bluuebunny/crossref_metadata_2025 huggingface dataset:
Digital Picture Analysis In Microphotometry Of Biological Materials
Authors: Zimmer Hans‐Georg
Published: August 2011

Automatic evaluation of two‐dimensional thin‐layer‐chromatograms exemplarily demonstrates the applicability of signal processing, image analysis, and pattern recognition to microphotometry by an enlarged dynamic range of the photometer, improved reproducibility by noise reduction, discrimination between objects and background, feature extraction and automatic classification of the results. Emphasis is put on simple procedures which are tested in routine work and can be implemented on minicomputers.
Spectral Changes Of The Fluorescent Acriflavine‐(Feulgen) Dna Dye Complex At Different Temperatures: Application Of A New Cryostat For Measurements In Reflected And Transmitted Light
Authors: Tiffe H. W. , Matzke K. H. , Thiessen G.
Published: August 2011

The fluorometric behaviour of cellular objects is influenced during excitation by two nearly independent phenomena: (1) by the photochemical reaction of the DNA/AF dye complex, and (2) by the energy transfer among several DNA/AF dye complexes. Both processes show a distinct temperature‐dependent behaviour and can therefore be characterized by the analysis of the fluorescence spectra at different temperatures.

All microfluorometric measurements were performed with a self‐constructed cooling device. The cryostat permits measurements of the cellular fluorescence within a range of temperatures between 4 K and 300 K. The cooling unit operates in accordance with the‘Continuous Flow Principle’ and allows the application of objectives up to a numerical aperture of 0·6.
An Account Of Electron Optics In The Cavendish Laboratory And Related Developments
Authors: Hawkes P. W.
Published: August 2011

Contributions to many aspects of geometrical optics and digital image processing have originated in the Electron Microscopy Section of the Cavendish Laboratory. An account of these is given in the opening sections, in the context of related contemporary work, with particular attention to quadrupoles and high frequency lenses and to the phase problem and image handling. Subsequent developments, with especial emphasis on very recent progress, are briefly reviewed in the penultimate section.
X‐Ray Microanalysis
Authors: Duncumb P.
Published: August 2011

Over the past 30 years the electron probe microanalyser has progressed from a laboratory concept to a highly sensitive and well‐engineered instrument in use in various forms all over the world. Ellis Cosslett has been associated with this development for 25 of these years, with a particularly fruitful period in his laboratory during the late 1950s and the decade following. The subject has always illustrated well the interdisciplinary nature of instrument development, with the engineering design complemented by the physical theory of analysis and by the user disciplines represented in its application. This paper traces briefly some of the developments in the technique at various stages from designer to end‐user, with particular reference to the part played by Cosslett's laboratory and by others in the Cambridge area.
Electron microscopic appearances of rapidly frozen lung
Authors: Mazzone Robert W. , Durand Carlita M. , West John B.
Published: August 2011

Analysis of pulmonary structure‐function relationships by microscopy requites that the lung be fixed under carefully controlled physiological conditions, since lung structure is extremely responsive to the relationship between airway and vascular pressures. Unfortunately, standard post‐mortem fixation techniques leave some doubt as to the exact relationship between these pressures during fixation. This problem can be circumvented by stabilizing lung structure by rapid freezing under carefully controlled physiologic conditions. Using ethylene glycol in a freeze substitution technique we have developed procedures which yield a degree of preservation compatible with the high degree of resolution of the electron microscope. These can be used to obtain a more detailed understanding of pulmonary structure‐function relationships under well‐defined physiological conditions.
A method for polishing shale for vitrinite reflectivity measurements
Authors: Simpson W. B.
Published: August 2011

A method of polishing friable rock samples is proposed in which the usual lubricating agent, water or isopropyl alcohol, is eliminated and the sample polished dry.

The plucking usually seen with water‐sensitive samples is not seen and scratching of the surface is not a problem.

The reflectivity of vitrinite in a coal is compared when polished dry and by the usual wet method and is found to be similar in both cases.
A computerized graphical method for analysing stereo photo‐micrographs
Authors: Minter F. J. , Piller R. C.
Published: August 2011

A method has been devised for presenting three‐dimensional information from stereo pairs of electron micrographs in a graphical form. From this it is possible to obtain information about the spatial distribution of defects or precipitates, the existence and depth of denuded zones, the thickness, and the surface topography of thin foils examined by transmission electron microscopy.
Cross‐Sectional Transmission Electron Microscopy For Polycrystalline Silicon Films
Authors: Oppolzer H. , Falckenberg R. , Doering E.
Published: August 2011

The grain structure and the surface roughness of undoped polycrystalline silicon films were studied by TEM of cross‐sectional specimens. The grain diameter parallel to the surface could be directly determined as a function of the distance from the interface. An almost linear increase was found for all deposition temperatures between 923 K and 1123 K. For low deposition temperatures, the grains are columnar, while for high temperatures they are equiaxed. Cross‐sectional TEM‐specimens further allow a quantitative determination of the surface roughness. The roughness is not correlated with the grain diameter at the surface. A sharp increase of the roughness at 1123 K can be explained by the disappearance of a preferred orientation of the grains.
The preparation of calibration standards for sodium, potassium and chlorine analyses by analytical electron microscopy
Authors: Harvey Diana M. R. , Flowers T. J. , Hall J. L. , Spurr A. R.
Published: August 2011

