bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis by confocal scanning laser microscopy imaging of the spatial distribution of intermediate filaments in foetal and adult rat liver cells
Authors: Vassy Jany , Rigaut Jean Paul , Hill Anne‐Marie , Foucrier Jean
Published: August 2011

Confocal scanning laser microscopy has been used to make three‐dimensional observations of the spatial distribution of cytoskeleton intermediate filaments in rat liver hepatocytes, at various stages during foetal development and in the adult. Single and double immuno‐labelling with fluorescein and Texas Red fluorescence have been used to study the intracellular spatial distribution of C18 cytokeratin and vimentin. Simultaneous confocal imaging with double‐fluorescence emission requires an image processing step for the correction of ‘contamination’ effects due to the overlap between fluorescein and Texas Red emission spectra.

At the pre‐natal period (day 20 of gestation) each type of intermediate filament labelling is only present in a certain cellular category, C18 cytokeratin in hepatocytes and vimentin in mesenchymal cells. However, at the earliest developmental stages (day 12 of gestation), vimentin and cytokeratin seem to be found in the same type of cells, probably mesenchymal cells.

Some striking developmental changes, associated with the differentiation of the liver parenchyma, are observed for both C18 cytokeratin and vimentin. In earlier foetal stages, C18 filaments are scarce, hazily labelled and randomly distributed inside the hepatocytic cytoplasm. Late during foetal development (days 18–20 of gestation), hepatocytic cytokeratin filaments are abundant, well individualized and sharply labelled. The hepatocytes are arranged in a muralium duplex architecture (two‐cell‐thick sheets) and the labelling intensity measured in the hepatocytic cytoplasm at the basal pole is double that measured at the sinusoidal pole, while, in the adult, hepatocytes are arranged in a muralium simplex architecture (one‐cell‐thick sheets) and cytokeratin filaments have a symmetrical distribution in relation to the nuclear region.
Three‐dimensional electron microscopy of entire cells
Authors: Bron Cph. , Gremillet Ph. , Launay D. , Jourlin M. , Gautschi H. P. , Bächi Th. , Schüpbach J.
Published: August 2011

The digital processing of serial electron‐microscope sections containing laser‐induced topographical references allows a three‐dimensional (3‐D) reconstruction of entire cells at a depth resolution of 40–60 nm by the use of novel image analysis methods. The images are directly processed by a video‐camera placed under the electron microscope in TEM mode or by the electron counting device in STEM mode. The deformations associated with the cutting of embedded cells are back‐calculated by new computer algorithms developed for image analysis and treatment. They correct the artefacts caused by serial sectioning and automatically reconstruct the third dimension of the cells. Used in such a way, our data provide definitive information on the 3‐D architecture of cells. This computer‐assisted 3‐D analysis represents a new tool for the documentation and analysis of cell ultrastructure and for morphometric studies. Furthermore, it is now possible for the observer to view the contents of the reconstructed tissue volume in a variety of different ways using computer‐aided display techniques.
A combination of topographical contrast and stereoscopy for the reconstruction of surface topographies in SEM
Authors: Beil W. , Carlsen I. C.
Published: August 2011

The reconstruction of three‐dimensional surface topographies can be done in two principally different ways: conventional stereoscopy and ‘shape from shading’. In conventional stereoscopy the depth information is obtained from two perspective views of the specimen. For that, perspectively corresponding features have to be identified in both views to determine depth from perspective shift. Conventional stereoscopy normally results in a relatively sparse set of irregularly distributed points whose elevations are known precisely. The shape‐from‐shading approach determines the local surface orientation from the local surface luminosity. Over a limited range of surface inclinations the emission of secondary (SE) and back‐scattered (BSE) electrons depends uniquely on the angle between electron beam and local surface normal of the specimen. Shape from shading uses this relationship to determine the surface normal with multiple detectors mounted in different take‐off directions. Contrary to conventional stereoscopy shape from shading yields depth information from each surface point, but this method is less accurate than stereoscopy.

In this paper we propose a combination of both approaches, in which the dense, but less accurate results from shape from shading are used to fill the gaps in the sparsely distributed, but very accurately known, depth information obtained from stereoscopy.
Methods for obtaining high quality stereoscopic images of microscopic objects
Authors: Sroczyński S. F.
Published: August 2011

Over 100 years ago Ernst Abbe developed the so‐called method of half‐moon diaphragms, placed in the exit pupil plane of a binocular microscope, for stereoscopic viewing of microscopic objects. Simplicity of the method being a great advantage, the obvious disadvantages were reduced resolution, azimuthal errors of imaging and failure to control plasticity of the images viewed. Using modern microscopic optics it is possible to avoid these complications, and a complete control of the plasticity of the stereoscopic image as perceived by the observer is achievable while working with medium, strong and the strongest microscope objective lenses. A number of formulae, a nomogram, and a set of tables for calculation of optimal settings are presented in order to promote a range of practical applications, including photomicrography.
Ultrastructural preservation of nuclei and chromatin: improvement with low‐temperature methods
Authors: Horowitz R. A. , Giannasca P. J. , Woodcock C. L.
Published: August 2011

