bluuebunny/crossref_metadata_2025 huggingface dataset:
Sputtered platinum films on colloidal gold particles: a calibration specimen for quartz film thickness monitors
Authors: Johansen B. V. , Namork Ellen
Published: August 2011

A method for calibrating quartz crystal thickness monitors is described, which makes use of 20 nm platinum sputtered colloidal gold particles. The actual thickness is measured on transmission electron micrographs of gold particles protruding from the edge of fractured, rolled up, carbon films. In the range up to ∼10 nm, the measured thickness for platinum films is ∼17% larger than the figures calculated from mass thickness equations.
Electron microscopy of melamine‐embedded frog retina: evidence for the overall crystalline organization of photoreceptor outer segments
Authors: Bachhuber Karlheinz , Frösch Dieter
Published: August 2011

Melamine‐formaldehyde, a widely used technical aminoplast, was recently introduced as a water‐soluble embedding‐medium for electron microscopy (Bachhuber & Frösch, 1983).

When processed, using this new technique, the membranes and associated cytoplasmic structures of rod and cone discs show a high degree of structural organization. The membranes are symmetrical and composed of arrays of a 2–2·5 nm particle with a very regular spacing of 3 nm. A network of delicate filaments of less than 2 nm is superimposed on to this framework of membranes, running perpendicularly between the discs and across the disc membranes.

The outer segment as a whole shows a crystal‐like organization.
Epifluorescence microscopy with a pulsed nitrogen tunable dye laser source
Authors: van Geel F. , Smith B. W. , Nicolaissen B. , Winefordner J. D.
Published: August 2011

This paper describes an epifluorescence system that uses a pulsed laser source as an excitation source for quantitative fluorescence measurements. A 1 μm diameter portion of the sample can be illuminated with high intensity, short light pulses and the fluorescence can be measured with a fast detection system. Selective excitation is possible at 337 nm and from 357 to 710 nm by using different dyes in the laser system.

The spectral bandwidth is 0·1–3 nm. Several advantages over the commonly used systems with continuum spectral sources are indicated, especially when measured intensities are low, as in the case of intrinsic fluorescence of the sample, and where reflection of unwanted excitation light may cause appreciable errors when using conventional light sources.
A combined light and electron microscopic method for the visualization of the same in vitro neuron by radioautography and serial sections
Authors: Vuillet J. , Montety M.‐C. Daguet‐de , Autillo‐Touati A. , Glowinski J. , Prochiantz A. , Seïte R.
Published: August 2011

We report here on a technical improvement which makes it possible to study, at the ultrastructural level, a dopaminergic neuron which has been previously identified by light microscopy. Primary cultures of virtually pure mesencephalic neurons from mouse embryos were obtained. These cultures were kept for 6 days, then incubated with tritiated dopamine. fixed and embedded in Epon. The dopaminergic neurons were firstly visualized by radioautography directly through Epon blocks in toto by light microscopy. In a second step, ultrathin sections of the identified dopaminergic cells were prepared and the neurons observed at the electron microscopy level. The dopaminergic nature of these neurons was regularly checked by radioautographic control on some selected ultrathin sections.
An anti‐roll plate system for Ralph knives
Authors: Richards Peter R.
Published: August 2011

At thicknesses above 4 μm, an anti‐roll plate is necessary when cutting frozen sections for light microscopy with long‐edged glass (Ralph) knives. The structure of the plate and a system which allows the use of such plates without dismantling or interfering with present systems for steel knives is described.
A computerized microspectrophotometer using fibre optics for transmission and detection of light
Authors: Neely John E. , Townend William J. , Combs John W.
Published: August 2011

A computerized microspectrophotometer was developed to provide rapid and accurate sequential measurements on cells, including nuclear DNA content, by fluorescence staining, photometric grain counting from autoradiographs, and nuclear size estimated by measurement aperture area. To alleviate bulk and vibrations caused by additional stepper motors, the excitation light and filters as well as the emission filters and photomultipliers were detached from the main microscope frame. The light was directed to and from the microscope through fibre optic bundles. This configuration provided an excellent light gathering faculty and made optical alignment easier. The machine provided highly accurate sequential measurements on a relatively large number of cells by static photometry.
Chromoxane cyanine R. I. Physical and chemical properties of the dye and of some of its iron complexes
Authors: Kiernan J. A.
Published: August 2011

Chromoxane cyanine R (Colour Index No. 43820, Mordant blue 3; also known as eriochrome cyanine R and solochrome cyanine R) is a valuable biological stain. The dyestuff is supplied as a powder containing a little less than 50% by weight of the monosodium salt of the dye, mixed with colourless crystalline and amorphous fillers. The tetrabasic colour acid was prepared and purified for study of the chemical and spectral properties of the dye. Chromoxane cyanine R is an acid‐base indicator, with five different colours corresponding to the colour acid and the four anions. The most conspicuous colour change, from yellow to blue, occurs with ionization of the phenolic hydroxyl group at pH 11–12. The dye is assayed by measuring the absorbance of a strongly alkaline solution at 585 nm, with reference to a standard solution prepared from the purified colour acid.