Attempts were made to prepare sodium and potassium thin section standards for analytical electron microscopy by introducing, in serial concentrations, a complex between the appropriate salt and macrocyclic polyether ([15] crown‐5 for sodium and [18] crown‐6 for potassium) into epoxy resin. Of the tested salts, sodium tetraphenylborate, tetrafluoroborate and cyanide failed to produce homogeneous standards, and those standards containing sodium or potassium iodide or bromide were analytically inhomogeneous at concentrations greater than 20 meq 1−1. Only sodium thiocyanate and potassium cyanide standards were analytically homogeneous at concentrations up to 600 meq 1−1. A chlorine standard, which was analytically homogeneous at concentrations up to 1000 meq 1−1, was prepared by incorporating 1,2,4‐trichlorobenzene into an epoxy resin.
Dimensions of active cytochrome c oxidase in reconstituted liposomes using a gold ball shadow width standard: a freeze‐etch electron microscopy study
Authors: Ruben George C. , Telford John N.
Published: August 2011

The preparation and characterization of a distribution of gold balls on a thin, flat carbon film is described. The relation of the platinum carbon shadow width distribution means to a gold ball size is reported.

Freeze‐etched cytochrome oxidase vesicles and gold ball calibration grids were simultaneously shadowed with platinum/carbon. The shadow width distribution of the cytochrome oxidase located in and spanning the membrane was measured. The membrane fracture face edge and cross‐fractured bilayer membrane edge were also measured.

Dimensions of the cytochrome oxidase were found to be 5·8 ± 0·3 nm in diameter parallel to the membrane and 8·2 ± 0·3 nm long across the membrane. The bilayer membrane dimensions were 3·0 ± 0·3 nm for the half bilayer and 5·8 ± 0·3 nm for the cross‐fractured bilayer membrane edge thickness. The length of the cytochrome oxidase was observed to span the bilayer membrane. Previous X‐ray diffraction measurements on similar hydrated liquid crystalline artificial membranes were found to be in good agreement with the freeze‐etched results.

Membrane widths from thin‐sectioned cytochrome oxidase vesicles were measured and found to be 5·8–5·9 nm in non‐post‐stained sections. Post‐staining with uranyl acetate and/or lead citrate was shown to increase this average thickness.

The technique of freeze‐etching electron microscopy in conjunction with the gold ball shadow width calibration experiment has been shown to provide accurate and precise measurements of membranes and a functional intramembrane protein in a hydrated non‐crystalline sample.
Surface studies in a UHV field emission gun scanning electron microscope
Authors: Venables J. A. , Janssen A. P. , Akhter P. , Derrien J. , Harland C. J.
Published: August 2011

Recent instrumental advances have enabled scanning electron microscopy using a field emission gun (FEG) to be combined with an ultra high vacuum specimen chamber. The resulting instrument, the UHV‐FEG‐SEM, allows high spatial resolution microstructural and micro‐analytic information to be obtained reliably from clean surfaces for the first time.

Such an instrument has been installed and developed at the University of Sussex. It incorporates SEM at ∼5 nm resolution, scanning Auger microscopy at ∼30 nm resolution, and various diffraction techniques, including RHEED and electron backscattering pattern (EBSP) techniques, at (lateral) resolutions which can approach 10 nm. The work function can also be measured microscopically as demonstrated in this paper. Combined with facilities for in situ cleaning and molecular beam deposition, these techniques represent a considerable improvement in our ability to study surface and near‐surface phenomena on a microscopic scale.

Examples of recent work on technique development, including work‐function measurement and backscattering patterns, are described. Recent applications to metal and semiconductor systems are described, including the crystal growth of Ag on Si (111) and Mo (100), the composition profiles of GaAs lasers, and adsorbed Cs layers on tungsten.
Observations on deformed electrolytically deposited Ni3P
Authors: Donovan E. , Stobbs W. M.
Published: August 2011

Electrodeposited amorphous Ni3P was deformed in situ on a brass substrate. The films, when observed in the electron microscope, showed both light and dark bands in bright field images which were not seen in undeformed material. It is proposed that the origin of this contrast is a change of both density and structure of the material within the deformation bands.
Internal and platinum initiated crystallization in Na2O.2 CaO. 3 SiO2 glasses: early stages of growth
Authors: Gonzalez‐Oliver C. J. R. , James P. F.
Published: August 2011