The ultrastructure of chromatin has been examined in nuclei prepared by a variety of low‐temperature methods. Embedding glutaraldehyde (GA)‐fixed nuclei in Lowicryl K4M or K11M following dehydration by the progressive lowering of temperature (PLT) method, or in K11M following spray freezing and freeze substitution (FS), produces chromatin fibres that have, in situ, a diameter close to the in vivo state, and show internal structural details consistent with patterns of nucleosome packing previously observed only in preparations of isolated fibres. This is a temperature‐dependent effect; fibres conventionally dehydrated and embedded in Lowicryl at 0°C or in conventional epoxy resin at 60°C have lower and less uniform diameters, and lack internal structural details. Of the techniques used, spray freezing followed by FS resulted in the most notable improvement over conventional methods. Inclusion of GA during FS of rapidly frozen, unfixed nuclei in methanol does not result in cross‐linking of nuclear proteins. In acetone, however, cross‐linking by GA occurs at — 45°C, or at lower temperatures if the water content of the acetone‐based FS media is kept deliberately high. Substitution regimes employing GA alone or in combination with uranyl acetate and/or osmium tetroxide do not result in fibre morphologies comparable to either prefixed or unfixed nuclei substituted in additive‐free substitution media. Whole fibroblasts show excellent preservation of nuclei and the nuclear/cytoplasmic interface after spray freezing followed by FS and low‐temperature embedding.
Morphogenesis of the brain as studied by 3‐D computer graphics simulation
Authors: Fujita Setsuya
Published: August 2011

Morphogenesis of the brain is analysed by 3‐D computer graphics simulation. The initial form of the brain, i.e. a neural tube, is created in the computer as a small tubular figure in 3‐D Cartesian coordinates which are subsequently transformed into curvilinear coordinates by a set of growth functions. By this method the morphological development of the human brain is simulated. The same computer program can be applied to simulate the development of the brains of mouse, rat and monkey simply by changing the parameters slightly. The result of the analysis reveals that the smaller brains are produced by faster growth of the matrix cell population at the onset of development, coupled with faster rate of retardation of the growth during ontogeny. Thus, smaller brains in smaller animals are produced in a shorter period during their ontogeny.
Fluorescence histochemical techniques for catecholamines as tools in neurobiology
Authors: Felten David L. , Felten Suzanne Y. , Sladek John R. , Notter Mary D. , Carlson Sonia L. , Bellinger Denise L. , Wiegand Stanley J.
Published: August 2011

Formaldehyde‐induced and glyoxylic‐acid‐induced fluorescence histochemistry permits the tissue localization of catecholamines in the central nervous system (CNS) and peripheral nervous system (PNS), and in culture. Counterstains such as ethidium bromide provide excellent background identification of specific innervated regions in both the CNS and the periphery. Use of fluorescence histochemistry with immunocytochemistry can elucidate catecholamine‐peptide relationships. Gelatin‐ink perfusion used with fluorescence histochemistry permits the investigation of neuro‐vascular relationships and documentation of vascular and parenchymal compartmentation of innervation. Combined use of fluorescence histochemistry and retrograde tracing methods demonstrates the specific cellular sources of innervation of target regions. Micropunch neurochemical analysis provides quantitative data for correlation with fluorescence histochemistry within a target region of innervation, and micro‐spectrofluorometric analysis provides a semi‐quantitative evaluation of the amount of fluorophore within a target region or within specific subcellular compartments such as the cell body or terminals.
Receptor autoradiography and 2‐deoxyglucose techniques in neuroscience
Authors: Wiederhold K.‐H. , Palacios J. M.
Published: August 2011

The use of the receptor autoradiography and 2‐deoxyglucose (2‐DG) techniques in neuroscience are reviewed. Receptors and other binding sites can be visualized autoradiographically in microtome tissue sections after labelling with radioligand in vivo or in vitro. Autoradiograms are generated by apposition of the labelled tissue to photographic emulsions. Combined with computerized image analysis, this technique can be used to analyse and quantify the microscopic distribution of receptors and receptor alterations associated with lesions or disease in human and animal tissues. The 2‐DG technique permits microscopic analysis of modifications in brain glucose utilization induced by physiological and pharmacological manipulations. Limitations of these techniques and attempts to optimize their resolution are also discussed.
Estimating the surface density of fissures in clay soil from deterministic parallel sections
Authors: Scott G. J. T. , Webster R. , Nortcliff S.
Published: August 2011

In much clay soil the pore space consists of 3‐D networks of narrow, approximately planar fissures. The density of the interface between fissures and solid soil is important; a method for its estimation from the horizontal and vertical cuboid samples that are taken conventionally is described here.

Undisturbed samples are impregnated with resin containing a fluorescent dye. Parallel plane faces are cut, and the exposed surfaces are photographed under u.v. light. Pairs of superimposed photographic negatives are projected onto a screen, and the lateral displacement of each interface from one section to the next is measured at points of intersection of the interface with linear probes through the soil. These distances are used to infer the local orientation of the interfaces, which are then used with the point counts to estimate the interface areas.

