bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron phase microscopy at 1 MeV with graphite crystal supports
Authors: Hines R. L.
Published: August 2011

Thin phase objects can be seen with good contrast with 1 MeV electrons if they are supported on a graphite crystal approximately half an extinction distance thick and viewed near 101 or 112 bend contours. The shape of unstained tobacco mosaic virus is easily seen with crystal phase contrast although it is almost invisible in a normal bright field micrograph taken with 1 MeV electrons. Phase contrast images of both TMV and 109 nm diameter latex spheres are observed to be bright against a grey background on one side of a 101 or 112 bend contour and dark against a grey background on the other side of the bend contour. For strong contrast the crystal diffraction vector g must have a component parallel to the beam. Contours such as 100 and 110 do not give strong phase contrast. The phase contrast images for biological material gradually increase in contrast as the specimens are irradiated by the electron beam. An electron exposure of about 104 electrons nm−2 is required to obtain maximum contrast.
Dye binding mechanisms in G‐banding of chromosomes
Authors: Sumner A. T.
Published: August 2011

The mechanisms whereby Giemsa is bound to chromosomes to produce G‐banding patterns have been studied. The magenta colour produced in chromosomes by Giemsa staining appears to be due to the same 2:1 thiazine‐eosin compound that precipitates from Giemsa solutions. This precipitate is formed in chromosomes in regions in which DNA phosphates occur at the correct distance apart to bind two thiazine molecules, which are subsequently able to bind to the same eosin molecule. Banding appears to be produced as a result of this precipitation occurring preferentially in hydrophobic regions of chromosomes.
Principles of TEM contrast simulations of inclined interfaces in homogeneous crystals
Authors: Gratias D. , Portier R.
Published: August 2011

The scattering matrix formalism used for the cases of translation and inversion boundaries is generalized to any type of interface in homogeneous crystals. The present method needs no arbitrary decompositions of the diffracting sets in the lower crystal. It leads to conditions of stacking‐fault‐like contrast, different from those proposed by Sutton & Pond, in agreement with experiments.
Description of a coverslip holder for, and observations on, the critical point drying of cell cultures
Authors: Evers P. , Robinson K. , Maistry L. , Higginson E.
Published: August 2011

The elimination of artefact during the preparation of cell cultures for scanning electron microscopy is difficult. Collapse of cellular projections, cytoplasmic cracks, perforations and fracturing of cell‐cell processes and cell‐substrate attachments occur during fixation, dehydration and critical point drying. Coating and storage may cause further artefact. A specimen holder which serves to minimize turbulence in the critical point dryer and which allows for the simultaneous processing of up to five coverslips, as well as a reproducible technique for the preparation of cell cultures are described.
A digital computer technique for orientation analysis of micrographs of soil fabric
Authors: Tovey N. K.
Published: August 2011

Several methods exist for quantifying soil fabric information from electron micrographs, but hitherto little interest has been shown in digital computer techniques. This paper describes such a method for the quantitative analysis of the orientation of clay particles in electron micrographs. The intensity distributions across several micrographs have been digitized at up to two million picture points and the data stored on magnetic tape. By examining the gradient of the intensity variations in two orthogonal directions, the resulting intensity gradient vector at each point can be determined. The information from all points in a micrograph can then be plotted as a rosette diagram, or used in a regression analysis to determine the equation of the ellipse which most closely approximates the actual rosette distribution. Finally an index of anisotropy can be estimated from the major and minor axes of the ellipse.
The use of high resolution microscope photometry in the discrimination of chromosome bands
Authors: Wayne A. W. , Sharp J. C.
Published: August 2011

High resolution microscope photometry was used to discriminate between regions of differing density along the length of a banded metaphase chromosome. It has been demonstrated that photometry can be performed on such material to give a higher level of effective resolution than has hitherto been obtained. Using conventional visible light microscope photometry it is possible to obtain useful comparative densitometric information from measurements performed at a point separation equivalent to 0.05 μm in the object plane. The practical details of the equipment employed and the theoretical basis of the study are discussed.
Stereological formulae based upon planar curve sections of surfaces in space
Authors: Miles R. E.
Published: August 2011

Attention is paid to the random curve (or curves) of intersection of a fixed surface in a spatial specimen by a random plane through that specimen. A formula is given for the expectation of the integral of a general function of curvature along that curve. This in turn yields a large family of formulae for stereological ratios.
On the use of the geodesic metric in image analysis
Authors: Lantuejoul C. , Beucher S.
Published: August 2011

Let X be a phase in a specimen. Given two arbitrary points x and y of X, let us define the number dx(x, y) as follows: dx(x, y) is the greatest lower bound of the lengths of the arcs in X ending at points x and y, if such arcs exist, and + ∞ if not. The function dX is a distance function, called ‘geodesic distance’. Note that if x and y belong to two disjoint connected components of X, dx(x, y) = + ∞. In other words, dx seems to be an appropriate distance function to deal with connectivity problems.