Transmission electron microscopy at 100, 900 and 1000 kV was used to study the early stages of growth of internally nucleated crystals of the phase Na2O.2 CaO. 3 SiO2 (‘NC2S3’) in glasses close to the NC2S3 composition. Glass samples were partially crystallized by heat treatment in the range 833–873 K and thin foils for electron microscopy were prepared by ion beam thinning. The phase identified by electron diffraction from a number of small crystals (the low temperature form of NC2S3) was the same as that determined by X‐ray diffraction of fully crystallized glass ceramics. Selected area diffraction and bright and dark field microscopy showed that in the early stages of development the crystallization centres were essentially single crystals but contained a significant concentration of defects. These crystals were nucleated, probably homogeneously, at temperatures higher than the well‐established low to high reversible polymorphic transformation of the NC2S3 crystalline phase. In a glass of similar composition to which platinum was deliberately added there was clear evidence for heterogeneous nucleation of NC2S3 crystals on platinum particles distributed in the glass. Also the contact angle between NC2S3 crystals and the rounded platinum particles could be measured directly.
High resolution studies of glass‐crystal interfaces
Authors: Krivanek O. L.
Published: August 2011

High resolution electron microscopy has been used to image intergranular glassy phases in sintered and hot‐pressed nitrogen ceramics, and the Si‐SiO2 interfaces important in MOS devices.

The thin intergranular glassy phases were found at most grain boundaries in all liquid‐phase sintered or hot‐pressed ceramics examined. Silicon carbide sintered by a solid state diffusion process, however, contained no such phases.

Si‐SiO2 interfaces have been examined in cross‐section at about 0ṁ3 nm resolution. The images show that the transition from the Si crystal to the amorphous oxide was abrupt, but that there were 0ṁ3‐0ṁ5 nm high hills on the Si surface, separated by 2–4 nm, and long‐range undulations of 0ṁ6‐0ṁ8 nm height and 20–50 nm periodicity. Ultra‐thin (3 nm) oxides were also examined and were found to be uniform in thinkness, indicating that the roughness at the Si‐SiO2 interfaces is initially caused by irregularities on the original, unoxidized Si surface.
The initial stages of ordering in alloys studied by electron diffraction and high resolution microscopy
Authors: Tendeloo G. Van
Published: August 2011

The combined use of selected area electron diffraction and high resolution electron microscopy can provide important information about the microstructure of materials. Both techniques have been applied to study the ordering behaviour in a number of alloys. The ordering process is not always straightforward and mostly intermediate long period modulated structures are formed. Using dark field high resolution electron microscopy the ordering sequence as well as the structure of the intermediate phases have been established for Au4Mn and Au5Mn2. For Ni4Mo the high resolution observations provide new information about the ordering mechanism in its very earliest stages.
Protein staining methods in quantitative cytochemistry
Authors: Tas Johan , Ploeg Mels van der , Mitchell JohnP. , Cohn NormanS.
Published: August 2011

The chemical action and practical application of the Naphthol Yellow S, Alkaline Fast Green, Coomassie Brilliant Blue, Dinitrofluorobenzene and some lesser known protein staining methods have been surveyed with respect to their potentialities for quantitative cytochemical analyses. None of the dyes can be said to bind to any specific protein or group of proteins, but each may be used to analyse the presence of one or more particular amino acid residues. For the cytophotometric measurement of the ‘total protein content’ of individual cells and cell organelles the covalent binding Dinitrofluorobenzene and the electrostatic binding Naphthol Yellow S can properly be used. Fast Green FCF, applied at alkaline pH, binds electrostatically to the basic amino acid side chains of strongly basic proteins only but not in a quantitative (stoichiometrical) way. Coomassie Brilliant Blue, recently introduced to protein cytochemistry, may be useful for quantitative purposes. The combined Feulgen‐Pararosaniline(SO2)/Naphthol Yellow S and Dinitrofluoro‐benzene/Feulgen‐Pararosaniline(SO2) methods enable the simultaneous cytophotometric analysis at two different wavelengths for protein and DNA within the same microscopical preparation.
Preliminary oxidation in histochemical staining methods for cholesterol
Authors: Adams C. W. M. , High O. B. Bayliss
Published: August 2011

The need for preliminary oxidation with histochemical methods for cholesterol was investigated on silica‐coated sheets and in tissue sections. The techniques used were the Schultz reaction, perchloric acid‐naphthoquinone (PAN), Lewis & Lobban's ferric alum‐sulphuric acid reagent and Okamoto's iodine‐sulphuric acid. The oxidants assessed were ferric chloride, ferric alum, potassium permanganate, ammonium sulphamate and ultraviolet light. The best combinations amongst those tested in order of reactivity were FeCl3‐PAN, ferric alum‐Schultz, Lewis‐Lobban (no additional oxidant), iodine‐sulphuric acid (no additional oxidant). Authentic preparations of cholesterol oxidation products were stained with these methods, but the nature of the oxidized product in the preliminary stage could not be determined.
Transmission electron microscopy of organic pigments
Authors: Fryer J. R.
Published: August 2011

Preparative techniques and electron microscopy applicable to organic pigments have been illustrated by the phthalocyanines, also showing that particle ablation and changes in shape occur before extensive lattice degradation. Similar radiation damage mechanisms are shown by both the transmission electron microscope and the scanning transmission electron microscope. Lattice resolution in phthalocyanines permits a definition of the primary particle in pigment agglomerates.
Morphological characterization of liposome suspensions by stereological analysis of freeze‐fracture replicas from spray‐frozen samples
Authors: Guiot Pierre , Baudhuin Pierre , Gotfredsen Carsten
Published: August 2011

A method for size distribution analysis of liposome suspensions is described. Spray‐freezing was used in order to avoid cryoprotectants and fixatives. Light scattering showed that, depending on their composition, liposomes can be modified by the spraying procedure. Dipalmitoylphosphatidylcholine liposome suspensions display large modifications of light scattering upon spraying. The commonly used unilamellar phosphatidylserine/phosphatidylcholine (1:4) liposomes, with or without cholesterol, are, however, only slightly affected by the spraying procedure.