The method was applied to subsoil of the Windsor series. The interface density was estimated to be 9.69cm2/cm3 from one horizontal and one vertical sample. Point‐counting with isotropic uniform random linear probes applied to five randomly orientated samples from the same site gave an estimate of 8.78cm2/cm3. It seems probable that the error associated with the new method is no greater than that associated with the standard method.
Study of the early growth and branching of Streptomyces tendae by means of an image processing system
Authors: Reichl U. , Buschulte T. K. , Gilles E. D.
Published: August 2011

A method is described for directly observing submerged growth of streptomycetes, which can be easily adapted for investigations of filamentously growing fungi. The use of an image processing system allowed a fast and accurate calculation of important morphological parameters. Most of the images can be analysed on‐line. Some results are presented for early growth and branching of Streptomyces tendae. It seems that mechanisms for growth of streptomycetes were the same for both solid and liquid media.
Measurements of cells in culture by scanning acoustic microscopy
Authors: Litniewski Jerzy , Bereiter‐Hahn Jürgen
Published: August 2011

A novel method allowing the determination of thickness, attenuation and impedance of both living and fixed cells in culture using a single image recorded with a scanning acoustic microscope is described. The method is based on the recording of the image data locating the surface of the cell far below the geometric focus. In this way only the normal incident acoustic wave is used for image formation.

Higher values of impedance and attenuation coefficients were found in the cell periphery than in the central part of the cell. This phenomenon is suggested to be due to the different organization of cytoskeletal elements.
Three‐dimensional dynamic morphometry on a diffraction pattern in the focal plane
Authors: Ishizaka S.
Published: August 2011

A new type of morphometry, which shortens the scanning time of precise observation by confocal microscopy, has been investigated.

To analyse the 3‐D distribution of active sites on a living cell, microspheres of the same size were immunologically marked on specific sites on a cell. Incident coherent light was scattered on the microspheres and the scattered light from each microsphere superimposed upon each other giving a diffraction pattern of the examined cell. Several series of interference fringes were generated on the diffraction pattern, according to the phase difference of the microspheres. These interference fringes on the 2‐D diffraction pattern enable the (relative) 3‐D positions of the microspheres to be determined.

3‐D dynamic morphometry on a 2‐D diffraction pattern with interference fringes speeds up imaging in confocal microscopy.
A method for evaluating microscope objectives to optimize performance of confocal systems
Authors: Cogswell C. J. , Sheppard C. J. R. , Moss M. C. , Howard C. V.
Published: August 2011

A method for evaluating the performance of microscope objectives on two types of confocal scanning optical microscope is presented. Of these two confocal microscope types, off‐axis beam‐scanning systems are found to require microscope objectives which have been corrected for flatness of field as well as for spherical aberration and astigmatism in order to obtain maximum axial and laterial resolution. In the case of on‐axis specimen‐scanning microscopes, less highly corrected objective lenses (not corrected for flatness of field) may in practice prove to have superior resolving properties.
Automated image segmentation and serial section reconstruction in microscopy
Authors: Moss V. A. , Jenkinson D. McEwan , Elder H. Y.
Published: August 2011

A technique for automatic 3‐D reconstruction of specifically stained features in televised serial histological sections has been developed using an image analyser. Images which included these features were individually converted to binary images, compiled, displayed to show the 3‐D morphology and used to quantify the structure. The criteria necessary for producing valid reconstructions and the problems associated with the manipulation of images of fine detail, particularly those containing several thousand features, are illustrated by examples from skin.
Detection of doubly stained fluorescent specimens using confocal microscopy
Authors: Mossberg Karin , Ericsson Maria
Published: August 2011

Studies of doubly stained specimens were performed with a confocal scanning microscope. The instrument used provides the possibility of making separate detections of the fluorescent dyes. The optimal choice of excitation wavelengths and optical filters are discussed. The fluorphores that were used are Lucifer Yellow, Texas Red, fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate.
Methodical aspects of 3‐D reconstruction of chromatin architecture in mouse trophoblast giant nuclei
Authors: Montag Markus , Spring Herbert , Trendelenburg Michael F. , Kriete Andres
Published: August 2011

Precise 3‐D reconstruction of the spatial organization of murine trophoblast giant‐cell chromatin is a prerequisite for detailed investigations on the fine structural changes in chromatin‐fibre organization during the trophoblast endomitotic cell cycle. It appears very likely that sequential fine structural changes in the chromatin arrangement are concomitant with the changes in the gene expression pattern of these cells during the early phase of murine gestation.