Spectrophotometric evidence has been found for the existence of three dye‐metal complexes in solutions of the dye containing added ferric chloride at pH 1·5 (the pH of iron‐dye solutions most useful in histological staining). These have the postulated compositions [Fe2H(dye)]‐(red), [FeH2(dye)]‐ (red), and [Fe2(dye)]2‐ (blue). The first two are probably simple carboxylate complexes of low stability. Increase in pH or molar iron:dye ratio promotes formation of the more stable blue complex, which is a metal chelate. Other blue complexes have been described by other investigators in solutions less acid than those that are useful in microtechnique. The production of blue and various shades of red in tissues stained by solutions containing iron(III) and chromoxane cyanine R probably involves reactions of both the red and the blue complexes of the dye.
Chromoxane cyanine R. II. Staining of animal tissues by the dye and its iron complexes
Authors: Kiernan J. A.
Published: August 2011

The staining properties of chromoxane cyanine R (Colour Index No. 43820, Mordant blue 3; also known as eriochrome cyanine R and solochrome cyanine R) have been studied. Used alone, the dye imparted its red colour to nuclei, cytoplasm and collagen. The dye was extracted by mild alkali but not by acids. Stainability required ionized amino groups in the tissue, and there was also evidence for non‐ionic binding of the dye.

The colours obtained by staining with mixtures of chromoxane cyanine R and ferric chloride varied with the molar iron: dye ratio and with the pH. Useful staining was seen only between pH 1 and 2. The tissues were coloured either all blue (when Fe: dye was high), or both red and blue (when Fe: dye was low). Lower pH favoured the deposition of red, higher pH the deposition of blue colour. The red was mainly in cytoplasm, blue in nuclei and myelin. Collagen fibres were red or purple, depending on pH and iron: dye ratio. Red colours were differentiated by acid and changed to blue, but not extracted, by mild alkali. The red substance in the stained sections was clearly not the free dye, so it was probably an iron‐dye complex. From the effects of various differentiating agents, it was deduced that the red and blue dye‐metal complex molecules were bound to the tissue by the dye moiety, not by interposition of iron atoms. Staining by the complexes of iron(III) with chromoxane cyanine R did not involve nucleic acids or other polyanions or the amino groups of proteins. There was evidence for only non‐ionic binding of both red and blue complexes. It is suggested that the red colour in sections stained by solutions with low iron: dye ratio is due to a simple carboxylate complex, [Fe2H(dye)]−. The blue colour would then result from withdrawal of a proton from the red complex to give [Fe2(dye)]2‐. The bases that remove the protons may be arginine‐rich nucleoproteins of nuclei and phospholipid bases of myelin.

Techniques are described for informative simultaneous staining in two colours, and for the selective staining of either nuclei or myelin.
Incident light microscopy of surfaces of plastic embedded hard tissues
Authors: Bab I. , Ashton B. A. , Owen M. E. , Boyde A.
Published: August 2011

To eliminate sectioning artefacts, we devised a method for the preparation of surfaces of plastic embedded hard tissues and their examination by incident light fluorescence microscopy. Flat surfaces produced by cutting on a microtome or using a low‐speed saw were stained with dyes in aqueous solutions. Best results were obtained in tissues embedded in glycol‐methacrylate. This polymer allowed good penetration of the dyes and its optical properties render cutting defects invisible with the present method. The relationship between hard and soft tissues was well preserved and the integrity of the mineralized component maintained. Cellular details and the distinction between osteoid and bone could be clearly demonstrated. The procedure is simple, rapid and produces good results even in instances when histological sectioning is unsatisfactory.
A parametric partial cross section for ELS
Authors: Joy David C.
Published: August 2011

A parametric expression has been developed which permits the rapid calculation of partial ionization cross sections, for most of the elements and experimental conditions encountered in electron energy loss spectroscopy, without the need for a computer. Since the results are in good agreement with those obtained using the full hydrogenic model, this approximation should be useful for such requirements as rapid microanalysis, or in calculations such as those of detection sensitivity where an analytical approximation to the cross section is needed.
A device for area measurement by TV
Authors: Millington P. F. , Floyd S.
Published: August 2011

This paper describes a low‐cost area measurement device constructed from readily available components to provide a direct numerical output related to area. It can be used simply as an area measurement system calibration or, if linked to other devices, can be adapted to act as a source of data in more elaborate experiments. One example of its use is given.
Extraction of substances during glutaraldehyde fixation of plant cells
Authors: Coetzee J. , van der Merwe C. F.
Published: August 2011

Extraction of Ca, Mg, reducing sugars, amino acids and protein was monitored during fixation of bean leaves. Fixatives included 0.5–5% glutaraldehyde in water at pH 3.4 and at pH 7.2 and phosphate, citrate, Na‐cacodylate, tris, glycyleglycine, collidine and four ‘Good’ buffers, all with 2.5% glutaraldehyde. Least extraction was found in phosphate buffered glutaraldehyde. The optimal glutaraldehyde concentration is 2–3%. This causes least extraction of substances.
Quantitative X‐ray microanalysis of uranium alloys with the analytical electron microscope
Authors: Romig A. D.
Published: August 2011