For liposome suspensions stable upon spraying, size distribution analysis can be performed on freeze‐fracture replicas by standard procedures and average size, area and volume of particles are obtained. The number of particles per unit volume cannot be determined by stereological procedures, since the fracture face does not correspond to a plane section through the sample. By combining stereological data with phospholipid mass and specific molecular area of phospholipids, abundance of particles, fractional volume and total membrane area can nevertheless be evaluated.

Data on small unilamellar liposomes prepared by sonication are presented as an illustration of the method. In suspensions obtained by this procedure, vesicles may be considered at thermodynamic equilibrium in the aqueous phase. The experimental size distribution compared favourably with that obtained from self‐assembly theory for liposomes, considering natural lecithin heterogeneity. For comparison purposes, data on large unilamellar liposomes, obtained by reverse‐phase evaporation, are also presented.
A cathodoluminescent study of quartz sand grains
Authors: Tovey N. K. , Krinsley D. H.
Published: August 2011

Surfaces and cross sections of individual quartz sand grains have been examined in both the emissive and cathodoluminescent (CL) modes using a scanning electron microscope. The cathodoluminescent micrographs reveal sub‐surface information not seen in the emissive mode micrographs. The CL features are generally associated with previous stressing, fracturing or abrasion of the grains, and are probably related to the formation of disrupted‐lattice and/or amorphous quartz. The sequence of events during the formation of these features can be identified on many grains by studying the spatial distribution of the most common form of CL contrast—narrow dark bands. On other grains, irregularly distributed dark patches are generally observed to be a surface phenomenon, but the distribution, depth and intensity of these features can be studied by examining cross sections of grains. While it is common for the banded features to cross the entire specimen, it is rare for the diffuse areas to penetrate more than 30 μm. Some grains when experimentally heated above the α‐β transition temperature show a marked change and even a complete reversal in CL contrast.

Such studies of grains in cathodoluminescence can supplement the environmental information obtained from emissive mode micrographs of geological materials.
Improvement in microscope photometry by voltage to frequency conversion: analogue measurement and digital processing
Authors: Freitas M. I. , Giordano P. A. , Bottiroli G.
Published: August 2011

A system for digital measurement of microscope photometric signals, based on Voltage to Frequency Conversion (VFC) and suitable for automatic data processing, has been devised.

Advantages and disadvantages are illustrated with special emphasis on the comparison between photocurrent measuring and photon counting techniques. A statistical analysis of the results allows the calibration of VFC net counts versus detected photons. A biological application is reported; this shows the suitability of the system even to low light levels.
Stereological meaning of the inflection point count
Authors: DeHoff R. T.
Published: August 2011

The relationship between the inflection point count on a representative microsection and the integral‐asymptotoic line curvature of saddle surface in the system constitutes a new fundamental stereological equation. While the geometric meaning of this quantity cannot be readily visualized, it is unambiguous, and general for all structures. Combinations of the inflection point count with other descriptors of two‐dimensional structures provide potentially useful descriptors of particle roughness, scale, and some aspects of shape. Like all measures of boundary properties, the inflection point count is sensitive to resolution; it is thus essential to report the level of resolution in conjuction with any experimental determination of IA.
Electron beam current measurement in the electron microscope
Authors: Nicholson W. A. P.
Published: August 2011

Two devices for measuring the beam current in an electron microscope are described; a Faraday cage rod for accurate absolute measurements in the specimen position and a collector plate mounted in the viewing chamber, which may be used continuously during experiments, for relative current measurements. Results of experiments to determine the effect of secondary electrons on, and choice of material of, the collector plate are presented. An example of the use of the collector plate as a photometer is given.
Some modifications on freeze‐fracture replica preparation
Authors: Bosch M. Van den , Jacob W.
Published: August 2011

Freeze‐fracture replicas are freed from lipid material by saponification in a hot sodium hydroxide solution and subsequently rinsed with 10% ethanol in distilled water. All manipulations are carried out with a glass bead fashioned onto a pasteur pipette. A stable, two‐layered plastic support film has been developed to allow the use of grids with larger openings so that more of the specimen is observable.
Stereological estimation of volume ratios by systematic sections
Authors: Cruz‐Orive L. M. , Myking A. O.
Published: August 2011

Two related problems are explored. Firstly, a single opaque solid Ω1 of arbitrary size and shape, containing an arbitrarily shaped phase Ω2, is considered. The problem is to estimate the minimum number of systematic sections m, necessary to estimate the volume ratio v = V(Ω2)/V(Ω1) with a coefficient of error of at most γ0 with a probability 1 ‐ α. Secondly, we consider a population of such specimens. The second problem is to estimate the optimum number n of specimens to be sampled and the number m of systematic sections per specimen in order to estimate the mean volume ratio of the population with a relative error of at most ∍0 with a probability 1 ‐ α.