The complex intra‐nuclear chromatin organization of mouse trophoblast giant nuclei was investigated in permanent tissue preparations which had been stained with a DNA‐specific dye. The spatial chromatin arrangement was examined in fluorescence with a confocal scanning laser microscope. Serial optical sections were recorded and subjected to a computer‐based 3‐D reconstruction method which is suitable even for very complex biological structures.
3‐D model of vascular network in rat skin obtained by stereo vision techniques
Authors: Kim N. H. , Aggarwal S. J. , Bovik A. C. , Diller K. R.
Published: August 2011

The quantitative analysis of the depth of injury, penetration of therapeutic agents in tissues, and the regeneration of vascular patency after a graded degree of thermal injury requires a knowledge of the shape and spatial configuration of the vascular networks in the tissue. We have applied computational stereo vision techniques to describe the 3‐D configuration of microvessels in full thickness rat skin vascular casts produced by perfusion of Yellow Microfil latex solution through the aorta. The principal concern is to describe the 3‐D structure of vascular networks using a set of 3‐D space curves. This representation is computed by integrating monocular and binocular processing; the 2‐D curve representation of blood vessels computed through monocular analysis is integrated with disparity data to yield a space curve representation for each vessel. A connection diagram is also computed to indicate the connections existing among the computed space curve representations.
Towards light microscopic imaging of hydrated ‘native’ ribosomal RNA genes
Authors: Spring H. , Trendelenburg M. F.
Published: August 2011

Investigations of the higher‐order structure of ‘native’ genes as well as antibody localization to defined gene chromatin areas require elaborate light microscopic imaging of the chromatin structure of interest, such as visualization of unfixed and unstained gene transcription units.

Since all our present structural information on the, relatively, small nucleolar rRNA genes has been obtained using transmission electron microscopy of spread genes following fixation, staining and complete dehydration, this type of EM specimen was our first specimen for LM imaging. Using Nomarski differential interference contrast video microscopy, it was found that spread EM chromatin can be sufficiently visualized by light microscopy to allow precise correlation with the conventional TEM image.

In addition, it is demonstrated that video‐enhanced LM can provide enough contrast enhancement for rapid visualization of spread ‘native’ rRNA genes in slightly fixed, fully hydrated, unstained gene chromatin.
Primary considerations for image enhancement in high‐pressure scanning electron microscopy
Authors: Farley A. N. , Shah J. S.
Published: August 2011

In the preceding paper the effect of the primary beam scattering on contrast in high‐pressure SEM techniques such as moist‐environment ambient‐temperature SEM and environmental SEM was examined. Here, the effect of the scattering of the emissive components on the image formation and contrast in high‐pressure SEM was considered and evaluated experimentally. A specimen current amplifier was used to detect the absorbed current signal together with the ionic signal components generated by the emissive and primary electrons. It was shown that space charge accumulations of ionic carriers can build up above the specimen when imaging in the presence of a gas and that this can act to suppress the secondary electron emission. The ionic carrier concentration is also discussed. Full image contrast can be restored by the application of an electric bias field above the specimen surface. Since ionic charge carriers contribute to the signal current, new image‐contrast mechanisms were considered and a theoretical model for contrast generation is therefore proposed. The predictions of this model were tested experimentally for a specimen substrate of uniform emissivity under varying electric bias field strengths. It was also found that under sufficiently high electric fields low‐energy charge carriers gain enough energy to ionize the host gas molecules and therefore multiply the signal current. Micrographs are presented to illustrate these image formation processes as a function of electrode bias. The new detection system successfully extends the signal information of high‐pressure SEM and a range of applications is discussed to exploit these possibilities.
How fine a surface crack can you see in a scanning acoustic microscope?
Authors: Briggs G. A. D. , Jenkins P. J. , Hoppe M.
Published: August 2011

Surface cracks can be detected with great sensitivity in an acoustic microscope, even when the width of the crack is much less than the acoustic wavelength. This is because of the strong excitation and scattering of Rayleigh waves. Cracks in a variety of materials, including cracks from indents in several different ceramics, have been studied by acoustic microscopy, and the results compared with images from light and scanning electron microscopes. No crack or part of a crack that was seen by the other two techniques failed to be seen by acoustic microscopy. The contrast of a crack in an acoustic microscope showed no sign of fading as the crack became thinner towards its tip, and diffraction from tips suggests that they are acoustically well‐defined points on the surface. Although theoretically the acoustic contrast from a crack might be expected to diminish when the crack width is smaller than the shear‐wave decay length in the fluid, in practice this is so small that for many purposes it may be considered that there is almost no limit to how fine a crack can be and still be detected in an acoustic microscope.
A new non‐linear pseudo‐Laplacian filter for enhancement of secondary electron images
Authors: Oho Eisaku , Ogihara Akinori , Kanaya Koichi
Published: August 2011

A simple, yet effective, non‐linear pseudo‐Laplacian filter has been newly developed to enhance secondary electron (SE) images. This filter is a combination of the second derivative along the direction of the local gradient and a non‐linear weight factor. The filter can successfully enhance SE images without the undesirable effects of noise which are often seen in conventional Laplacian filtered images. Hence, the processed results with high image quality can make original SE images easier to interpret. The effectiveness is especially useful in low‐voltage scanning electron microscopy, because SE images taken at low voltages are not so likely to have good image sharpness.
An improved technique for flattening semithin sections of epoxy‐embedded tissues used for high‐resolution light microscopy
Authors: Sierra Felix , Hegele‐Hartung Christa , Beier Henning M.
Published: August 2011