In the metric space (X, dx), all the classical morphological transformations (dilation, erosion, skeletonizations, etc.) can be defined. The geodesic distance dx also provides rigourous definitions of topological transformations, which can be performed by automatic image analysers with the help of iterative algorithms.

All these notions are illustrated with several examples (definition of the length of a fibre; automatic detection of cells having at least one nucleus, or having exactly a single nucleus; definitions of the geodesic centre and of the ends of a particle without holes, etc.). As an application, a general problem of segmentation is treated (automatic separation of balls in a polished section).
Resolution effect on the stereological estimation of surface and volume and its interpretation in terms of fractal dimensions
Authors: Paumgartner D. , Losa G. , Weibel E. R.
Published: August 2011

Estimating surface and volume density of subcellular membrane systems at different magnifications yield different results. As the magnification is increased from × 18,000 to × 130,000 the estimates of surface density of endoplasmic reticulum and inner mitochondrial membranes increase by a factor of 3, whereas that for outer mitochondrial membranes increases only by 20%. The estimate of volume density of endoplasmic reticulum also increases by a factor of 3. No further increase is observed at magnifications above × 130,000 which is therefore called critical magnification. The findings are interpreted on the basis of the concept of fractals proposed by Mandelbrot, and the fractal dimensions of the membrane systems considered are estimated. This can lead to the derivation of resolution correction factors which permit measurements obtained at any magnification to be converted to estimates at critical magnification. These findings may explain, at least in part, the large discrepancy in the estimates of the surface of cytomembranes found in the literature.
Optimizing sampling efficiency of stereological studies in biology: or ‘Do more less well!‘
Authors: Gundersen H. J. G. , ØSterby R.
Published: August 2011

The aim of the sampling design for stereology is to obtain the maximal amount of quantitative structural information at a given total cost or effort. Principles of such optimal designs are discussed and methods for generating them are illustrated by a biological example. In general, the variation between different individuals—the biological variation—is the major determinant of overall efficiency, whereas the variation between single microscopic features is unimportant. It follows that the expenditure of time and/or money in order to increase the precision of the individual measurements is irrational in almost all studies where the emphasis is on the biological results.
Morphological analysis of sintering
Authors: Chermant Jean‐Louis , Coster Michel , Jernot Jean‐Paul , Dupain Jean‐Louis
Published: August 2011

In this paper mathematical morphology is applied to describe the microstructural changes of metal powders during sintering. We show which morphological parameters are accessible to rapid and automatic methods. Of these parameters the change in the connectivity number and the concavity number enable the various stages of sintering to be distinguished. Moreover the growth of the necks between powder particles can be followed using a morphological method of analysis to reveal them. This method can be applied to the investigation of the microstructure of any material consisting of convex grains.
Automated pattern analysis of growing rat lung
Authors: Keller Hj. , Burri P. H.
Published: August 2011

This study discusses the use of automatic image analysis equipment in quantifying the structural changes during growth of rat pulmonary parenchyma. At birth the latter consists of primary septa delineating a coarse structure of alveolar sacs which are then subdivided into alveoli by outgrowth of secondary septa from day 4 onwards. This process is followed by internal restructuring of the septa in the third week which further increases the surface area by formation of small irregularities.

A partial quantification of these processes was obtained by measuring the full set of parameters available with the Quantimet 720 using light microscopy on rat lung sections at the ages of 4, 10 and 21 days. This yielded basic data, e.g. volume and surface densities of air space and tissue. Using closure and opening as preprocessing operations made it possible to split up such parameters into contributions from different ranges of detail resolution.

The mass of eighty‐four variables obtained could be reduced to six independent factors by Factor Analysis techniques. Interpretation through their correlation with the original variables yielded septal surface, septal volume, minor and major axes of the large air spaces, and surface detail as important structural descriptors explaining 70% of total variance. The remainder is attributed to between‐field variations.
Microemulsions and electron microscopy
Authors: Biais J. , Mercier M. , Bothorel P. , Clin B. , Lalanne P. , Lemanceau B.
Published: August 2011

The replicas obtained from various microemulsions prepared under different freezing conditions were observed by electron microscopy. The difficulties encountered in obtaining proper freezing may be related to the peculiar properties of such systems. A proposal is made for using systems with particular physical characteristics, which preserve the micellar structure during freezing, instead of looking for perfect freezing conditions. This proposal proved to be successful.
Adhesion of particulate specimens to support films for electron microscopy: a model system for assessing the surface properties of support films, and its application to chromatin particles
Authors: Woodcock C. L. F. , Frado L‐L. Y. , Green G. R. , Einck L.
Published: August 2011