X‐ray microanalysis in the analytical electron microscope (AEM) is a powerful tool for studying elemental segregation on a submicrometre scale. This technique has been applied to uranium and its alloys. Materials such as uranium are strong electron scatterers due to their high density and high atomic numbers. Hence, when performing AEM X‐ray microanalysis on such materials special attention must be given to effects such as electron scattering and X‐ray absorption. The objective of this paper is to describe the techniques which are required to perform quantitative X‐ray microanalysis in these heavy metal alloys (U‐Nb, U‐Mo and U‐Ti). Cliff‐Lorimer k‐factors determined experimentally and theoretically are in good agreement. Electron scattering has been studied experimentally and the results are compared to those obtained by Monte Carlo calculations and the single scattering model. X‐ray spatial resolutions of better than 100 nm are attainable. With the proper selection of characteristic X‐ray lines, X‐ray absorption can be avoided in most cases to the limit of electron transparency, ∼ 100 nm.
Gallium: a new supporting medium for specimen preparation
Authors: Warner Ronald R.
Published: August 2011

Gallium offers a useful alternative to conventional plastic and paraffin embedding media. Samples can be inserted into or removed from gallium cleanly and intact in minutes. Gallium can be polished, has good sectioning properties, is electrically conductive, tolerates high beam currents, is usually not a detectable sample component, and probably would not be a solvent for sample constituents. Samples can be immobilized in gallium within a few degrees of room temperature. Gallium has low toxicity, can be obtained in ultrapure form, is not expensive, and is reusable. Gallium has been used to prepare thin and thick sections and to expose bulk sample cross sections for STEM, SEM, microprobe and light microscope analysis. Gallium does not infiltrate; consequently it is not likely to be useful for soft biological samples.
Understanding thin film X‐ray spectra
Authors: Chapman J. N. , Nicholson W. A. P. , Crozier P. A.
Published: August 2011

Formulae suitable for predicting X‐ray production cross‐sections in thin foils are described. A modified Bethe‐Heitler equation seems well suited to describe the production of bremsstrahlung photons and, for fixed experimental conditions, may be expressed in a simple parametric form. Simple equations for calculating characteristic photon production are less satisfactory and the predictions of different formulae differ markedly. For microanalytical purposes, however, where relative rather than absolute cross‐sections are required, the differences become less pronounced. Finally, some uses for the cross‐sections in X‐ray microanalysis are considered.
Electron energy loss spectra and reflection images from surfaces
Authors: Howie A. , Milne R. H.
Published: August 2011

There is renewed interest in the interaction of high energy electron beams with surfaces at glancing angles of incidence, due partially to recent improvements in, and consequential resurgence of, reflection electron microscopy. In this paper electron energy loss of beams running parallel to a surface or at glancing incidence to it are studied and compared with predictions arising from the classical theory of dielectric excitation by a moving charge. There is good agreement for both the Cu and MgO surfaces studied here. It is pointed out that small angle energy loss from high energy electrons is insensitive to surface reconstructions or surface steps. Reflection energy filtered micrographs of the Cu surface are shown and the similarity between the zero and various energy loss images shows the preservation of contrast mechanisms for elastic and inelastic electrons.
The role of carbamino compounds in histological staining
Authors: Cook D. J.
Published: August 2011

The effects of high CO2 concentration on tissue staining with buffered eosin, methylene blue and azure A were investigated to determine if carbamino compound formation could alter dye binding. Only in the case of eosin staining were the differences in results with high and low CO2 concentrations compatible with the formation of carbamino compounds. The differences observed with basic dyes were statistically significant but could not be accounted for by carbamino compound formation.
Impregnation of biological material by ZnI2‐OsO4, KI‐OsO4 and NaI‐OsO4 mixtures for electron microscopic observations: Chemical interpretation of the reaction
Authors: Carrapiço F. , Madalena‐Costa F. , Pais Maria Salomé S.
Published: August 2011

Alkaline iodide‐osmium tetroxide mixtures were used in an electron microscopic study of plant cells and gave better results in the preservation of the cell structure in contrast to the cell structure observed when ZnI2‐OSO4 mixtures are used.

The electron density observed in the reactive sites of the cell by OsO4‐iodide mixtures is controlled by the presence and oxidation state of iodine that acts as a regulator of the potential redox of the reaction of OsO4 with the biological material.
Blue molybdenum oxides: A stain for light and electron microscopy
Authors: Ferrer J. M. , Tato A. , Stockert J. C.
Published: August 2011

Blue molybdenum oxides (molybdenum blues) have been prepared from aqueous phosphomolybdic acid solutions and applied to thin and semi‐thin sections of glutaraldehyde‐fixed, epoxy‐embedded tissues. A light blue colour and high electron opacity were found in mast cell granules, the secretion content of goblet cells, and cytoplasmic granules in Drosophila salivary glands. The possibility that binding of blue molybdenum oxides to polyhydroxylic components accounts for the staining and contrasting reactions in some cell structures is briefly discussed.
Surface roughness and the use of a peak to background ratio in the X‐ray microanalysis of bio‐organic bulk specimens
Authors: Boekestein A. , Thiel F. , Stols A. L. H. , Bouw E. , Stadhouders A. M.
Published: August 2011