The usefulness of sodium ethoxide as an agent for flattening thick sections (0.5‐2.0μm) of epoxy‐embedded material was studied. It was found that sodium ethoxide conditioned by its resin‐resolving properties leads to elimination of folds and crumples known to be present to various degrees in sections of resin‐embedded tissue. Two minutes of incubation leads to a complete section flattening without any damage to the cellular structures. With regard to large sized sections of epoxy‐embedded tissue, this simple and fast method represents an improvement in the production of useful and qualitatively satisfactory sections for high‐resolution light microscopy
Letter to the Editor
Authors: van Heel Marin
Published: August 2011

In a recent paper Frank et al. (1988; J. Microsc. 150, 99–115) proposed the use of a hybrid clustering technique to sort macromolecular images. In contrast to the claims made in that paper, however, the proposed technique is less sophisticated than the enhanced hierarchical ascendant classification technique proposed by us a few years earlier. The hybrid approach will lead to partitions which are less optimal in terms of minimizing the total intra‐class variance for a predetermined number of classes, irrespective of whether the data show distinct clusters or, rather, possess a quasi‐continuous intrinsic structure.
Three‐dimensional reconstruction of porous media from serial section data
Authors: Kwiecien M. J. , Macdonald I. F. , Dullien F. A. L.
Published: August 2011

Software has been developed, with the aid of data for a Berea sandstone sample, to partition the irregular, interconnected pore space of a porous medium into its constituent pores. Output includes the sizes (volumes) of the individual pores, the sizes (area and radii of curvature) of the individual necks (windows) between the pores, the order in which the pores and necks are connected, and the degree of interconnectedness.

Data are entered into the computer by digitizing a series of closely spaced serial sections of a pore cast of the sandstone. The pixels represent either rock matrix or pore space which are represented in binary format by ′0′ and ′1′, respectively. Each pixel is assumed to be the top surface of an associated volume element (voxel).

The data array is analysed from seven orientations. For each orientation, a feature map is created which identifies the interconnected pore channels through the array. Then a set of potential necks is identified by locating local minima in the radius of curvature of pore space features along the pore channels.

The seven sets of local minima are merged, overlaps among the minima are identified, and a consistent set of non‐overlapping necks is created by systematically deleting the larger overlapping minima. The necks are then ‘closed’ and the data set re‐analysed to identify each cluster of pore space voxels bound by rock matrix and ‘closed’ necks as a pore.
Mechanisms of detection and imaging in the ESEM
Authors: Danilatos G. D.
Published: August 2011

For proper understanding of image formation using charge carriers, it is shown that signal detection by means of induction must be considered. This explains the possibility of imaging insulators as well as other phenomena especially in the conditions of the environmental scanning electron microscope. In addition, a basic principle and method to separate the secondary and backscattered electrons is demonstrated.
The measurement and calculation of the X‐ray spatial resolution obtained in the analytical electron microscope
Authors: Michael J. R. , Williams D. B. , Klein C. F. , Ayer R.
Published: August 2011

The X‐ray microanalytical spatial resolution is determined experimentally in various analytical electron microscopes by measuring the degradation of an atomically discrete composition profile across an interphase interface in a thin‐foil of Ni‐Cr‐Fe. The experimental spatial resolutions are then compared with calculated values. The calculated spatial resolutions are obtained by the mathematical convolution of the electron probe size with an assumed beam‐broadening distribution and the single‐scattering model of beam broadening. The probe size is measured directly from an image of the probe in a TEM/STEM and indirectly from dark‐field signal changes resulting from scanning the probe across the edge of an MgO crystal in a dedicated STEM. This study demonstrates the applicability of the convolution technique to the calculation of the microanalytical spatial resolution obtained in the analytical electron microscope. It is demonstrated that, contrary to popular opinion, the electron probe size has a major impact on the measured spatial resolution in foils < 150 nm thick.
A rapid preparation method for ultrastructural investigations of conifer needles
Authors: Ebel B. , Hamelmann U. , Nieß C.
Published: August 2011

The method discussed here for preparing conifer needles for transmission electron microscopy combines three important components in a new way: chilling (273 K) during fixation to prevent autolysis, HEPES buffer to protect cytoplasm against washing out and VCD/HXSA as an ultralow viscid and therefore rapidly infiltrating medium. Ultrastructural analysis of Picea abies and Pinus silvestris shows very satisfactory tissue preservation.
Assessment of particle retention and clearance in the intrapulmonary conducting airways of hamster lungs with the fractionator1
Authors: Geiser Marianne , Cruz‐Orive Luis M. , Hof Vinzenz Im , Gehr Peter
Published: August 2011

A modified version of the fractionator was used to estimate the total number of polystyrene microspheres retained in the airways of hamster lungs at two different time points after inhalation. A systematic three‐stage subsampling procedure with known sampling fractions was adopted. First, each lung was cut into slices, from which primary disectors were sampled systematically with a known sampling fraction. From each primary disector, smaller sub‐disectors were subsampled, and the corresponding sampling fraction was estimated by point counting. Finally, a few particles were counted at the microscopic level in the sub‐disectors, and the final estimate of total particle number (which is unbiased irrespective of any tissue deformations) was easily computed as a product of the counted number times the reciprocal of the successive sampling fractions. The error variance of each estimate was assessed from the data using a new estimator.