Silica microspheres bearing a known surface charge were used to test the adhesive properties of support films and support film treatments commonly used in the electron microscopy of particulate specimens. Adhesion was strongly correlated with surface charge, negatively charged microspheres binding well only to positively charged support films and vice versa in solutions of low ionic strength. This charge dependency could be overcome by increasing the ionic strength to about 100 mmol with monovalent cations; under these conditions, it was not necessary to provide an oppositely charged film surface to obtain adhesion. Chromatin particles (nucleosomes) which have a net negative charge, behaved very much like the negatively charged silica with respect to adhesion, confirming that the microspheres provided an accurate indication of support film surface properties. The chromatin particles showed dramatic structural changes under conditions when adhesion was either poor, or very strong, indicating the need for careful selection of binding conditions for delicate biological specimens. A new and simple method for pretreating carbon films to improve adhesion was developed, and a preliminary account of this technique is presented.
The examination of fresh or living plant material in an environmental scanning electron microscope
Authors: Danilatos G. D.
Published: August 2011

An environmental scanning electron microscope (ESEM) has been developed which allows the examination of specimens at pressures up to 6 kPa. This new technique has been applied to the examination of live and wet plant specimens. The results are compared with those obtained using similar material that has been dehydrated or prepared by conventional techniques. The plant materials can survive the hypobaric pressure and beam irradiation, especially if the latter is carefully controlled.
A method for determining the three‐dimensional form of active flagella, using two‐colour darkground illumination
Authors: Woolley D. M.
Published: August 2011

Using darkground illumination, and placing colour filters over half the field, one can make a single, highly resolved, colour micrograph of a filamentous object (such as a mammalian sperm tail) from which it is possible to get a complete and unambiguous three‐dimensional reconstruction.
A simple method for the use of gallocyanin‐chrome alum as an electron stain*
Authors: Sandritter W. , Riede U. , Kiefer G.
Published: August 2011

A method has been elaborated for the demonstration of DNA in the electron microscope. The method uses glutaraldehyde fixed tissue pieces from which RNA has been removed by incubation with RNase. DNA is stained by gallocyanin‐chrome alum in the tissue block. Embedding and cutting is done in the usual manner. The method is based on histochemical observations at the light microscope level which show sufficient specificity and a good stoichiometry of the staining reaction.
Preparation and assessment of frozen‐hydrated sections of mammalian tissue for electron microscopy and X‐ray microprobe analysis
Authors: Ross A. , Sumner A. T. , Ross A. R.
Published: August 2011

A system is described for preparing and examining frozen‐hydrated sections of mammalian tissue, using commercially available equipment. An essential part of the system is a liquid nitrogen cooled vacuum transfer system. Minor modifications were required to interface the various manufacturers' equipment, and also to facilitate handling of specimens in the cryoultramicrotome. A variety of criteria indicates that our sections are truly frozen‐hydrated, but negligible image contrast in the sections is a serious problem both for morphological work and X‐ray microanalysis.
The microscopes of Antoni van Leeuwenhoek
Authors: van Zuylen J.
Published: August 2011

The seventeenth‐century Dutch microscopist, Antoni van Leeuwenhoek, was the first man to make a protracted study of microscopical objects, and, unlike his contemporary Robert Hooke, he viewed by transmitted light. Leeuwenhoek made over 500 of his own, curious, simple microscopes, but now only nine are known to exist. The exact nature of the lenses Leeuwenhoek made, has for long been a puzzle. The existing microscopes have now been examined in detail, and their optical characteristics measured and tabulated. It is proposed that the lens of highest magnification, x 266, was made using a special blown bubble technique.
Minimizing sampling error in transmission electron microscopy
Authors: Lewin Jackie F.
Published: August 2011

The last few years have seen an increase in the use of electron microscopy, both for research and as a diagnostic tool in pathology. With the increase in the number of specimens being used for diagnostic purposes, limitations due to sampling error have become apparent. The method described is designed to help overcome this problem.
A simple, inexpensive apparatus to allow sequential perfusion of an animal with several different fluids without an intervening drop in perfusion pressure
Authors: Bower A. J.
Published: August 2011

An inexpensive, versatile perfusion apparatus which allows the sequential perfusion of several fluids into an animal without an intervening drop in perfusion pressure is described.
An integrated electron and optical metallographic procedure for the identification of precipitate phases in type 316 stainless steel
Authors: Slattery G. F. , O'Riordan P. , Lambert M. E. , Green S. M.
Published: August 2011

A sequential and integrated metallographic procedure has been developed and successfully employed to differentiate between carbide, sigma, chi, Laves and ferrite phases which are commonly encountered in type 316 austenitic steel. The experimental techniques of optical and electron microscopy to identify these phases have been outlined and provide a rapid and convenient method of characterizing the microstructure of the steel. The techniques sequence involves selective metallographic etching, Nomarski interference microscopy, scanning electron microscopy, energy dispersive microanalysis, transmission electron microscopy and electron diffraction.
A freeze‐etch electron microscopic study of liquid propane jet‐frozen human erythrocyte membranes
Authors: Espevik Terje , Elgsaeter Arnljot
Published: August 2011

