cited as areas of statistics presently used by quantitative microscopists. The potential role of statistics in quantitative microscopy is discussed. Examples from particle sizing and characterization of alveolar lung structure illustrate the value of statistical ideas. Automatic image analysers, while making the collection of large amounts of data feasible, have complicated statistical estimation. An investigation of the size distribution of random chord lengths through alveolar chambers illustrates how statistical methodology must be modified for valid inference.
Special Specimen Preparation Methods For Image Processing in Transmission Electron Microscopy: A Review*
Authors: Horne R. W.
Published: August 2011

One of the important developments in quantitative electron microscopy has been the application of optical and computer imaging methods to electron micrographs. In general these techniques of image analysis have been applied to electron micrographs from isolated biological structures prepared in the presence of various negative stains. To make full use of image processing techniques there are obvious advantages in preparing suitable specimens containing large areas of repeating features. However, the number of naturally occurring biological specimens exhibiting crystalline or paracrystalline features suitable for high resolution electron microscopy and subsequent image analysis is relatively small.

Some recent experiments on the in vitro formation of crystalline and paracrystalline arrays from highly concentrated and purified isometric, filamentous and rod‐like viruses is reviewed. The problems associated with the preparative procedures for producing two‐dimensional and three‐dimensional crystalline arrays are discussed together with the possibility of extending the negative staining‐carbon film method for studying the gradual dissociation or assembly of viral components.
A simple laser microbeam system for cell irradiation
Authors: Marchant Harvey J.
Published: August 2011

A simple adaptation to a vertical illuminator is described to allow a laser beam to be directed into the light path of a microscope where it is focused by the objective lens and used for cellular microsurgery. Any microscope to which a vertical illuminator can be fitted could similarly be used to micro‐irradiate specimens with a laser.
Quantitation in biological X‐ray microanalysis, with particular reference to histochemistry
Authors: Sumner A. T.
Published: August 2011

Thin specimens consisting of various light and heavy elements in gelatine have been subjected to X‐ray microanalysis to determine the relationship between the number of X‐ray counts for a specific element expressed as a percentage of the continuum (the percentage counts) and the concentration of that element. For light elements, the relationship between the percentage counts and concentration is strictly linear. For heavier elements, the relationship is not linear, because of the increase of the continuum counts with . If a correction is made for the effect of , heavy elements also show a linear relationship between percentage counts and concentration.

Within the limits of atomic number (Z = 56) and concentration (∼ 10%) studied here, it is shown that when X‐ray microanalysis is carried out on bulk specimens consisting of various elements in gelatine, the relationship between X‐ray counts and concentration for a particular element is linear.

The problems in quantitation of the results of X‐ray microanalysis caused by exogenous continuum and mass loss induced by irradiation are discussed. It is pointed out that when X‐ray microanalysis is used to study histochemical and other staining procedures, allowance must also be made for the reduction in concentration of other elements in the specimen as a result of the addition of the stain to the specimen.
Estimation of the mean caliper diameter of cell nuclei
Authors: Greeley Donald , Crapo James D. , Vollmer Robin T.
Published: August 2011

In morphometric studies of lung tissue, accurate determination of the total number of any specific type of cell, such as the endothelial cell, requires knowledge of the shape of the cell nucleus. This knowledge of shape is necessary to calculate the mean caliper diameter, which, in turn, is an indispensable element of the equation for the number of nuclei (therefore cells) per unit volume (Nv).

Nine human lung endothelial cell nuclei were therefore reconstructed in dental wax from serial sections and were found to be pleomorphic triaxial ellipsoids. Five of these approached an oblate shape. The axes of these nine nuclear models were directly measured and a computer program using numerical integration was written to determine the mean caliper diameters of these ellipsoids.