The use of a net peak intensity and of a peak to background (P/B)‐ratio of sulphur and chlorine is examined in the X‐ray microanalysis of a 2·4% w/w S bulk standard in Spurr's epoxy resin. In calculating the P/B‐ratio, the background intensity is calculated for the same energy region as for the net peak. Analyses were carried out on the flat top of the standard and on the slope running down from the top on the side not facing the X‐ray detector. The results obtained for the peak to local background ratios from the top and the slope yielded a relatively small mean deviation (11%) while net peak intensities ultimately were reduced to 7% or less of the initial value for the flat top. This indicated that a peak to local background ratio is to be preferred in the quantitative analysis of bulk specimens which have poorly defined local tilt and takeoff angles. A second advantage is the inherent correction for beam current fluctuations.
A simple inexpensive cryogenic storage device for microscopy specimens
Authors: Rigler M. W. , Patton J. S.
Published: August 2011

Cryogenic storage devices for microscopy specimens are costly and may require a particular size or type of dewar. Described here is a simple inexpensive storage container for small specimens (< 5 mm in diameter) which can be constructed in less than 15 min from common laboratory materials. The unit is modular in design, can fit into any thermos type dewar and is reusable.
Accurate method of measuring grain misorientations and analysing grain boundary planes in TEM
Authors: Voice W. E. , Faulkner R. G.
Published: August 2011

A description of a TEM method for accurately determining the misorientation across grain boundaries, and identification of the grain boundary plane is given. The method is equally applicable to thin or thick foils and possesses the accuracy of Kikuchi line analysis. A computer program is available to process the TEM data to yield directly the grain boundary misorientation parameters. Examples of the application of the method to grain boundary structure determination in a nickel base superalloy, Nimonic 80A, are quoted.
Improved imaging of phase gradients in scanning optical microscopy
Authors: Hamilton D. K. , Sheppard C. J. R. , Wilson T.
Published: August 2011

Previous work has used a circular large area split detector for phase gradient imaging; it is shown that substituting an annular split detector offers improvements in frequency response, particularly for weak phase gradients. Transfer functions are calculated and transmission and reflection micrographs demonstrate the different imaging properties. Other forms of detector are also considered, some of these possessing certain advantages.
Variations in fungal spore dimensions in relation to preparatory techniques for light microscopy and scanning electron microscopy
Authors: Beckett A. , Read N. D. , Porter R.
Published: August 2011

The volumes of urediospores of Uromyces viciae‐fabae and ascospores of Sordaria humana in the critical point‐dried, freeze‐dried, osmium vapour‐fixed/freeze‐dried, frozen‐hydrated and ‘wet’ states were measured. Dried spores shrank and were significantly smaller than frozenhydrated and ‘wet’ spores. Urediospores exhibited a greater range of dimensional changes in relation to the preparatory treatment than did ascospores. Ascospores, dried in different ways, were not significantly different from each other but were significantly smaller than frozenhydrated ones. Both urediospores and ascospores dehydrate naturally and collapse. On wetting they imbibe water rapidly and swell. Low temperature scanning electron microscopy may be suitable for investigating fungal spores in their ‘natural states' on the leaf surface.
A generalization of atom location by channelling enhanced microanalysis
Authors: Krishnan Kannan M. , Thomas Gareth
Published: August 2011

The technique of atom location by channelling enhanced microanalysis (ALCHEMI), has limitations in practical application. A generalization of this technique, with the underlying idea that additional relationships can be generated by performing these experiments at an appropriate number of orientations depending upon the stoichiometry of the original compound has been developed. The precise number of orientations required to solve the distributions in a whole class of related compounds is expressed in terms of the stoichiometry of the original compound along with the alloying additions.
Stereo presentation of Monte Carlo electron trajectory simulations
Authors: Bright David S. , Myklebust Robert L. , Newbury Dale E.
Published: August 2011

Electron trajectory data from Monte Carlo simulation techniques is three dimensional in nature, and thus is best represented by methods that most preserve the spatial information. Stereo plotting is a method that gives the three‐dimensional illusion effectively while not requiring any special equipment beyond what is required to make standard two‐dimensional plots. Stereo plots of electron trajectories are presented that illustrate the advantages of the spatial illusion in the context of examining in detail some of the interactions of the electron beam with planar bulk meta lic samples.
The comparison by simulation of solutions of Wicksell's corpuscle problem
Authors: Blödner R. , Mühlig P. , Nagel W.
Published: August 2011

In this paper six distribution‐free (nonparametric) stereological methods for the solution of Wicksell's corpuscle problem—i.e. the determination of the distribution of diameters of spheres embedded in an opaque specimen from the diameters of their profiles on plane sections—are compared as regards their numerical stability, sensitivity to underlying distributions and certain error criteria. The study is based on the results of simulation studies for several types of distribution (one‐point, normal, exponential, logarithmic normal) of sphere diameters. Recommendations are suggested for the choice of methods, sample size and the optimal number of classes for grouping sample data.
Fracturing of melamine‐embedded cells and tissues: a new technique for studying cell membranes
Authors: Westphal Christel , Frösch Dieter
Published: August 2011

A new technique is presented for studying cell membranes by scanning electron microscopy. It is based on the observation that cells and tissues, embedded in a water‐compatible melamine resin, are as hard as glass and consequently can be fractured with ease. Fracture faces so exposed are either sputter‐coated for studying the surface topography or re‐embedded for thin‐sectioning, or both.

