bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of directly frozen macromolecules and tissues in the field‐emission STEM
Authors: Leapman Richard D. , Andrews S. Brian
Published: August 2011

A VG Microscopes HB501 field‐emission high‐resolution scanning transmission electron microscope (STEM) was used to image and analyse rapidly frozen, isolated macromolecules and small organelles in tissue cryosections. Dark‐field images were obtained from frozen‐hydrated microtubules demonstrating that sufficient contrast is available to reveal structural information. The samples were subsequently freeze‐dried in the STEM and low‐dose (≅ 103 e/nm2) dark‐field mass maps were recorded with single electron sensitivity. Elemental analysis of individual macromolecules was achievable at high dose using parallel‐detection electron energy‐loss spectroscopy, albeit with some structural degradation. Detection of copper (320 atoms) in di‐decameric haemocyanin molecules was easily within the limits of sensitivity. Elemental analysis of hydrated cryosections is limited by radiation damage to a resolution of approximately 1 μm2. For freeze‐dried sections, however, the high probe current and stable cold stage of the HB501 STEM allow energy‐dispersive X‐ray (EDX) microanalysis of low elemental concentrations in highly localized subcellular volumes. EDX spectra from cryosections of cerebellar cortex show that a 100‐s analysis time is sufficient to quantify the calcium content of 400‐nm2 regions within Purkinje cell dendrites with an uncertainity of ± 2 mmol/kg dry weight, equivalent to ± 12 atoms.
Freeze‐fracturing for conventional and field emission low‐temperature scanning electron microscopy: the scanning cryo unit SCU 020
Authors: Müller T. , Guggenheim R. , Düggelin M. , Scheidegger Ch.
Published: August 2011

A dedicated cryopreparation system, the SCU 020 (Balzers), is introduced and described in detail for use in low‐temperature scanning electron microscopy (LTSEM). The basic unit consists of two parts: (i) a high‐vacuum preparation chamber equipped with a cold‐stage, motor‐driven fracturing microtome, planar magnetron (PM) sputter source, quartz‐crystal thin‐film monitor, Meissner cold trap, and turbo molecular pump stand; and (ii) a second part (separated from the first by a sliding, high‐vacuum valve) residing in the SEM chamber. This is equipped with an anti‐contamination cold trap, a fully movable goniometer cold stage (having motor drives for x, y, and rotation) and replaces the SEM's original stage (Raith). The SCU 020 is entirely self contained allowing independence from, and synchroneity with, the SEM of choice.

LTSEM micrographs of specimen (that are fully frozen hydrated or partially freeze‐dried) surfaces or fracture faces, without or with various metal coatings, can be examined over a broad temperature range (‐150 to +50°C). This is made possible by the combined application of the two, independently controlled, cold stages and the on‐line, high‐vacuum, specimen cryo transfer between them. In‐situ etching is simple and straightforward. Intramembranous particles and membrane fracture steps, typically imaged in transmission electron microscopy (TEM), are resolved by PM sputtering with platinum at low specimen temperature and high‐resolution LTSEM in a field emission microscope.
Freeze‐fracture cytochemistry: a simplified guide and update on developments
Authors: Severs N.J.
Published: August 2011

A wide variety of methods by which cytochemistry and freeze‐fracture can be successfully combined have recently become available. All these techniques are designed to provide information on the chemical nature of structural components revealed by freeze‐fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied. Colloidal gold labelling is the most widely used cytochemical technique in freeze‐fracture cytochemistry, and for many of the methods it is indispensable. In principle, there are four points in which the cytochemical labelling step may be integrated into the standard freeze‐fracture procedure: (i) before the specimen has been frozen, (ii) after it has been fractured and thawed, (iii) after platinum shadowing or (iv) after completion of the full replication sequence. Retention of the gold label so that it can be viewed with replicas can be achieved by depositing platinum and/or carbon upon the labelled surface, thereby partially entrapping the marker particles within the replica, or by retaining, attached to the replica, fragments of fractured membrane (or other cellular components) that would normally have been lost during the replica cleaning step. Another approach to visualizing the label is to use sections, either with portions of a replica included face‐on, or for examining the fracture path through the sample (without replica). Recent developments have centred on the use of replicas to stabilize half‐membrane leaflets; not only may these and associated attached components be retained for labelling just before mounting, but they provide a means for manipulating the specimen— specifically, turning it over during processing—so that additional structural information can be obtained.