Freeze‐etch electron micrographs of haemolysing erythrocytes and isolated erythrocyte membranes frozen using a liquid propane jet‐freezer reveal fracture faces very different from those seen after conventional freezing by dipping the specimens into partly solidified Freon 22. Instead of the rather smooth extracellular fracture faces found after conventional specimen freezing, extracellular fracture faces exhibiting large amounts of fibre‐like structures are seen after liquid propane jet‐freezing of these specimens. No such structures were found in normal red blood cells. When isolated erythrocyte membranes are frozen under conditions favouring spectrin‐actin release, freeze‐etch micrographs reveal an apparent continuity between the fibrelike structures on the extracellular fracture face and the long fibre‐like structures which extend from the protoplasmic surface of the erythrocyte membrane. These results suggest that liquid propane jet‐freezing is capable of revealing a structural difference between the membrane of haemolysing and nonhaemolysed red blood cells, and that this difference is related to the fibrous, peripheral proteins of the membrane.
Distribution curves of sectional areas through some families of convex particles
Authors: Paul J. Lawrence
Published: August 2011

We outline the results of a computer program which calculates frequency distributions of functions of cross‐sectional regions of convex planar‐faced polyhedra, ‘sphero‐polyhedra’, and populations of perturbed polyhedra. This is done by ‘cutting’ a computer representation of the figure by a large number of random planes. The procedure is applied to cubes, dodecahedra, truncated octahedra, and approximate β‐tetrakaidecahedra. Certain aspects of the shape of the object, e.g. sharpness of corners, angularity, compactness, and degree of symmetry are related to aspects of the shape of the distribution curves, e.g. beginning and ending points of the curves, rate of rise of the peak, relative location of the peak, and height and width of the peak.
Preparation of P‐47 scintillators for STEM
Authors: Baumann W. , Niemietz A. , Reimer L. , Volbert B.
Published: August 2011

Uniform P‐47 powder scintillators can be prepared with reproducible mass thicknesses by sedimenting the powder in an 0·01% solution of formvar in chloroform. A reflecting coating can be glued by floating a 60 nm aluminium film on the scintillation layer in an 0·1% solution of gelatine in water. The optimum thickness of P‐47 for 20–100 keV electrons increases slower than the electron range due to the absorption and scattering of light with increasing thickness. An aluminium coating increases the light output by a factor 1·85 for 20 keV and 1·4 for 100 keV electrons. The noise of the emitted light quanta and the photomultiplier detection system shows a minimum for the optimum thickness of largest light output and is only a factor 1·3–1·6 larger than the calculated shot noise of the incident electron beam.
Excitation under the scanning electron microscope of DNA‐associated fluorescence from chicken erythrocyte nuclei, polytene chromosomes and adenovirus 2 virions
Authors: Hough Paul V. C. , McKinney Wayne R.
Published: August 2011

Biological structures not seen by conventional light microscopy, such as longitudinal striations in polytene chromosomes, and, at the limit of sensitivity, virions of adenovirus 2, have been detected via DNA‐associated fluorescence excited under the scanning electron microscope. The maximum sensitivity realized, about 1 detected photon per 700 base pairs, falls short by about an order of magnitude of that required to achieve, in unreplicated specimens, the 2 nm intrinsic resolution of the method. A combination of D2O‐H2O substitution with freeze‐drying provides the best unquenching procedure found for in situ DNA.

DNA‐associated fluorescence for light microscopy can be created by moderate exposure of the specimen in the electron microscope.
The characterization of periodic dislocation arrays from the fine structure of electron diffraction patterns
Authors: Morton A. J.
Published: August 2011

The accuracy of the technique of using the fine‐structure observed in electron diffraction patterns to determine the periodicity and characteristic direction associated with a periodic strain field is assessed. This is done by applying the technique to dislocation multipoles which have a strain field with only a single periodic component and which can be well characterized by other means. It is shown that, for such a single‐component strain field, precision measurement of the diffraction patterns combined with numerical analysis of the data allows the determination of the direction and spacing characterizing the strain field with an accuracy of about 0.25° to 1.0° in direction (depending upon periodicity) and ± 2% in spacing. This is better than can usually be achieved from measurements of corresponding features in the electron micrographs.
Sampling designs for stereology*
Authors: Cruz‐Orive Luis‐M. , Weibel Ewald R.
Published: August 2011

The purpose of this paper is to propose the necessary sampling techniques for estimating a global parameter defined in a solid opaque specimen (e.g. the total volume of mitochondria in a given liver, the total capillary surface area in a given lung, etc.). The geometry of the specimen often suggests a multi‐level or cascade sampling design at different magnifications, whereby the object phase at one level becomes the reference phase in the next level. The final parameter is then estimated as the product of the intermediate ratios with the volume of the specimen, which is estimated independently. Each level can be regarded as an independent sampling design; a given stereological project may be planned in terms of one or more of these designs.