Scanning electron microscopy shows that the fracture faces of a variety of tissues reveal cell membranes and associated structures with remarkable detail. Re‐embedding and thin‐sectioning of fractured frog retina, mycoplasma and red blood cells indicate that membranes become divided into their exo‐ and protoplasmic leaflets during fracturing. In this respect, the results reported here must be compared with conventional freeze‐fracture techniques.
Interferometric determination of the optical properties of fibres with irregular transverse sections (Ramie)
Authors: Hamza A. A. , Sokkar T. Z. N. , Shahin M. M.
Published: August 2011

The thickness of fibres and their irregularity were determined from the forward scattering of a He‐Ne laser beam. Two methods have been used for the measurement of refractive indices and birefringence of Ramie fibres. The first of these methods is the application of multiple‐beam Fizeau fringes in transmission and at reflection. The second is the use of the double refracting interference microscope designed and developed by Pluta. In each method, refractive index values have been determined using two different analyses of the interference pattern. To overcome the difficulty of the irregular transverse section of Ramie fibres, the area enclosed under the fringe shift is considered as representing the path difference integrated across the fibre.
A technique for comparative light and scanning electron microscopy of the same deparaffinized microtomed section*
Authors: Pattee H. E. , Mohapatra S. C. , Agnello E. K.
Published: August 2011

Evaluation of various types of clear plastic support material for light and scanning electron microscopy of the same section was conducted. GelBond was found to be the most satisfactory among seven different types of plastic materials tested. It offers all of the advantages of glass slides for light microscopy while being thin and flexible enough to cut and sputter coat for SEM.
Application of frustrated total reflection for studying cells in vitro
Authors: Akatov V. S.
Published: August 2011

A mathematical analysis of the dependence of the light flux scattered upon frustrated total reflection (FTR) on the angle of incidence of the light beam is presented for monolayers of spread and spherical cells. It is shown that using these relationships it is possible to estimate the refractive index of hyaloplasm at cell‐substrate contacts and to study the flattening of the cell surface facing the glass. Analysis of FTR for single cells is presented. A condition is given that must be fulfilled for the brightly shining regions of the cell to be interpreted as the sites of attachment of the cell surface to glass. The condition defines the range of angles of incidence at which FTR does not depend on the optical properties of the cytoplasm portion being at a distance from the glass. The fulfillment of this condition is necessary for spectroscopic and ellipsometric studies of cell surface contacting the glass, by means of FTR. The conclusions are confirmed by experimental data. A peculiarity of light scattering by cells, associated with the coherence of the incident light beam is discussed.
Analysis of coal blends by automated microscopy
Authors: Lee James B.
Published: August 2011

The relative efficiency of coal blend analyses by automated reflectance measurement techniques is compared with an optimum point counting method. The influence of overlapping reflectance distributions and the advantage of image analyser systems over point reflectance measurements are evaluated. Automated point reflectance measurement techniques are feasible for coal blend analysis provided the measurement time is sufficiently small, while image analyser instruments can gain an advantage by reducing the particle top size in the specimen preparation.
Critical‐point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes
Authors: Nordestgaard B. G. , Rostgaard J.
Published: August 2011

Critical‐point drying and freeze drying were compared both quantitatively and qualitatively as preparative procedures for scanning electron microscopy. Isolated hepatocytes were used as model cells.

Nomarski differential interference contrast microscopy was used for light microscopic measurements of the hepatocytes in the unfixed, the glutaraldehyde fixed, the glutaraldehyde + OsO4 fixed, the critical‐point dried and the freeze dried states. Critical‐point dried hepatocytes were found to shrink to 38% of glutaraldehyde + OsO4 fixed volume, whereas optimal freeze dried hepatocytes (frozen in water saturated with chloroform and freeze dried at 183 K for 84 h) were found to shrink to 51% of glutaraldehyde + OsO4 fixed volume. Transmission and scanning electron micrographs of the critical‐point dried cells showed well‐preserved ultrastructure and surface structure. Micrographs of the freeze dried cells showed ultrastructure destroyed by internal ice crystals and surface structure destroyed by external ice crystals.

Double‐fixed isolated hepatocytes were shown to swell during storage in buffer and to shrink during storage after critical‐point drying.