General guidelines for solving the two problems are presented. Practical results applicable to two populations of mouse and guinea‐pig lymph nodes, exhibiting a wide variation in size and shape, are obtained.
Trace fossils as revealed by staining: a light microscope/scanning electron microscope study of the ancient microbiota from carbonate matrix
Authors: Karkhanis Suresh N.
Published: August 2011

Previously invisible fossil remains of ancient iron‐bearing microbiota from the carbonate matrix have been detected by staining the petrographic thin sections by weakly acidic solutions of potassium ferricyanide. The chemical staining technique imparts a deep blue colour—Turnbull's blue—in the presence of ferrous iron to these fossil remains, which are considered to be an example of authigenic preservation process, whereby the microbiota are preserved as carbonate molds or casts.

The technique was found to be suitable for Proterozoic rocks and has been further extended to Archaean rocks. Hence its universality.
The myosin filament
Authors: Ashton Francis T. , Pepe Frank A.
Published: August 2011

Different multistage fixation procedures patterned after the procedure described by Reedy (1976) have been evaluated with regard to preservation of subfllament structural organization along the length of the vertebrate skeletal myosin filament. The criteria used for evaluation of structural preservation include (a) the precision of subfllament organization as reflected by the quality of the optical diffraction patterns obtained from images of transverse sections of the myosin filament and (b) the clarity with which structural differences in serial transverse sections along the myosin filament could be defined.
Scanning immuno‐electron microscopy of human leukaemia and lymphoma cells: a comparative study of techniques using immunolatex spheres as markers
Authors: Gamliel Haim , Leizerowitz Rachel , Gurfel Dorit , Polliack Aaron
Published: August 2011

In this study scanning immuno‐electron microscopic (SIEM) techniques were used to identify human leukaemia‐lymphoma cells. Monodispersed polystyrene (latex) beads were conjugated to specific antisera using glutaraldehyde, in an attempt to detect surface antigenic components on a variety of cells of known origin. Antisera, mostly immunoglobulin fractions, against human thymus (T) derived cells, common type acute lymphoblastic leukaemia cells (C/ALL) and surface immunoglobulin (sIg) bearing cells were used to coat latex spheres, while rabbit anti‐mouse Thy‐1 antiserum or whole human‐IgG (γ‐globulin) bound to latex were used as controls in some experiments. The use of SIEM techniques in the direct mode as a simple and sensitive method for labelling surface antigens is described. The disadvantages of the SIEM methodology are also summarized while the requirements for optimal cell preparation using this technique are stressed. The experiments were designed to ascertain whether prolonged fixation of cells could be used prior to incubation of the cells with the marker. In this respect, repeated neutralization of the glutaraldehyde with glycine is essential. SIEM labelling of cells is random and unreliable without adequate quenching with glycine. The heteroantisera used in this study proved to be adequate and insignificant non‐specific attachment and cross reactivity were seen. SIEM adds a further dimension to ultrastructural aspects of immunology and is a potentially useful tool in the study and identification of leukaemia and lymphoma cells.
Use of a multichannel pulse height analyser for the analysis of back‐scattered SEM images
Authors: Hall M. G. , Skinner G. K.
Published: August 2011

The use of a simple electronic switch to convert the video signal from a scanning electron microscope to a series of pulses is described. A multichannel pulse height analyser may then be used to perform quantitative image analysis. Examples are given showing how composition analysis, area fraction measurement and image contouring may be performed using the atomic number contrast signal from a back‐scattered electron detector. Other detector systems such as scintillators or specimen current imaging could also be used.
The calibration of standards for the measurement of specular reflectance with a microscope photometer
Authors: Piller Horst
Published: August 2011

Solid standards with reliable reflectance references are of fundamental importance for exact measurements of specular reflectances of microscopical objects. The liquid surface is the utopia of surfaces sought as reference for the calibration of solid standards. However, there are difficulties in setting the measuring equipment and adjusting the liquid surface for the comparison with the solid body. In this paper, the ‘liquid’ method is described clearly for microscopists to consider.
Application of morphometrical methods in studies of the mandibular condyle
Authors: Luder H. U. , Weilenmann W. , Schroeder H. E.
Published: August 2011

Two mandibular condyles of a growing monkey (Macaca fascicularis) were used for developing and testing a morphometrical model system suitable for generating quantitative data about the structure of and the growth activities in the articulating surface layers. The animal received 3H‐proline (1 mCi/kg body weight) 24 h and 3H‐thymidine (0.5 mCi/kg body weight) 3 h prior to killing. Using standardized conditions, 1–2 μm thick histological sections were processed for autoradiography. The morphometrical analysis was carried out using light microscopy at two levels of magnification. Sampling of tissue fields was performed at twenty‐four (level I) and twelve (level II) different sites, respectively. These sites were distributed systematically in the anteroposterior and lateromedial dimension of the cartilaginous condylar covering. Using a multivariate analysis of variance, a parameter comparison revealed significant differences between single sampling sites in the anteroposterior as well as in the lateromedial direction, suggesting that the articulating condylar surface is inhomogeneous with respect to both structures and growth activities. The sampling mode eventually arrived at in this study was shown to be sufficient for the recording of gradients within the condylar surface layers. The model system presented permits simultaneous quantitative characterization of structure and growth activities such as mitotic cell division and extracellular matrix production of the condylar cartilage including the zone of its replacement by bone.
Propane jet‐freezing: A valid ultra‐rapid freezing method for the preservation of temperature dependent lipid phases
Authors: van Venetië R. , Hage W. J. , Bluemink J. G. , Verkleij A. J.
Published: August 2011