Our development is a blend of practical experience and recent theoretical advances on sampling for stereology with well‐known sampling techniques previously developed with different purposes in mind.
Spatial resolution and detection sensitivity in microanalysis by electron energy loss selected imaging
Authors: Adamson‐Sharpe K. M. , Ottensmeyer F. P.
Published: August 2011

An electron energy filter of the Castaing‐Henry type in a high resolution transmission electron microscope was tested for sensitivity and spatial resolution at a specific electron energy loss in energy selected images of a murine leukaemia virus. Electron spectroscopic images of phosphorus within the virus membrane bilayer demonstrated a best spatial resolution between 0·3 and 0·5 nm and a sensitivity of 2 × 10−21 g.
In situ liquid propane jet‐freezing and freeze‐etching of monolayer cell cultures
Authors: Espevik Terje , Elgsaeter Arnljot
Published: August 2011

A procedure for in situ liquid propane jet‐freezing of unfixed and unglycerinated monolayer cell cultures is described. We have used the method to study monolayer cultures of rat Sertoli cells and human monocytes. The described procedure gives a cooling rate during specimen freezing high enough to yield adequately frozen specimens even in the absence of any cryo‐protectant. Use of the procedure therefore eliminates artefacts usually caused by cell fixation and addition of cryoprotectives.
Scanning electron microscopy, autolysis, and irradiation as techniques for studying small intestinal morphology
Authors: Carr K. E. , Hamlet R. , Watt C.
Published: August 2011

Examination of autolysed control mouse small intestine using scanning electron microscopy has revealed details of the connective tissue components of the mucosa. The cores of the villi are seen collapsed across the intervillous basin. Crypts of Lieberkuhn are seen as tubular channels stretching down from the intervillous basin. Sometimes the crypts are split in two by a connective tissue septum. The mouths of the crypts of Lieberkuhn are, in general, arranged in double rows between the single rows of villi. The ratio of number of crypts to numbers of villi was calculated as 5.01:1. This is close to the figure of 4.53:1, as quoted by Smith & Jarvis (1980) who used differential interference contrast microscopy to investigate the crypt to villus ratio. After radiation, the severe drop in the number of crypt mouths can be clearly seen by the combination of autolysis and scanning electron microscopy: the rows of crypt mouths between the villi have been lost, and many crypt mouths have been occluded by stromal tissue. The arrangement of the crypt mouths and the observation of mucosal abnormalities after irradiation have led to the postulation that cells leaving the crypt mouths move in a spiral manner towards and then up the villous surface: this postulated movement might imply an asymmetry in some properties of enterocytes. The use of scanning electron microscopy in conjugation with autolysis and irradiation has thus forced a critical re‐examination of the relationships between crypts and villi.
Cell surface distribution of fibronectin in cultures of fibroblasts and bladder derived epithelium: SEM‐immunogold localization compared to immunoperoxidase and immunofluorescence
Authors: Trejdosiewicz L. K. , Smolira M. A. , Hodges G. M. , Goodman S. L. , Livingston D. C.
Published: August 2011

Expression of cell surface fibronectin in cultures of untransformed fibroblasts is well documented, but little is known of its presence and distribution in cultured epithelial cells. Using species monospecific anti‐fibronectin antibodies, the distribution of fibronectin in untransformed fibroblasts and in normal and neoplastic bladder epithelial cells was characterized by indirect labelling experiments using immunogold scanning electron microscopy (SEM). The surface matrix of fibronectin expressed in rodent and human fibroblast cell lines was demonstrated with ease by SEM of gold‐tagged second antibodies. However, no fibronectin could be detected on any of the mouse and human bladder epithelium‐derived cells studied in single or in mixed epithelial‐fibroblast cultures. These SEM‐immunogold observations were compared to and confirmed by immunofluorescence and immunoperoxidase microscopy. Immunofluorescence and SEM localization of the fibronectin in the extracellular matrix presented similar distribution patterns but the higher resolution of the SEM provided a more detailed analysis.
The effect of chromium and nickel on the γ→α phase transformation in steels and iron‐base alloys
Authors: Ricks R. A. , Southwick P. D. , Howell P. R.
Published: August 2011

STEM X‐ray microanalysis has been used to determine the redistribution of chromium and nickel during the austenite to ferrite and reverse ferrite to austenite transformation. This paper reports results concerning three particular systems which are representative of a large number of alloy steels and iron‐base alloys. It is shown that, in general, no partitioning of the substitutional solute is observed in the transformable alloy steel or the carbon‐free iron‐base alloy such that during the austenite to ferrite transformation the austenite maintains the same chemical composition. Conversely, for the duplex stainless steel, partitioning of the solute does occur within certain temperature ranges.

The results also suggest that solute enrichment may occur at the austenite/ferrite interface which can be interpreted in terms of the existence of a ‘solute drag’ effect.
Crazes in osmium tetroxide stained styrene polymers
Authors: Kruse Robert L.
Published: August 2011

Crazes in polystyrene and rubber modified polystyrene became opaque in the electron microscope after films of the polymers are exposed to osmium tetroxide. The osmium tetroxide with the polystyrene in the crazes is shown to form a flexible insoluble material that is easily folded and twisted without breaking.
Uranyl sulphate: A new negative stain for electron microscopy
Authors: Estis Leonard F. , Haschemeyer Rudy H. , Wall Joseph S.
Published: August 2011

Uranyl sulphate is a negative stain of high quality for electron microscopy of macromolecules below their isoelectric point. The stain results in good contrast and high resolution as demonstrated by optical diffraction of periodic structure. Analysis of scanning transmission electron microscopic data reveals that uranyl sulphate is lower in background noise level than uranyl acetate and is dramatically more resistant toward granularization upon continued exposure to electron irradiation.