The estimated mean semi‐axes of the nine nuclei were (±SD) 5·98 ± 1·61, 3·61 ± 0·70, 1·36 ± 0·34; and the estimated overall mean caliper diameter for the total population of human lung endothelial nuclei was 7·97 ± 1·27 μm.
Estimation of the mean caliper diameter of cell nuclei
Authors: Crapo James D. , Greeley Donald A.
Published: August 2011

Morphometric studies of lung tissue in which the number of cell nuclei (therefore cells) are determined require the determination of the mean caliper diameter for each type of cell nucleus studied. We determined the mean caliper diameter for the five most common classes of cells found in the gas exchange portion of rat lung. This was done by serially sectioning through ten nuclei from each class of cells and then constructing wax models of the nuclei. Most of the nuclei were found to be triaxial ellipsoids and direct measurements taken from the models were used to calculate the following estimated overall mean caliper diameters (±SE): alveolar type I nuclei, 7·97 ± 0·2 μm, alveolar type II nuclei, 6·77 ± 0·19 μm, interstitial cell nuclei, 7·64 ± 0·35 μm, endothelial cell nuclei, 7·22 ± 0·19 μm, and alveolar macrophage nuclei, 7·65 ± 0·17 μm.
A film holder for use with the Vickers M85/M86 microdensitometer
Authors: Sumner A. T. , Finlayson D.
Published: August 2011

A film holder is described which allows the Vickers M85/M86 microdensitometer to be used to make transmission and density measurements on images on photographic film. This facility is particularly useful for applications such as making profiles of fluorescent banded chromosomes.
The use of simple image analysers in lung morphometry
Authors: Langston Claire , Thurlbeck William M.
Published: August 2011

We have used several commercially available image analysers to measure the inter‐alveolar wall distance on histologic sections of animal and human lungs. Inter‐ and intra‐observer variations using these machines are small, and very similar results are obtained using different machines. The recorded result depends on the magnification used since higher resolution produces more intercepts. The plane in which the measurement is made affects the result, representing the difference in shrinkage from fixed tissue to stained slides in the horizontal and vertical planes. Machine‐measured inter‐alveolar wall distances are smaller than those measured by humans because of the spurious intercepts made by the image analysers. However, the measurements are highly correlated and human‐measured inter‐alveolar wall distances can be closely predicted from the machine‐measured ones. The number of alveoli per unit volume is less well predicted from the average inter‐alveolar wall distance. Image analysers may possibly play a useful role in making similar simple measurements in other organs.
A simple method for maintaining relative positions of separate tissue elements during processing for electron microscopy
Authors: Stirling Charles A.
Published: August 2011

Molten (328 K) 20% gelatin is used as a ‘glue’ to hold together separate tissue elements or tissue elements that may be separated when cutting small blocks of tissue for plastic embedding. Standard aldehyde and osmium fixation, dehydration and epoxy embedding are compatible with this as is semi‐thin sectioning for light microscopy or thin sectioning for electron microscopy.
A practical electron spectrometer for chemical analysis
Authors: Joy David C. , Maher Dennis M.
Published: August 2011

This paper describes the basic parameters which have been considered in the design of a practical electron spectrometer optimized for micro‐chemical analysis in a 100 kV transmission electron microscope fitted with a scanning unit. Constructional factors and the operation of this spectrometer are discussed, as well as data acquisition. Typical results are presented to illustrate the operative modes. Both the prototype spectrometer and subsequent copies of it now are being utilized sucessfully in studies related to materials and biological sciences.
The microstructure of glass fibre reinforced polyester resin composites
Authors: Guild F. J. , Silverman B. W.
Published: August 2011

The microstructure of a composite material is commonly defined by the volume fraction occupied by the fibres. An attempt is made to gain a more quantitative definition of the microstructure of unidirectional glass fibre reinforced polyester resin using data derived from a Quantimet 720 Image Analysing Computer.

Sections to be examined under the Quantimet were cut perpendicular to the fibre direction and polished and etched such that the fibres appeared black. Values of the area covered by the fibres inside a small test cell, moved around the cross‐section in a contiguous grid, were determined; values for larger cells, made up of several adjacent test cells, were calculated from these.

Two methods of analysis are presented. The first method involves the determination of the variance of the values of fractional area covered for different cell sizes. Information regarding the nature of the fibre distribution is sought both from comparing the variance values with those predicted for the null hypothesis, a random distribution, and from examining the shape of a surface defined by a variance matrix.