We have developed a temperature controlled specimen stage on the existing jet‐freezing apparatus, in order to quench samples from elevated temperatures up to 330 K. We have shown that the phase transition both from the liquid‐crystalline to solid phase for pure phosphatidylcholines and from hexagonal II to lamellar phase for phosphatidylethanolamine/4% egg phosphatidic acid is prevented. Moreover, cooling rates exceeding 10,000 K s—1 were recorded indicative of ultra‐rapid freezing capacity. Cryoprotectants can be omitted, since phase separation due to ice crystal growth does not occur.
Precipitation induced by ion beam thinning
Authors: Njegic A. , Williams D. B.
Published: August 2011

Ion beam thinning of aluminium–lithium alloys under conventional conditions required to produce electron microscope thin foils is shown to induce significant microstructural changes, equivalent to heating the specimen in excess of ∼400 K.
An electron microprobe study of the influence of beam current density on the stability of detectable elements in mixed‐salts (isoatomic) microdroplets
Authors: Morgan A. J. , Davies T. W.
Published: August 2011

This paper shows that a means of accurately measuring beam current during microprobe analysis of inorganic fluid microdroplets is essential, since certain elements were sublimated from such specimens under easily achieved beam current densities, i.e. S at 1·8 nA/μm2, K at 2·5 nA/μm2, Na at 3·5 nA/μm2, P at 5·3 nA/μm2. In comparison, Cl was volatilized even under the mildest conditions used (0·35 nA/μm2), and Ca, Mg and Co were stable under the severest operating conditions (7·1 nA/μm2). Elements were less stable in large (3 μm diameter) droplets than in small (1 μm) droplets under identical irradiation conditions. The onset of volatilization is a direct function of the current delivered per unit area and not of the total integrated dose.

The addition of 50 g/l of urea to the mixed‐salts (isoatomic) solution, or (a) the mounting of the droplets so that the carbon‐celloidin support film was interposed between them and the electron source, and (b) top‐coating the droplets with carbon, did not, in general, raise the threshold of volatilization of a given element, but did effectively retard the rate of loss at current densities above the volatilization threshold. A literature survey confirmed that similar losses can occur from biological tissue specimens, albeit at higher beam current densities.

Finally, the possibility that local specimen heating during electron/specimen interaction is a cause of element loss during microprobe analysis is discussed.
Cryopreparation of mammalian tissue for X‐ray microanalysis in STEM
Authors: Zierold Karl
Published: August 2011

A cryopreparation technique for studies of ultrastructure and distribution of diffusible elements in biological tissue is described. Electron microscopical contrast and characteristic X‐ray spectra are found to be poor in completely frozen‐hydrated ultrathin cryosections of fresh chemically untreated tissue. Both STEM contrast and detection of characteristic X‐rays are enhanced by careful freeze‐drying in the microscope. Although the ultrastructure is affected by ice crystals, intracellular compartments can be identified by STEM without staining and studied by X‐ray microanalysis.
Intramembrane particle movement revealed by study of an intercellular junction
Authors: Green Colin R.
Published: August 2011

Comparison of results from thin section and freeze‐fracture techniques on the echinoderm epidermal septate junction has shown that a large degree of intramembrane particle translocation is possible during fixation and cryoprotection. The intramembrane and intermembrane components of the junction are apparently separated from each other in the process. Lanthanum tracer studies show that septa of this junction are double, straight, unbranched structures running around the cell apex. Freeze‐fracture of fixed tissue reveals junctional particles or pits in a broad band, also around the apical circumference of cells. However, they do not line up as would be expected after viewing septa in tangential sections and form a belt of randomly dispersed structures. In contrast, replicas of unfixed tissue show particles that clearly align in rows, often double, as are the septa in tangential views. It appears that intramembrane structural changes revealed by freeze‐fracture do not necessarily reflect corresponding intercellular changes.
Specimen preparation for electron microscopy using low temperature embedding resins
Authors: Armbruster B. L. , Carlemalm E. , Chiovetti R. , Garavito R. M. , Hobot J. A. , Kellenberger E. , Villiger W.
Published: August 2011

Studies using polar and non‐polar methacrylate‐based resins (Lowicryl® K4M and HM20) suitable for low temperature embedding are described. We present the first applications of the system to various membrane structures in glutaraldehyde‐fixed, uranyl acetate‐stained thin sections of bacteria, mitochondria and cell‐cell contact regions.
Freezing patterns in quench frozen, freeze‐dried polyvinylpyrrolidone (PVP)
Authors: Scheie Paul , Dalen Helge , Saetersdal Thorvald , Myklebust Reidar
Published: August 2011