Electron microscopic images of most macromolecules contrasted with uranyl sulphate were indistinguishable from those obtained with uranyl acetate. However, electron microscopic images of Reo virus contrasted with uranyl sulphate are always readily distinguished from those obtained with uranyl acetate.
Stereological ratio estimation based on counts from integral test systems
Authors: Jensen E. B. , Gundersen H. J. G.
Published: August 2011

The stereological practice of using integral test systems in the estimation of the fundamental stereological ratios is studied in the light of recent theoretical developments in sampling. The estimation of a ratio is based on counts only, obtained from two, in general different, test sets constituting the integral test system. The ordinary ratio‐of‐sums estimator based on counts from uniformly positioned integral test systems is compared with two estimators based on non‐uniform, weighted sampling. It is shown that the estimators based on weighted sampling are not, in general, unbiased. Furthermore, it is pointed out that the mean‐of‐ratios estimator based on replicated weighted sampling needs not have smaller MSE than the ordinary ratio‐of‐sums estimator based on replicated uniform sampling.

The fact that the estimation is based on counts as opposed to complete 2‐d observations does not necessarily mean a reduction in information. For certain types of stereological ratios, the ordinary ratio‐of‐sums estimator based on complete observation is shown, counter to intuition, to be less accurate than that based on simple and fast counting.
Improved spatial and temporal resolution in an apparatus for time‐lapse phase contrast ciné light micrography of cells in vitro
Authors: Veselý P. , Malý M. , ČUmpelík J. , Pluta M. , Tůma V.
Published: August 2011

An apparatus for time‐lapse cinemicrography of living animal cells in vitro is described. It has an increased information capacity in comparison with conventional types of equipment in that successful combination of the highest possible spatial resolution with phase contrast microscopy and an improved temporal resolution provided by flash light illumination has been achieved. The interval between exposures of 1/200 s approximately can be reduced to 1/4 s using a negative phase‐contrast objective of NA 1·3. Negative phase‐contrast also appeared to be the best technique for imaging of tiny cell surface structures. Thus the new apparatus is suitable for the study of the patterns of cell surface motility in vitro.
The use of quadrats and test systems in stereology, including magnification corrections
Authors: Cruz‐Orive Luis‐M.
Published: August 2011

At present a model‐free, design‐based theory of unbiased estimation, and a model‐based one of linear unbiased estimation of minimum variance, are available for stereology. The main developments rest upon the nested scheme {section (quadrat)}, whence the raw data are expressible in terms of area, length and number. The main aim of this paper is to complete the available model‐based theory by introducing the step in which sections are analysed by point‐counting via coherent test systems (CTSs). Using this development, the stereologist should be able to handle raw point and intersection counts optimally, in order to find the best estimator of a ratio R in a given specimen in a wide range of circumstances. The latter include, for instance, the use of different CTSs on different sections and of double CTSs on each section, as well as the case—(not uncommon in electron microscopy)—in which different sections from the same sample are observed at slightly different magnifications but analysed by quadrats (via automatic or semi‐automatic image analysers, for instance), or CTSs of fixed sizes. The main conclusion pertaining to the latter case is that the estimators obtained via section‐wise magnification corrections are in general superior to those corrected via a global, average magnification.

In order to illustrate the methodology, a synthetic numerical example, and a real one, are given.
Experiments on freezing damage with freeze substitution using moth antennae as test objects
Authors: Steinbrecht Rudolf Alexander
Published: August 2011

Insect antennae can be easily frozen by immersion into propane at 90 K, freeze substituted at 194 K and, after warming up, embedded and sectioned at room temperature. Freezing damage (f.d.) may occur during cooling the specimen down (primary f.d.) or due to re‐crystallization, during warming up, if substitution was not complete (secondary f.d.). Experimental evidence suggests that secondary freezing damage is not likely to occur with freeze substitution, provided the acetone is water free (by adding molecular sieve) and the rate of warming up is low. All freezing damage observed in the specimens therefore most probably is due to primary freezing damage during cooling. Propelling the specimen with high speed into the coolant, instead of merely dropping it in, does not improve the quality of preservation obtainable but increases the yield of well‐frozen specimens.
Thermal and radiation damage to frozen hydrated specimens
Authors: Talmon Yeshayahu
Published: August 2011

Although low temperature fixation seems to be superior to chemical fixation with respect to structure preservation, complex interactions between the electron beam and frozen hydrated specimens can limit its applications. A review is given of the present knowledge of beam heating and radiation damage with emphasis on recent developments in the field.