The second method of analysis is based on the concept that a structure may be defined by its relationship to a structuring element. In the present case, structuring elements are squares and rectangles of empty space; the probability of finding such empty space is estimated. Information regarding the nature of the fibre distribution is sought from the probability matrices.
An iterative approach to the analysis of EM autoradiographs
Authors: Williams M. A. , Downs Angela
Published: August 2011

The errors inherent in EM autoradiography are discussed and certain of them deemed to be of particular practical significance in the quantitative assessment of preparations. A method is described for estimating the standard errors attributable to each of several sources of variation and thence for obtaining the overall standard error value to be attached to relative activity estimates obtained in the method of Downs & Williams (1978). In an appendix, a fully worked example is given illustrating clearly the strategy of the method and the magnitudes of error estimates that are to be attached to the final specific activity values.
A rapid preparation method for scanning electron microscopy of Lugol preserved algae
Authors: Bistricki T. , Munawar M.
Published: August 2011

A method that utilizes samples preserved and stored in Lugol solution is described. The samples are postfixed in osmium tetroxide, rapidly dehydrated in absolute ethanol and dried by the critical point drying technique. Aliquots of several samples can be processed simultaneously. Surface details of algae stored in Lugol and processed by this method are comparable to those found in conventional preparative procedures. No loss of nannoplankton or other small sized fractions have been observed.
Negative staining of freeze‐fractured envelopes of Escherichia coli K12
Authors: Nermut M. V. , Burdett I. D. J. , Williams L. D.
Published: August 2011

Envelope fragments of E. coli K12 have been produced by freeze‐fracturing ‘by hand’ and negatively stained after thawing. The outer leaflet of the plasma membrane disintegrated upon thawing whereas the outer leaflet of the outer membrane did not. Negative staining revealed the following structural features on the outer membrane fragments: (i) ‘grooves’ 4–6 nm wide, (ii) spherical particles 6–8 nm in diameter, (iii) ‘black dots’ 3–8 nm in diameter.

Treatment of cells with EDTA before freeze‐fracturing caused dilatation of grooves into holes eventually leading to fragmentation of the outer membrane.

A mutant strain deficient in two outer membrane proteins fractured exclusively through the outer membrane. The outer leaflets so obtained disintegrated upon thawing similarly as observed for the outer leaflet of the plasma membrane.
Practical approach to the estimation of the overall mean caliper diameter of a population of spheres and its application to data where small profiles are missed
Authors: Greeley Donald A. , Crapo James D.
Published: August 2011

In the morphometry laboratory a practical, accurate, and computationally simple procedure is needed when the estimation of the number of spherical particles per unit volume (Nv) is pursued. In addition, this procedure should be able to deal with the very real problem of profiles too small to be counted.

Two computationally simple methods, the size class analysis method and the mean profile diameter method, were examined in detail. Computer‐generated random profiles from various size‐distributions of spheres were analysed by both methods. The percents error to be expected with various sphere distributions with a relatively large number of missed small profiles were determined for both methods. The accuracy of the size class analysis method was poor when a significant number of small profiles were missed. In this situation the accuracy of this method could be greatly improved by applying a simple modification to the procedure. A size class was identified which was larger than the largest missed profile but smaller than the diameter of the smallest sphere in the population. All contributions of this size class and all smaller size classes were deleted from the computation of Nv.

The mean profile diameter method was found to be difficult to apply to distributions containing missed small profiles. Missed small profiles were handled more predictably by the modified size class analysis method.
The estimation of neuronal population density by a robust distance method
Authors: Aherne W. A. , Diggle P. J.
Published: August 2011

A new ‘nearest‐neighbour’ or ‘distance’ method of estimating neurone population density is introduced. The method was originally developed for ecological studies but can be imported into histology without significant modification; changes in population density can be estimated by inverting the measure of area per unit cell (the so‐called mean area). Its advantages include tests of randomness for the spatial distribution of the cells at issue and a robustness which can tolerate some departure from a random distribution pattern. To illustrate how the method is applied estimates of neurone density, in terms of ‘mean area’ per cell‐point, are made on a montage tracing of the human cerebellar dentate nucleus.
Preparation of resin embedded unicellular organisms without the use of fixatives and dehydration media
Authors: Pfaller Walter , Rovan Erwin
Published: August 2011

A method is presented for processing single cells for conventional ultrathin sectioning without the use of fixatives and dehydration media.