For low magnification scanning electron microscopy (up to about 3000 times) both critical‐point drying and freeze drying can be used. However, for high magnification scanning electron microscopy, critical‐point drying is superior to freeze drying.
Laser scanning microscopy of surface spread polytene chromosomes
Authors: Kalisch W.‐E. , Whitmore T. , Siegel A.
Published: August 2011

A new type of a Laser Scan Microscope (Zeiss) was used for the analysis of the band‐interband pattern of polytene chromosomes in Chironomus. In contrast to the previously used techniques of transmission light and electron microscopy, we used differential interference contrast (DIC) in incident light to depict the pattern. Instead of using common squash preparations, we carried out this investigation with surface spread polytene (SSP) chromosome preparations of salivary glands. The combination of techniques used enabled a more detailed light microscopic presentation of polytene structures in individual preparations than conventional techniques used so far for chromosome mapping.
A modification of the chromic tartrate silver impregnation technique for block impregnation of the central nervous system and paraffin wax embedding
Authors: Abadía‐Fenoll F. , Garrido M. V. Ostos , López‐Lendínez J. L. Caballero , Martos R. Carmona
Published: August 2011

This paper describes a block silver impregnation technique for the CNS. The procedure, which is quite simple, yields highly consistent and reproducible results.

After fixation during 6–10 days in 10% saline formaldehyde, 4 mm thick blocks of brain are treated with chromic anhydride and sodium potassium tartrate solution for 4 days. After this period the specimens are rinsed in 0.75% silver nitrate solution to which 8–10 drops of pyridine per 100 ml of solution have been added. This is followed by impregnation for 4 days at 37°C in silver nitrate‐pyridine solution identical to that used in the previous rinsing step. The impregnated blocks are reduced during 20–26 h in 1% pyrogallol to which 6 ml commercial formaldehyde per 100 ml of solution have been added, followed by dehydration in dioxan and paraffin embedding. Sections no thicker than 30 μm are then cut for histological study.

This fundamentally neurofibrillar method reveals: (a) neuronal somata and their processes; (b) synaptic structures; (c) fibre bundles; and (d) cell nuclei and nucleoli.
Comments on the computation of electron wave‐propagation in the slice methods
Authors: Ishikuza K.
Published: August 2011

The slice methods developed to calculate dynamical electron diffraction involve an interaction of electrons with each slice and the wave‐propagation between the successive slices alternately. Thus an accurate evaluation of the propagation effect, which is mathematically described in terms of a convolution integral, is indispensable. Three main slice methods are examined from the viewpoint of their accuracy and efficiency on the numerical evaluation of the wave‐propagation. Some difficulties of two real‐space methods are discussed. In conclusion, the FFT multisclie method is the fastest way to obtain the most accurate evaluation of the wave‐propagation at present.
Light microscope based analysis of three‐dimensional structure: Applications to the study of Drosophila salivary gland nuclei. I. Data collection and analysis
Authors: Mathog David , Hochstrasser Mark , Sedat John W.
Published: August 2011

Many biological structures of interest are large enough that they may be viewed by light microscope methods, yet they are sufficiently complicated that interpretation of what is seen is quite difficult. The salivary gland nuclei from Dipterans are an example of this. Previous attempts at determining the path of the giant chromosomes in these nuclei have depended on the laborious construction of models by hand. A unified Computer Aided Modelling and Analysis system (CAMA) has been implemented, allowing data collection and analysis of structures visible by light microscopy. This system is extendible to the analysis of electron micrographs of serial sections or of other data consisting of images present in a stack.
Light microscope based analysis of three‐dimensional structure: Applications to the study of Drosophila salivary gland nuclei. II. Algorithms for model analysis
Authors: Mathog David
Published: August 2011

In Drosophila melanogater there are significant differences in the way the polytene chromosomes are arranged in different nuclei from the same salivary gland (Mathog et al., 1984). Visual inspection of the models of these nuclei was inadequate to delineate all of the features conserved in their structures. In order to bypass this limitation, models of these nuclei were constructed in a format compatible with computational manipulation (Mathog et al., 1985). By applying some simple algorithms to these models, quantitative comparisons were made which revealed otherwise cryptic features common to many nuclei. Given here are the details of several algorithms for describing and comparing the arrangement of the chromosomes within the nuclei.
A computational test for the removal of plural scattering from angle‐resolved energy loss spectra
Authors: Sölkner G. , Schattschneider P.
Published: August 2011

A recently developed method based on matrix analysis for the removal of plural scattering from angle‐resolved energy loss spectra is tested. A single loss function, Lorentzian in the energy and Gaussian in the angular variable is assumed as input for the test. Multiple scattering probabilities are simulated by summing up n‐fold self‐convolutions of the input function according to the Poisson distribution for incoherent n‐fold scattering. The simulated profile serves as input for the retrieval algorithm, the result of which is compared with the original single‐loss probability. It is concluded that the method is feasible, but not likely to be suited for routine investigations.
Analysis of the irregular planar distribution of proteins in membranes
Authors: Hui S. W. , Frank Joachim
Published: August 2011