An average of 6% of the deposited particles were retained on the epithelial surface of the intrapulmonary conducting airways shortly after the inhalation, from which at least one‐third was already phagocytosed by macrophages. After 24 h, an average of 87% of the particles retained shortly after the inhalation had been cleared. The proportion of particles ingested by macrophages had increased to at least 87%, in three out of four animals studied.
An introduction to singular systems with applications to confocal scanning microscopy
Authors: Pike E. R. , Davies R. E. , Walker J. G. , Young M. R.
Published: August 2011

An introduction to both the classical and the singular‐systems theory of resolution is given. Singular‐system analysis is applied to the case of the coherent confocal scanning microscope. Ultimate resolution is predicted to be half the Rayleigh distance. The results of a low‐numerical‐aperture experiment are presented and compared with the theory. The predicted increase in resolution is accurately found.
Imaging in scanning microscopes with slit‐shaped detectors
Authors: Wilson T. , Hewlett S. J.
Published: August 2011

We consider the imaging in scanning microscopes employing point and slit‐shaped detectors in both the bright‐field and fluorescent mode. In particular we consider the three‐dimensional aspects of the imaging and show inter alia that acceptable, albeit asymmetrical, images result from a slit detector system at low levels of defocus. The situation becomes worse as the defocus is increased although acceptable extended‐focus and isometric images are still possible in some cases.
High‐resolution electron microscopy of human enamel crystals
Authors: Brès E. F. , Voegel J.‐C. , Frank R. M.
Published: August 2011

The structure of enamel crystals obtained from four human premolars has been studied by high‐resolution electron microscopy (HREM) in the [0001], [***2110], [***1540], [***0110] and [***1213] crystallographic directions at various microscope defocus and crystal thickness values. The resolution obtained has not previously been reported for human enamel crystals. In all cases, it was possible to match the experimental images to images calculated using the atomic positions of mineral hydroxyapatite. However, a deviation from hexagonal symmetry characterized by marked (***1010) planes of intensity different from the one of the (***3030) and {***1010}‐type planes was observed. In this work, we present an improvement of Scherzer resolution of 0.25‐0.20 nm over previous work on biological enamel crystals. This improvement of resolution has permitted the incorporation of crystallographic reflections of higher spatial frequencies into the imaging process of the microscope and has led to a more precise structure determination of the crystals studied.
Transmission electron microscopy of lead scandium tantalate thin‐films
Authors: Reaney I. M. , Barber D. J.
Published: August 2011

Preliminary results are reported on the examination of lead scandium tantalate (PST) ferroelectric thin‐films by transmission electron microscopy (TEM). The samples were grown on sapphire substrates by the solution‐gel route and were examined in both plan and transverse section.

Scanning electron micrographs of the surface of the samples of PST on sapphire showed that the PST films were invariably cracked. TEM examination of the PST on sapphire in plan section revealed that two distinct distributions of PST grains had grown on the sapphire (100 and 1500 nm diameter) and that nucleation of PST on the sapphire surface was associated with surface flaws and dislocations. TEM samples prepared in transverse section revealed that ordering of the Sc and Ta cations was greater at the PST‐sapphire interface than within the interior of the thin‐film. Small crystallites of an unidentified second phase were also observed to have formed in the centres of PST grains.
Low‐temperature scanning electron microscopy in biology
Authors: Read Nick D. , Jeffree Christopher E.
Published: August 2011

A review of low‐temperature scanning electron microscopy (LTSEM) with regard to preparation protocols, specimen preservation, experimental approaches, and high‐resolution studies, is provided. Preparative procedures are described and recent developments in methodologies highlighted. It is now well established that LTSEM, for most biological specimens, provides superior specimen preservation than does ambient‐temperature SEM. This is because frozen‐hydrated samples retain most or all of their water, are rapidly immobilized and stabilized by cryofixation, and are not exposed to chemical modification or solvent extraction. Nevertheless, artefacts in LTSEM are common and most arise because frozen‐hydrated specimens contain water. LTSEM can be used as a powerful experimental tool. Advantages of employing LTSEM for this purpose and ways in which it can be used for novel experimentation are discussed. The most exciting development in recent years has been high‐resolution LTSEM. The advantages, problems and requirements for this approach are defined.
Low‐temperature scanning electron microscopy of birch leaves after exposure to ozone
Authors: SCHEIDEGGER C. , GÜNTHARDT‐GOERG M. , MATYSSEK R. , Hatvani P.
Published: August 2011

Comparison of different harvesting and preparation pathways showed that low‐temperature SEM is an adequate method to conserve the stomatal aperture for SEM. Both critical point drying and freeze drying cause considerable artefacts.