Beam heating is found not to be a major limitation to cold stage microscopy. But radiolysis of ice and the formation of free radicals that subsequently attack organic matter in contact with ice can severely alter the specimen structure before a useful image is recorded. Use of low dose techniques may help in some cases, but the problem cannot be overcome in X‐ray microanalysis of very small regions, when high beam doses must be used. The use of liquid helium stages operating near 4 K may reduce damage also in frozen hydrated specimens.
Mercuric chloride in alcohol and chloroform used as a rapidly acting fixative for contracting muscle fibres
Authors: Brown L. M. , Hill L.
Published: August 2011

A fixative which preserves the correct alignment of the filaments in actively contracting muscle fibres must prevent sliding during fixation. Glutaraldehyde is unsuitable because the fibres are capable of relaxation and contraction for several seconds. A modification of Carnoy's fixative rapidly fixes tetanized fibres without relaxation or further activity.

The fixative contained ethanol and chloroform saturated with mercuric chloride. Fibres were thereby fixed and dehydrated at the same time. They were then stained with uranyl acetate, embedded, and stained with lead in the usual way.

Isolated fibres from R. temporaria were tetanized with low voltage pulses in air, and normal tetanic tensions were recorded prior to fixation. At least 50% of the tension remained after fixation. Unstimulated fibres were activated by the fixative, but fibres depolarized with K+ developed no tension.

Embedded fibres were examined intact in polarized light and closely resembled fresh fibres.

Electron micrographs showed that, although the membranes were disrupted, the myofibrils were well preserved to 15 μm below the surface and the band pattern was maintained throughout the fibre. There was some local variability in the width of the overlap zone, especially where myofibrils branched. The fine structure of the filaments was similar to that seen after conventional fixation. The cross‐bridges were visible, but the alcoholic dehydration probably interfered with their orientation.

Measurements of filament length, and of troponin and cross‐bridge periods by optical diffraction of calibrated electron micrographs, indicated differential shrinkage between the I‐band, overlap zone and H‐zone similar to that seen with other fixatives.

Criteria for satisfactory fixation of contracting muscle are discussed. The fixative is considered to be suitable for examining the longitudinal alignment of the filaments, and also for periodicity measurements if the small shrinkage artefact is allowed for. It is unsuitable for studying the reticulum, or for measuring the volume ratio of the myofibrils.
Quantitative low temperature optical microscopy of biological systems
Authors: Diller Kenneth R.
Published: August 2011

A comprehensive review is presented of low temperature optical microscopy techniques as applied to the study of freezing processes in biological systems. Emphasis is placed on analysis of physical and physiological parameters which were measured and/or controlled and the procedures for effecting such operations. Quantitative analysis of photomicrographs by digital computer processing is also discussed.
Organic mass loss at 100 K and 300 K
Authors: Egerton R. F.
Published: August 2011

Electron energy‐loss spectroscopy has been used to measure the loss of light elements from thin organic samples during exposure to 80 keV electrons. Cooling the specimen from 300 to 100 K is found to reduce the rate of removal of carbon, nitrogen and oxygen by factors up to 100.
Understanding the artefact problem in freeze‐fracture replication: a review
Authors: Sleytr U. B. , Robards A. W.
Published: August 2011

Freeze‐fracture and freeze‐etching techniques do not provide artefact‐free images of native in vivo or in vitro cells and tissues. Each preparation stage can produce specific artefacts which must be recognized and understood if these methods are to contribute meaningful information to cell biology. This paper reviews the latest information available on artefacts in freeze‐fracture replication (and etching) methods and points to possibilities for avoiding some of them. Different specimens show different sensitivity to artefactual changes and the final images must be interpreted carefully with regard to the multi‐event process that has led to their production.
Protein‐A gold particles as markers in replica immunocytochemistry: high resolution electron microscope investigations of plasma membrane surfaces
Authors: Mannweiler K. , Hohenberg H. , Bohn W. , Rutter G.
Published: August 2011

Due to their high atomic number contrast in transmission electron microscopy, gold particles are ideal markers in surface replicas of cultured cells. The suitability of protein‐A‐coated gold particles in replica immunocytochemistry for labelling surface antigens is demonstrated using measles virus‐infected cells as a model system. Labelled areas can easily be distinguished from unlabelled areas, and even markers positioned in the evaporation shadow of large structures can be accurately identified, which is a prerequisite for an exact quantification and mapping of antigen. In addition, the ultrastructure of labelled areas can still be visualized because of the small size of the marker.
Effect of substrate on freeze‐dried and shadowed protein structures
Authors: Trinick J. , Elliott A.
Published: August 2011

Muscle thick filaments have been prepared for electron microscopy by a method involving freeze‐drying and shadowing. The same method applied to filaments on three different substrates gives rise to quite different appearances.

The filaments on one of the substrates, hydrophilic carbon, show new detail not previously observed.
Electron dense granules and the role of buffers: artefacts from fixation with glutaraldehyde and osmium tetroxide
Authors: Hendriks Hans R. , Eestermans Inge L.
Published: August 2011

Electron dense granules may appear in tissues after glutaraldehyde prefixation and osmium tetroxide postfixation. In order to determine the conditions under which the granules are formed various vehicles in pre‐ and post‐fixatives were tested on lymph node, thymus and heart.