Methods to characterize the irregular but non‐random planar distribution of proteins in biological membranes were investigated. The distribution of the proteins constituting the intramembranous particles (IMP) in human erythrocyte membranes was used as an example. The distribution of IMPs was deliberately altered by experimental means. For real space analyses, the IMP positions in freeze fracture micrograph S were determined by an automatic procedure described. Radial distribution and autocorrelation analysis revealed quantitative differences between experimental groups. These methods are more sensitive than the corresponding optical diffraction or Fourier‐Bessel analyses of the same IMP distribution data, due to the inability of the diffraction methods to separate contrast and distribution effects. A method to identify IMPs on a non‐uniform background is described.
A low cost image and data acquisition system for use with the Vickers M85 scanning microdensitometer
Authors: Glynn P. J. , Larbalestier G.
Published: August 2011

A simple and inexpensive interface has been constructed between the Vickers M85 microdensitometer and a BBC model B microcomputer. The interface incorporates three sensitivity ranges and enables the production of pseudocolour images of the specimen using the two‐dimensional scanning mode of the M85. The operator can select a 160times256 pixel image with eight colours or a 320times256 display using only four colours. Each colour represents a defined range of transmittance which is software controlled. The image histogram can be displayed and the interval between colours redefined so as to enable contrast stretching. Intervals between colours can be either linear or logarithmic and the images thus obtained can be stored on disc or videotape, or a hard copy can be obtained using a screen dump routine. Two‐dimensional absorption images can thus be obtained at any single wavelength from 400 to 700 nm at normal magnifications of the light microscope.

In addition, the system can be used to acquire, store and process data from one‐dimensional scans to obtain quantitative information about variations in optical density within the specimen, so considerably increasing the usefulness of the instrument. Although obviously limited in its capabilities, the system produces images of very high quality and one‐dimensional data of high sensitivity. The interface can be constructed for less than £40. A small modification to one of the M85 circuit boards is necessary to obtain maximum resolution.
A simple device for low temperature polymerization of Lowicryl K4M resin
Authors: Völker W. , Frick B. , Robenek H.
Published: August 2011

Difficulties concerning low temperature polymerization of Lowicryl K4M have been overcome by constructing and using a simplified polymerization chamber with plastic syringe. Five Beem capsules without cover containing the resin together with the specimen can be inserted into this syringe. For polymerization it is filled with nitrogen gas and closed tightly. Then the device is exposed to long wave UV‐light at 253 K. Using this polymerization chamber Lowicryl K4M resin blocks hardened completely and homogeneously.
Quantitative assessment of three‐dimensional roughness, anisotropy and angular distributions of fracture surfaces by stereometry
Authors: Exner Hans Eckart , Fripan Michael
Published: August 2011

Techniques for characterizing the geometrical nature of rough surfaces by three‐dimensional descriptors are developed. Measurement of surface coordinates from SEM stereopairs by semi‐automatic stereometry is used to establish the raw data. The surface area, the distribution of size and angles of triangular surface facets, the anisotropy of surface geometry, and surface roughness parameters can be assessed by various computing algorithms. Some typical results obtained for fracture surfaces in a ceramic material are shown. Problems, alternative methods and future developments are discussed.
Contacts in random packings of spheres
Authors: Pomeau Y. , Serra J.
Published: August 2011

The first section considers a packing of spheres with the same radius, whose contact zones are reduced to points. The number n of contacts per unit volume is derived from the histogram of the closest distances between couples of discs induced on a plane section. The second section extends the results of the preceding one to the case of slight elasticity. The new formulae differ noticeably from the previous ones. Finally, the case of a population of spheres with different radii is treated, and the two previous approaches are generalized. Once more, two‐dimensional estimates of the 3‐D contacts are provided.
Unbiased estimation of particle density in the tandem scanning reflected light microscope
Authors: Howard V. , Reid S. , Baddeley A. , Boyde A.
Published: August 2011

The tandem scanning reflected light microscope has the property of being able to obtain information from ‘inside’ solid objects by taking a thin optical section at the focal plane of the objective lens. This plane can be focused up and down through the specimen.

We describe an unbiased 3‐D counting rule for the TSRLM, which is applied to the estimation of osteocyte lacunar density in whole bone. This is shown to be an extremely efficient way of making such an estimate. Further possibilities for the application of the microscope in the field of stereology are discussed.
Manufacture and tests of objective zone plates for use in a scanning transmission X‐ray microscope
Authors: Michette A. G. , Burge R. E.
Published: August 2011

A method of making high resolution zone plates for use as the focusing elements in soft X‐ray microscopy is briefly described. Tests carried out on these zone plates indicate a first‐order diffraction efficiency of ∼0.3% rather than the calculated value of ∼0.9%. This indicates that the zones are not positioned as accurately as expected, a conclusion also drawn from tests at optical wavelengths on electron micrographs of the zone plates. Modifications to the manufacturing method to enable zone plates with improved imaging properties to be made are described.
The origins and development of scanning electron microscopy
Authors: Stewart A. D. G.
Published: August 2011