Exposure to site‐relevant concentrations of ozone led to reduced width of the stomatal aperture. Moreover, unetchable droplet‐like exudates were found on the outer face of mesophyll cells of leaves where the trees had been exposed to ozone. These exudates were later followed by collapsed mesophyll cells and ended in necrotic zones before premature leaf loss.
Radiation damage in dry and frozen hydrated organic material
Authors: Lamvik M. K.
Published: August 2011

Electron‐induced radiation damage can cause errors in interpreting electron micrographs. Radiation damage is distinguished from contamination (polymerization of hydrocarbons) and etching (radiolysis in the presence of water), both of which can be controlled by a proper specimen environment in the microscope. While temperature has little effect on the primary interactions of fast electrons with matter, most secondary radiation‐damage processes are temperature dependent. Because damage mechanisms differ so greatly among materials, there is no simple factor by which specimen stability is improved as a function of temperature (some cases improve fivefold, others improve 100‐fold). While some specimens are stable to almost arbitrarily high doses, some tests reveal damage at 1 e/nm2. This paper surveys damage rates and temperature dependencies of various materials as a guide for future electron microscopic studies of organic specimens.
Freeze‐substitution and the preservation of diffusible ions
Authors: Edelmann Ludwig
Published: August 2011

Freeze‐substitution of biological material in pure acetone followed by low‐temperature embedding in the Lowicryls K11M and HM23 yields stable preparations well suited for sectioning and subsequent morphological and microanalytical studies. Transmission electron microscopy of dry‐cut sections shows that diffusible cellular thallium ions (Tl+) of Tl+‐loaded muscle are localized at similar protein sites in freeze‐substituted as in frozen‐hydrated preparations. A comparison of X‐ray micro‐analytical data obtained from freeze‐dried cryosections and sections of freeze‐substituted normal (potassium‐containing) muscle shows that K+ ion retention in the freeze‐substituted sample is highly dependent on the freeze‐substitution procedure used; so far, in the best case, about 67% of the cellular K+ is retained after freeze‐substitution in pure acetone and low‐temperature embedding. It is concluded that the retention of diffusible cellular ions is dependent on their interactions with cellular macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze‐substitution and low‐temperature embedding.
Cryosputtering—a combined freeze‐drying and sputtering method for high‐resolution electron microscopy
Authors: Lindroth Margaretha , Fredriksson Bengt‐Arne , Bell Paul B.
Published: August 2011

Preparing cellular structures for visualization by high‐resolution scanning electron microscopy (SEM) is a multi‐step process which includes fixation, dehydration, drying and metal coating. Drying and metal coating are limiting for high‐resolution work. Commonly, the dried samples are exposed to the air before they are inserted into a metal coating apparatus, thereby exposing them to moisture and the accompanying risk of rehydration, which may cause changes in the supramolecular structure. We have modified a freeze‐dryer to accommodate a magnetron sputtering head, in order to sputter‐coat the frozen‐dried samples while still in the drying chamber in the cold, a process we call cryosputtering. A layer of 1·5 nm of tungsten was cryosputtered onto whole mounts of cytoskeletons from detergent‐extracted human glioma cells or fibroblasts and the specimens were examined by high‐resolution SEM and transmission electron microscopy (TEM). To reduce the effects of backstreaming oil from the vacuum system, a turbomolecular pump backed by a two‐stage rotary vane pump was connected to the drying‐coating chamber. This pump system provides a high vacuum, making it possible to dry the specimens at — 90°C/183 K, thus reducing the risk for recrystallization of water. Furthermore, the high vacuum minimizes the negative effects of contaminants, which can be deposited onto the specimen surface and affect the quality of the metal coat formed during sputtering.
Preparation of ultrathin amorphous ice films for cryo‐electron microscopy
Authors: Jakubowski U. , Mende M.
Published: August 2011

Electron microscopy of biological macromolecules embedded in vitrified ice films suffers from serious problems caused by excessive inelastic scattering, beam‐induced movements of the specimen, deformation of the molecules by adsorption at the water‐air interface and insufficient mechanical stability of the films. We have built an environmental chamber to control temperature and humidity independently in order to produce ultrathin water films (< 20 nm) spanning holes with diameters of 20 μm to 1 mm. The surface tension of the water films was reduced by adding lipid monolayers, thus prolonging the usable time for thinning of the film and avoiding adsorption artefacts in the embedded material. After cryofixation in ethane a carbon film was evaporated on each side of the specimen to stabilize the ultrathin ice—lipid film. Mechanical stability and charging effects could successfully be reduced by this preparation method. Collapsing water films could be cryofixed and the shape of the hole was analysed. By the eccentricity of the elliptical holes an estimation could be made of the burst velocity of the rim of the hole and of the cooling rate of the cryofixation process.
Image analysis and processing of an imperfect two‐dimensional crystal: the surface layer of the archaebacterium Sulfolobus acidocaldarius re‐investigated
Authors: Lembcke G. , Dürr R. , Hegerl R. , Baumeister W.
Published: August 2011