The cells were fixed by a physical method— spray freezing— which provides extremely high cooling rates, needs no pretreatment with cryoprotective agents and is therefore assumed to maintain the in vivo morphology of the cell. Hitherto cells prepared in this way have been investigated exclusively by freeze etching. To combine the advantages of this method with those of conventional ultrathin sectioning we have processed spray frozen cells with widely varying water contents (spermatozoa and lymphocytes) by freeze drying at 188 K and vacuum embedding.

When compared to conventional chemical fixation the differences found in ultra‐structural preservation of spermatozoa using this kind of preparation were confined to the arrangement of spermhead membranes and middlepiece structures. Lymphocyte structure was much closer to that known from chemical preparation, the only differences being a denser cytoplasm, denser mitochondrial matrices and thicker plasma membranes. These differences are probably due to the absence of eluating and dissolving effects present in conventional chemical preparations.

The ultrastructural preservation of spray frozen cells is not different after freeze etching or after freeze‐drying and vacuum embedding. This indicates clearly that drying and resin embedding does not produce artefacts and that structural preservation is therefore limited by the quality of cryofixation.

Therefore this method is considered a contribution to the problem of preservation of the in vivo assembly of cellular substructure. Furthermore it seems to be a potential basis for preparation of soluble or diffusible substances or cellular compounds which would be influenced by fixatives and dehydrating agents.
A simple time‐lapse unit
Authors: Briarty L. G. , Ramsey R. L.
Published: August 2011

A circuit is described for a time‐lapse unit which controls exposure interval, exposure time and an external light source.
The direct measurement of temperature changes within freeze‐fracture specimens during rapid quenching in liquid coolants
Authors: Costello M. J. , Corless J. M.
Published: August 2011

We have evaluated the cooling rates of specimens mounted in a variety of freeze‐fracture holders when plunged into a series of liquid coolants. These rates were measured using miniature thermocouples placed within the mounted specimens. The most rapid cooling rates were obtained using propane at 83 K as the coolant. When mounted on a newly devised ‘copper sandwich’ holder, specimen cooling rates in excess of 4500 K/s have been recorded.

A simple guillotine‐like device for quenching freeze‐fracture specimens under reproducible conditions is presented.
Low temperature scanning electron microscopy: a review
Authors: Echlin Patrick
Published: August 2011

Low temperature scanning electron microscopy is useful for morphological and analytical studies both in situations where low temperature techniques are used during specimen preparation and where low temperature stages are used for specimen examination and analysis. Examples are given of different low temperature specimen preparation techniques and how they may be applied to different types of specimen. There are still a number of problems associated with morphological identification in fully frozen‐hydrated samples and it is important to carry out parallel studies using more conventional transmission electron microscopy and light microscopy preparation techniques. A number of criteria are presented, some or all of which may be used to establish the existence of the frozen‐hydrated state.
Quantitative X‐ray microanalysis of diffusible ions in the skeletal muscle bulk specimen
Authors: Zierold K. , Schäfer D.
Published: August 2011

X‐ray microanalysis in the scanning electron microscope (SEM) is used to obtain information about the distribution and mobility of electrolyte ions in the skeletal muscle tissue. In order to localize the diffusible ions in their physiological state, so far as possible, any chemical fixative is avoided. A special ‘cooling chain’ preparation method is applied which enables studies on cryofractured bulk specimens at low temperatures.

Quantitation of the obtained X‐ray spectra is done by the use of glycerol‐treated tissue specimens which are incubated with electrolyte solutions of defined ion concentration.
Low temperature microspectrofluorometry: design of a ‘cold chamber‘
Authors: Giordano P. , Bottiroli G. , Prenna G. , Doglia S. , Balnini G.
Published: August 2011

Low temperature microspectrofluorometry allows an improvement of spectral resolution and an increase of fluorescence intensity. Suppression of fluorescence fading, or at least a marked reduction, is also obtained. A cooling chamber for microspectrofluorometric measurement is described which allows the cooling, under a microscope, of a biological sample down to liquid nitrogen temperature. Objectives with numerical apertures better than 1.0 and a magnification power up to 100 × can be used. Low temperature measurements on a histological sample are presented and discussed.
Structure determination of frozen, hydrated, crystalline biological specimens
Authors: Taylor Kenneth A.
Published: August 2011