An SEM investigation of the effects of freezing and freeze‐drying on 2 mm diameter columns as well as on 10 mm diameter thick discs of PVP was carried out using standard techniques of quench freezing and cryofracturing followed by freeze‐drying. Quench freezing in liquid Freon 12 or liquid propane led to patterns of ice formation that depended on the position in the sample, the orientation of the fracture plane observed relative to the geometry of the sample and the concentration of PVP. Vitrification did not occur. Quench freezing in liquid N2 resulted in larger ice crystals and the pattern of the crystals was much less predictable.
Transfer, observation and analysis of frozen hydrated specimens
Authors: Hax Werner M. A. , Lichtenegger Siegfried
Published: August 2011

Hitherto, the observation of frozen hydrated specimens in transmission electron microscopes has been inhibited due to the technical difficulties experienced in transferring the specimen to the microscope and maintaining it at a low temperature during observation. This has resulted in loss of the primary advantage of freezing since the frozen water had to be removed from the specimen before it could be introduced into the electron microscope.

The cryo‐transfer system overcomes these objections and provides a means to transfer frozen hydrated specimens from any preparation equipment into the microscope without ice condensation on the specimen. The cryo‐transfer system consists of a cryo‐transfer unit, a cryo‐specimen holder and a temperature control unit.
A television system for point counting morphometry in high resolution light microscopy
Authors: Nielsen H. Winther
Published: August 2011

High resolution light microscopical images that can be superimposed on a test system for the estimation of stereological parameters are obtained by a television system modified for point‐counting morphometry. The full resolving power of the light microscope is exploited and a test system applied in the same plane as the microscopical image displayed on the monitor screen. Volume fraction estimates of nuclei and zymogen granules from the rat pancreas made with the television microscopical system and with electron‐microscopic micrographs rendered the same precision, and showed that the volume fraction of objects as small as zymogen granules was measurable with the light microscope. The measuring with the television microscopical system was about 15 times faster than with electron‐microscopic micrography and about 5 times faster than with conventional photomicrography and the light microscope.
Multiple image integration: a new method in electron microscopy
Authors: Wrigley N. G. , Chillingworth R. K. , Brown Elaine , Barrett A. N.
Published: August 2011

An optical ‘flicker’ method is described for the precise azimuthal and translational co‐registration of many noisy but identical molecular images. Starting with a real micrograph of known biological objects showing no visible detail below 4 nm, a lattice of images of individual objects was synthesized by computer and translationally filtered, using real experimental data throughout. Detail was recovered conforming with known structural features of the object down to about 1·5 nm, and rotational analysis showed that the registration accuracy of the lattice elements was better than 0·5 nm on the object. Application to the straightening of real two‐dimensional lattices with long‐range distortion is discussed.
Point projection imaging of unstained ferritin molecules on tungsten*
Authors: Panitz J. A. , Ghiglia D. C.
Published: August 2011

Point‐projection microscopy has been used to obtain the first images of closely spaced (but isolated) unstained ferritin molecules on a tungsten substrate. The microscopy, in conjunction with a digital processing algorithm, produces images which display a striking three‐dimensional quality usually associated with scanning electron micrographs. From these images, an apoferritin diameter of 13 nm has been deduced. This is identical to the diameter of apoferritin obtained from a reconstruction of the molecule using X‐ray coordinate data, but almost 70% larger than that deduced from TEM images of ferritin in which only its iron rich core can be seen.
A grinding method for producing sections of large areas of plastic embedded tissues
Authors: Weber Dennis F. , Eisenmann Dale R. , Zaki Abd El‐Moneim
Published: August 2011

We have developed a method utilizing relatively thick ground sections of plastic embedded tissue which affords the resolution obtained with 0·5 μm cut sections. The sections, which are permanently affixed to plastic microscope slides, are much larger in area than ultramicrotome sections. Additional advantages are: sections can be destained and restained and selected areas can be examined with various forms of electron microscopy. Autoradiographic studies are also possible. Although the method has a broader application, it is particularly useful in examining the interface between hard and soft tissues.
Microscopic observations on endogenous fluorochromes within a nerve fibre excited by a 325 nm He‐Cd laser
Authors: Cummins J. T. , Rahn C. L. , Rahn R. S.
Published: August 2011

A microscopic procedure to observe endogenous fluorochromes in superfused tissues is described. In this procedure, a 325 nm beam from a He‐Cd laser is directed through the objective lens of a microscope onto the surface of a brain slice. Fluorescent structures within the tissue can be observed, photographed and measured. A nerve fibre running from the corporis callosi to the dorsal cerebral cortex was studied with this approach. The 325 nm laser beam excited material with an emission peak at 420 nm within a fibre on the cut surface of a rat brain slice superfused with Krebs‐Ringer. A similar fibre was observed in vivo in the exposed mouse brain. Packets of the fluorescent material appear to move within the fibre in vitro. The fluorescence of the fibre was rapidly diminished by cyanide, colchicine, rotenone and the withdrawal of Ca2+. Elevation of [K+] in the superfusate caused a ‘haloing’ of the fibre's granularity without decreasing the total fluorescence. This phenomenon is possibly related to the depolarization by [K+] of neurones. Although the structure of the fluorochrome was not established, it is suggested that it may be a neurotransmitter‐related substance undergoing axonal movement.
The simultaneous use of Hanks' and Hepes buffers in the preparation of human leucocytes for SEM observation
Authors: Lalague E. D. , Haviernick S. , Corvellec M.‐R. , Pelletier L. , Derghazarian C. , Cousineau G. H.
Published: August 2011