If granules appeared they were found in all cell types investigated, particularly in the nuclei. There was no difference in the distribution of the granules in the different compartments of these organs.

The granules probably consist of complexes of glutaraldehyde, osmium and phosphate. The ultimate phosphate concentration in the tissues during the postfixation was shown to determine whether or not the artefacts appeared. Local conditions in the tissues also contributed to the appearance of the granules. It is concluded that phosphate buffers can be used in the double fixation procedure, but to avoid the granules in lymph node, thymus and heart, a concentration of 0·1 mol/l or less phosphate should be used. For brain and kidney other conditions apply.
Physical randomization of tissue architecture: an alternative to systematic sampling
Authors: Stringer B. M. J. , Wynford‐Thomas D. , Williams E. D.
Published: August 2011

A method is described for the rapid and accurate quantitation of tissue morphology using rat thyroid as a model. Perfused‐fixed rat thyroid was diced into approximately thirty small pieces which were then randomly embedded in epon. Sections were taken at 500 μm intervals and stained with toluidine blue. Epithelial, follicular lumen and stromal components were quantified by a point counting technique. An analysis of variance was then performed on the data to determine whether there was any significant variation in the distribution of components between sections cut at different levels.

No significant variation in any component was seen between sections for normal diced thyroid. This was also the case for diced thyroids of animals chronically treated with goitrogen to create a different physiological state. The method is therefore reliable even when marked changes in component distribution are induced.

The physical randomization of tissue architecture by dicing, prior to embedding, greatly reduces the number of sections needed for the accurate morphometry of non‐randomly distributed tissues.
An improved method for combined autoradiography and histochemistry: the simultaneous detection of 6‐3H thymidine and acid phosphatase activity in cryostat sections of mouse thymus and duodenum
Authors: Lewis G. H. J. , Bowen I. D.
Published: August 2011

A new method is described that enables the simultaneous detection of 6‐3H thymidine incorporation and acid phosphatase activity in the same tissue section. Histochemically, naphthol AS B1 released by tissue based acid phosphatase activity from the substrate naphthyl AS B1 phosphoric acid is coupled with a range of diazonium salts to produce insoluble azo dyes. The azo dye tests result in a particulate localization of lysosomal acid phosphatase and also label diffuse sources associated with cell death. The tests selected permit the application of photographic emulsion without the necessity of an inert barrier layer to separate the emulsion from the histochemically treated cryosections.

The localization of 6‐3H thymidine incorporation and cell death in mouse thymus and duodenum is demonstrated and comparative counts estimating the distribution of 6‐3H thymidine incorporation and hydrolase labelled cell death in the thymus are presented. Young mouse thymus (5 weeks) was found to contain 1·36 ± 0·12% dying cells and 6·78 ± 0·03% thymidine incorporating cells, whilst old mouse thymus (53 weeks) was found to contain 2·34 ± 0·6% dying cells and 5·29 ± 0·37% thymidine incorporating cells.
Rapid and reliable method for diagnostic electron microscopy of faeces
Authors: Gbewonyo Armstrong J. K.
Published: August 2011

Four methods are described for examining viruses in faeces by electron microscopy using negative staining. Faeces samples from a total of 180 patients with diarrhoea illnesses were processed for electron microscopy by the direct staining technique, pseudoreplica technique, microsolute concentration technique and by ultracentrifugation. Virus particles (including rotaviruses and adenoviruses) were found in fifty‐five (31%) samples when the results of all methods were combined. Bacteriophages were found in thirty of the samples which were read virus negative. Herpesviruses were also found in the faecal samples of two patients with diarrhoea illness. Parvovirus‐like particles were identified in one sample which was rotavirus and adeno‐virus positive. Calicivirus‐like particles were found in a sample which was adenovirus positive. With the direct staining technique thirty‐six (20%) samples contain virus particles; with micro‐solute concentration technique forty‐eight (27%) samples contain virus particles and with ultracentrifugation thirty‐eight (21%) contain virus particles.

It is believed the microsolute concentration technique is rapid and more reliable than the pseudoreplica technique and ultracentrifugation method, and is a more preferable method for the diagnosis of faecal sample by electron microscopy.
A simple method for the handling and orientation of small specimens for electron microscopy
Authors: Carson J. , Lee R. M. K. W. , Forrest J. B.
Published: August 2011

Cyanoacrylic glue (Eastman 910) was used to affix small pieces of nasal scrapings to lens paper immediately before fixation in the glutaraldehyde. The lens paper not only served to hold specimens together so that they were not lost during tissue processing, but also functioned as a ‘landmark’ for the specimens, so that specimens could be oriented in a specific manner during embedding and subsequent sectioning.
A time‐saving device for vacuum evaporation
Authors: Pelttari A. E. , Markkanen S. O.
Published: August 2011

An inexpensive device for vacuum evaporation is described. It saves time and work when there are a great number of specimens to be coated with evaporated carbon.