Low temperature transmission electron microscopy can be used to study the structure of biological materials in the hydrated state. Spatial averaging techniques are necessary to overcome the radiation damage problems, and for this reason the techniques described are most applicable to crystalline objects. However, with thin, crystalline biological specimens there are no difficulties with preserving periodicity during the freezing process. Improved specimen preparation methods are described which achieve the production of very thin aqueous films with the hydrated specimen embedded in them. Defocused bright field images of frozen, hydrated protein crystals possess a surprisingly high contrast, presumably due to the difference in density of the protein and the aqueous phase. The techniques described have been used to study the crystal structure of hydrated catalase and the outermost cell wall of Spirillum serpens.
Particle size‐shape distributions: the general spheroid problem: II. Stochastic model and practical guide
Authors: Orive Luis‐M. Cruz
Published: August 2011

Mathematical relationships linking the size‐shape probability density function (pdf) of an infinite population of variable spheroids uniformly and isotropically scattered in space on the one hand, with the size‐shape pdf of the ellipses produced by a plane of section on the other, have recently been published (Cruz Orive, 1976). In the present paper, an independent model is developed for estimating the size‐shape bivariate histogram of a finite population of variable spheroids (either prolate, or oblate) uniformly and isotropically scattered within an arbitrary specimen, from the corresponding histogram of the elliptical profiles produced by an arbitrary section (or sections) through the whole specimen. The approach is stochastic, allowing the estimation of the variance‐covariance matrix of the ‘unfolded’ size‐shape spheroid frequencies.

The numerical reliability of the methods is checked by means of an example, whereas a practical guide illustrates and summarizes the unfolding procedure.

Natural ways of estimating spheroid properties, alternative to those found in Cruz Orive (1976), are presented.
Structure and function of frozen cells: freezing patterns and post‐thaw survival
Authors: Nei Tokio
Published: August 2011

Freezing patterns and post‐thaw survival of cells varies with different cooling rates. The optimal cooling rates, indicating the highest percentage survival, were different in yeast and red blood cells. A difference of freezing patterns was also noticed in preparations frozen above and below the optimal cooling rate for each cell, namely, cell shrinkage at lower rates and intracellular ice formation at higher rates, which showed similar trends in both the cells, even though there was some shifting of the optimum.

Ultra‐rapid freezing and addition of cryoprotectants are useful ways to minimize ice crystal formation and to cause such ice formations to approach the vitreous state. Ice crystals are hardly detectable in yeast cells as well as in erythrocytes, when these cells are frozen ultra‐rapidly in the presence of cryoprotective agents in moderate concentration.
Description and comparison of a new cleaning method of diatom frustules for light and electron microscope studies
Authors: Ma Josten C. W. , Jeffrey Lela M.
Published: August 2011

A general review is made of some of the methods commonly used by phyto‐planktologists in preparing and cleaning siliceous diatom frustules for light and electron microscopy. A comparatively simple and safe method using potassium persulphate and oven heating is described. Procedures for rinsing and mounting field samples are also included.
Enhancement of three‐dimensional appearance of freeze‐fracture images by reversal processing of electron microscopy sheet film
Authors: Schulz Werner W. , Reynolds Rolland C.
Published: August 2011

A method for reversal processing of electron microscopy sheet film is described. The procedure permits printing of freeze‐fracture pictures with black shadows without the necessity of preparing an internegative.
Signal to noise enhancement in a study of cell wall structure of Bacillus subtilis by interactive computation
Authors: Barrett A. N. , Burdett I. D. J. , Paton K. A.
Published: August 2011

This paper describes a technique for obtaining values of the width and variation in optical density of sectioned bacterial cell walls by interactive computational methods. Background ‘noise’ prevents accurate determination of cell wall boundaries from the data in a single scan line but the noise may be suppressed by averaging several consecutive scan lines. Although application of the technique is explained for sectioned bacterial cell walls, it is equally valid for similar situations where single line scans of electron micrographs are inadequate for precise determination of measurement.