Leucocytes were isolated from the peripheral blood of normal individuals and prepared for SEM observation. The addition of the buffer Hanks and Hepes to the glutaraldehyde fixative was found to maintain the initial ambient pH throughout cell manipulation, and to allow satisfactory preservation of surface architecture.
A simple quantitative analysis of phase contrast microscopy, not restricted to objects of very low retardation
Authors: Goldstein D. J.
Published: August 2011

A simple vector analysis of the diffraction of light by an ideal, extended phase grating suggests that irrespective of the amount of retardation imposed on the light passing through a transparent microscopic object the diffracted light leaving the object plane is precisely 90° out of phase with the resultant direct (zero‐order diffracted) light. In positive phase contrast microscopy, with a 90° phase plate of transmittance T for the direct light, the image and halo respectively have intensities relative to the original illumination of (√T.cos 1/2Φ–sin 1/2Φ)2 and (√T.cos 1/2Φ+sin 1/2Φ)2. The empty background has an apparent intensity of T. Zero intensity of the image is seen if T=tan2(1/2Φ), and reversal of contrast if T≤tan2(1/4Φ). The same equations can be used to predict the intensities of the image and halo in negative phase contrast microscopy, if Φ is replaced by (360°‐Φ).

Unlike the ‘standard’ description of phase contrast microscopy, exemplified by Barer's vector method, the present account is consistent with the conservation of energy and is not restricted to very small object retardations. Barer's method is, however, theoretically valid for objects of any shape, and the two approaches may perhaps be regarded as complementary.
Improved low dose technique for high resolution electron microscopy
Authors: Chalcroft J. P. , Tavener A.
Published: August 2011

The use of low‐dose electron micrography can result in unacceptable focusing errors if the specimen plane between focus and micrography areas is tilted. The corrective method applied here involves the use of two focus areas symmetrically disposed with respect to the area to be recorded. A reasonable estimate of correct focus can be made by interpolation between the two known settings.

Annular illumination applied to the specimen immediately prior to recording is recommended to reduce any likelihood of specimen drift caused by changing thermal gradients.

The methods involve only minor modifications to existing circuitry, are easy to implement, and have resulted in a dramatically improved percentage of well‐focused micrographs.
Permanent slides of freshly collected algae: a review of techniques
Authors: Foote MaryAnn
Published: August 2011

A number of techniques are given for preparing permanent slides of freshly collected algae. The methods reviewed are suitable for phytoplankton, delicate species, filamentous algae and marine samples. Included are traditional techniques using corn syrup or glycerin and also a new technique with a commercial histological product.
Incident light microscopy of normal and transformed cultured fibroblasts stained with Coomassie blue R250
Authors: Paddock Stephen W.
Published: August 2011

Incident light microscopy of fibroblasts stained with Coomassie blue R250 results in images of the cytoskeleton with a similar high contrast to those obtained by specific immunofluorescence microscopy. This technique is especially useful for the routine visualization of the cytoskeleton of transformed cells.
The application of automatic image analysis in studies of bubble populations produced by radiation damage
Authors: Shaw M. P. , Kohler V. L. , Ralph Brian
Published: August 2011

This paper describes a method by which automatic image analysis can be applied to the study of size distributions of small (< 5 nm) radiation induced bubbles. Statistically valid results are obtained directly from transmission electron microscope negatives and the optimum imaging conditions for obtaining reliable data are discussed.
The preservation of mucus and surface‐associated microorganisms using acrolein vapour fixation
Authors: Garland C. D. , Nash G. V. , McMeekin T. A.
Published: August 2011

Three fixation schedules were devised and compared in terms of their influence on the preservation of mucus and surface‐associated microorganisms contained within it. Different mucus‐secreting epithelial tissues from normal and spoiled oysters and normal rats were examined by scanning and transmission electron microscopy and light microscopy. On all tissues, mucus was best preserved in specimens fixed by 10% acrolein vapour for 1 h then immersed in 3% glutaraldehyde—3% formaldehyde fixative containing 0·05% ruthenium red, cacodylate buffer pH 7·4, for at least 3 h. This fixation schedule also greatly increased the preservation of microorganisms in mucus in specimens from spoiled oysters and normal rats. In contrast, the retention of mucus and surface‐associated microorganisms was poor in tissues fixed either by 1% OsO4 vapour for 1 h followed by immersion in combined aldehyde fixative, or by direct immersion.

The quality of preservation of the mucus layer, epithelium and sub‐epithelium was also noted by transmission electron microscopy in tissues prepared by the different fixation schedules. Cellular preservation was satisfactory in directly immersed tissues but poor in vapour fixed specimens.