The surface layer of the archaebacterium Sulfolobus acidocaldarius has been re‐investigated exploring various image analysis and image processing strategies. Straightforward correlation averaging correcting for translational disorder only, and lattice unbending in conjunction with filtration in the Fourier domain gave equivalent results. Careful examination of cross‐correlation functions as well as multivariate statistical analysis of individual unit cells revealed the existence of interpenetrating domains with intrinsic p3 symmetry but rotated with respect to each other by 60° and separated by twin boundaries. Non‐discriminative averaging over twinned crystal patches generates a higher (p6) class of symmetry emphasizing the importance of selective averaging.
Quantitative light microscopy of combined perfusion and freezing processes
Authors: Walcerz Douglas B. , Diller Kenneth R.
Published: August 2011

The rational design of cryopreservation protocols for living tissues demands an understanding of the mechanisms of mass transport between cells and their environment throughout the entire process. We have developed a new microscope stage to enable a specimen to be viewed continuously during a preservation protocol, including the addition and removal of cryoprotective additives and freezing and thawing. The specimen is contained in a sealed chamber having inlet and outlet ports for admitting and collecting perfusate solution, the entire volume of which may be exchanged with a time constant of 1–5 s, depending on the solution viscosity. The temperature of the active area of the stage is regulated by the standard techniques of convection cryo‐microscopy over a range in excess of 50 to — 100°C.

A series of experiments has been performed on this system to measure the osmotic behaviour of rat pancreas islets during the addition and removal of dimethyl sulphoxide at temperatures between 25 and — 10°C. The technique involves mounting a single islet onto the low‐temperature stage so that it is constrained from lateral movement by a specially sized mesh. Both the system temperature and chemical composition are monitored and controlled simultaneously and independently; as a consequence, virtually any defined cryopreservation protocol may be imposed on the specimen. For making permeability measurements, the bathing medium of the specimen may be changed very rapidly to produce a defined osmotic stress. Alternatively, the specimen may be subcooled to a specific and fixed subzero temperature, at which point ice is nucleated in the extracellular medium, creating a near instantaneous change in composition. The temporal alteration in specimen size is monitored by video microscopy and quantified by computer vision analysis methods. One of several mass transfer models is fitted to the data to estimate the membrane permeability based on the assumption of either transport dominated by the movement of water or simultaneous coupled flows of water and cryoprotective agent.
Intracellular ice formation: cryomicroscopical observation and calorimetric measurement
Authors: Körber Christoph , Englich Sabine , Rau Günter
Published: August 2011

The formation of ice crystals within biological cells is generally deleterious and results in a severe loss of cellular viability and function. With the aim of circumventing this lethal event, the mechanisms of nucleation and their dependence on governing parameters such as temperature, cooling rate and solute and/or additive concentration, and the correlation with the osmotically induced water transport across the cell membrane were investigated. Quantitative low‐temperature light microscopy was used for this purpose as it offers the major advantage of studying the dynamics of the involved processes. To substantiate further the visual observations of the morphological changes associated with intracellular ice formation, supplementary studies by differential scanning calorimetry (DSC) were performed under comparable conditions to measure the quantity of water actually transformed into the crystalline state due to the evolution of latent heat.

Human lymphocytes were used as a biological model cell. In particular it could be shown that the twitching type of intracellular ice formation which is evident but difficult to observe under the cryomicroscope can be attributed to a liquid‐solid phase change within the cells as determined by DSC. Good agreement was obtained between the results measured by both techniques with respect to the following dependencies of governing parameters: the fraction of cells exhibiting intracellular ice determined as a function of the cooling rate shows a sharp demarcation zone with an increase from 0 to 100% at about the same threshold cooling rate. On the other hand, the temperatures at which intracellular ice forms were found to be only weakly dependent on the cooling rate. With respect to the effect of cryo‐additive concentration at a fixed value of the cooling rate, the crystallization temperatures were seen to decrease with concentration. The DSC results may hence be regarded as a validation of the microscopic observations.
Early results using high‐resolution, low‐voltage, low‐temperature SEM
Authors: Pawley James B. , Walther Paul , Shih Shian‐Jiun , Malecki Marek
Published: August 2011

Recent advances in the design of the scanning electron microscope (SEM) column, such as the coupling of a field‐emission gun to a low‐aberration immersion lens and the availability of a high‐stability cryo‐transfer stage, make low‐temperature, low‐voltage SEM (LTLVSEM) possible at very high resolution. We have used this combination to obtain results with uncoated biological specimens.

The trichocyst from a Paramecium was used as a test specimen to observe the shrinkage of this structure as the temperature is raised from 170 K to room temperature following freeze‐drying. High‐magnification stereo images were obtained of trichocysts that had been prepared by freezing, freeze‐substitution and critical‐point drying and which were subsequently viewed by LTLVSEM to reduce beam damage and contamination.