A short history of the factors which resulted in the Cambridge Instrument Company producing the first commercial scanning electron microscope and its effects on electron microscopy today. These early machines were primarily based on the research work done in the Cambridge University Engineering Laboratories and the Cavendish Laboratory.
The detective quantum efficiency of the scintillator/photomultiplier in the scanning electron microscope
Authors: Oatley C. W.
Published: August 2011

The efficiency of a scintillator for use with a photomultiplier in a scanning electron microscope has, in the past, been specified by its detective quantum efficiency (DQE). It is shown that this is likely to give misleading results since the DQE is the product of two factors, each of which may be influenced by the properties of the photomultiplier. It is further shown that there is reason to mistrust the theoretical basis on which the measurement of DQE rests. It is suggested that a better method of specifying the merit of a scintillator/photomultiplier system is by the mean number of electrons per pulse reaching the first dynode of the photomultiplier. A description is given of the way in which this quantity, ϕ, can be determined. Results of measurements of DQE and of ϕ for three commonly used scintillators are recorded.
Astigmatism correction and determination of resolving power in the SEM
Authors: Smith K. C. A.
Published: August 2011

Some of the early work carried out on the first scanning instrument constructed in the Cambridge University Engineering Department is recalled. The methods then used for astigmatism correction and the determination of resolving power are described and contrasted with later developments, particularly those relating to the use of on‐line digital computer techniques.
Assessment of electron irradiation damage to biomolecules using the Patterson function
Authors: Misra Manoj , Egerton R. F.
Published: August 2011

A method for the assessment of electron radiation damage to 5‐iodouracil is described which involves the use of the Patterson function. Changes in Patterson maps computed from the electron diffraction patterns recorded at increasing electron irradiation have been related to structural and chemical damage to 5‐iodouracil. The process of radiation damage is also discussed in terms of a disorder parameter which was found to increase with increasing irradiation.
Dilute aldehyde treatment on aldehyde‐fixed cells for observing chromosomes in situ with the scanning electron microscope
Authors: Maruyama Keizo
Published: August 2011

A new preparation method is introduced to reveal intracellular structures in the scanning electron microscope and its application to mitotic cells in root meristems of Vicia faba is demonstrated. The root tips are fixed with a mixture of formaldehyde and glutaraldehyde and the fixed tissues are frozen and fractured in liquid nitrogen. They are then incubated successively in dilute solutions of aldehyde (formaldehyde or glutaraldehyde) and osmium tetroxide. By this treatment, the excess cell‐matrix is removed from the fractured surface of the cell, and a deep view into the cell‐interior can be obtained with the scanning electron microscope. Varied levels of substructure are observed on the surface of chromosomes.
Glycol methacrylate embedding in histotechnology: factors which influence the evolution of heat during polymerization at room temperature
Authors: Gerrits P. O. , Leeuwen M. B. M. van
Published: August 2011

Benzoyl peroxide is widely applied as an initiator–accelerator system in combination with tertiary amines for the polymerization of water‐miscible methacrylates. However, this system is very sensitive to the ambient temperature and the evolution of exothermic heat is, especially at room temperature (293 K), difficult to control. Temperatures involved may increase beyond 373 K. A linear correlation between a graded series of benzoyl peroxide (0.2–1.0 g% benzoyl peroxide/glycol methacrylate) and the maximum temperature within the polymerizing plastic was established using a commercially‐available embedding system. Commercially‐available glycol methacrylates contain various concentrations of inhibitor in order to prevent spontaneous polymerization of the monomer. We tested a series of 200–300–400–800 ppm of the inhibitor hydroquinone and found a significant influence exerted upon the maximum temperature and the moment at which the maximum temperature was reached. Supplementary addition of inhibitors to the GMA monomer may effect a decrease of the maximum temperature within polymerizing GMA mixtures. Possibilities for standardizing the polymerization of glycol methacrylate by means of the benzoyl peroxide/tertiary amine initiator–accelerator system at room temperature are discussed.
Improved vacuum evaporation unit for beryllium coating for biological X‐ray microanalysis
Authors: Marshall A. T. , Carde D. , Kent M.
Published: August 2011

An improved vacuum evaporator is described for coating frozen‐hydrated biological samples with beryllium for X‐ray microanalysis. The evaporator permits repeated coatings without bringing the main chamber to atmospheric pressure and ambient temperature. The use of a glass sleeve in the evaporation chamber facilitates cleaning.
The relative merits of various cooling methods
Authors: Bald W. B.
Published: August 2011

Three different methods of rapidly cooling specimens prior to microscopical analysis are currently in use, namely, slamming, plunging and spraying.

The heat transfer processes and specimen cooling rates which are theoretically achievable by each method are discussed and compared with some of the published experimental data.

For the slamming method it is concluded that very little improvement in specimen freezing will be achieved by pre‐cooling the block below about 20 K and where pure silver is the most efficient block material.

The highest cooling rates and best microscopy by either the plunging or spraying method will be achieved using nitrogen at supercritical pressures. At subcritical pressures propane will produce the best results for metal samples whereas ethane is superior to propane for exposed biological specimens.

Future experimental work which relates ice crystal size to cooling rate must be based on the cooling rate at the instant of freezing or nucleation and not on some average cooling rate.