This article aims to explain how modern freeze‐fracture cytochemisty works, and how the various techniques differ in what they can tell us about membranes and other cellular structures. With the effectiveness of many of the techniques now demonstrated, freeze‐fracture cytochemistry is firmly established, alongside a range of related labelling techniques, for increasing application in cell and membrane biology in the 1990s.
Ice crystal damage and radiation effects in relation to microscopy and analysis at low temperatures
Authors: Echlin Patrick
Published: August 2011

There are several limitations to the low‐temperature techniques which are currently being used for the preparation, examination and analysis of biological and organic samples by means of high‐energy beam instrumentation. The low thermal conductivity of samples and the inadequacy of rapid cooling techniques means that, with the exception of thin‐film suspensions and the surface of impact‐cooled bulk specimens which may be vitrified, ice crystals of varying sizes will be present in nearly all samples which are quench cooled. Data are presented which indicate the depth to which adequate cryo‐fixation may be achieved for both morphological and analytical studies. Although dynamic processes may be time resolved in the outer parts of quench‐cooled samples, the decreased freezing rate below the surface makes resolution of these processes much less certain. The quality of information which may be obtained from quench‐cooled samples is limited by radiation damage. Low‐dose microscopy of vitrified thin‐film suspensions of macromolecules continues to provide valid structural information at the molecular level. The increased doses needed for X‐ray microanalysis present serious problems with the high spatial resolution analysis of thin frozen‐hydrated sections although much less damage is observed in dried samples. A case is presented for using the outer fracture faces of frozen‐hydrated bulk samples for low‐resolution analysis of cells and tissues.
The potential of cryofixation and freeze substitution: observations and theoretical considerations
Authors: Kellenberger E.
Published: August 2011

The theoretical and experimental evidence in favour of cryofixation and freeze‐substitution are critically reviewed. The solubility of macromolecules in water is due to the hydration shells. Their behaviour at different temperatures and the consequences of their removal during the processing for embedding are explained. Gelation prior to the transfer into solvents prevents macromolecules aggregating. During substitution at low temperatures, DNA is gelled, justifying the use of the term cryofixation. It is proposed that the preservation of hydration shells at the lowest temperature, and their transformation into minute gaps after a rise of temperature, facilitates the exhibition of epitopes.
Contrast analysis of cryo‐images of n‐paraffin recorded at 400***kV out to 2·1 Å resolution
Authors: Brink J. , Chiu W.
Published: August 2011

N‐Paraffin was used as a test specimen for evaluating the relative merits of 400‐kV versus 100‐kV electron microscopy in recording data for electron crystallographic analysis of beam‐sensitive materials. The parameter used for comparison, the relative contrast R, is the ratio of amplitudes from the computed Fourier transform of images and amplitudes from an electron diffraction pattern from the same crystal. R will thus be a measure of the contrast from an experimental image relative to that of a perfect image. Electron diffraction patterns and bright‐field images were recorded at 400 kV at a specimen temperature of −167°C. Using the flood‐beam imaging technique the best R‐value is 0 08 for all reflections in the resolution zone from 4 to 3 Å. This value is equivalent to that found at 100 kV. In the resolution zone from 3 to 2 Å we have found R — 0 02. Using the spot‐scan imaging technique, on the other hand, R was measured to be 0·42 for the reflections between 4‐ and 3‐Å resolution. This amount of relative contrast is 1·7 times that observed at 100 kV. Reflections at 3–2 Å displayed an R‐value of 0 05. Besides obtaining higher R‐values when applying the spot‐scan imaging technique at 400 kV, we observe a higher yield of images with isotropic diffraction and/or higher resolution reflections. Various contrast‐attenuating factors, including the modulation transfer function of the photographic film and the cryo‐holder, envelope functions for spatial and temporal coherence and lens and high‐tension instabilities, the contrast transfer function and lastly the radiation damage effects, have been considered in interpreting the observed image contrast. Overall, use of 400 kV in combination with spot‐scan does offer important improvements in contrast levels, which can be very useful in determining the three‐dimensional structure from protein crystals.
Ergodicity and integral range
Authors: Lantuéjoul Ch.
Published: August 2011

When attempting to estimate stereological parameters starting from measurements in a limited domain, the question has to be addressed of how large the domain size must be in order to ensure representative measurements. To answer that question, the concept of ‘integral range’ is introduced. It allows a comparison between the scale of the phenomenon under study and the scale of observation. The integral range can be assessed using curves of dispersion variance, which can also yield empirical laws of change of scale.
Direct visualization of colloidal gold‐bound molecules and a cell‐surface receptor by ultrahigh‐resolution scanning electron microscopy
Authors: Tanaka Keiichi , Mitsushima Akira , Yamagata Noboru , Kashima Yuzuru , Takayama Hisao
Published: August 2011

The structure of protein A‐coated colloidal gold particles, and of macrophage cell‐surface receptors conjugated with immunogold particles, was studied using an ultrahigh‐resolution scanning electron microscope. Protein A, when conjugated with 15‐nm gold, formed a coat completely surrounding the particle. Particles conjugated with both protein A and immunoglobulin G (IgG) were similar, but with additional protrusions formed by the IgG. IgG molecules directly bound to gold were resolved sometimes as complexes of three units, sometimes as more filamentous, V‐shaped structures.

On the cell surface of a macrophage reacted with a monoclonal antibody to Mac‐1 antigen (the murine C3bi receptor) followed by protein A‐gold, gold particles were seen to be linked via the IgG to the receptor, visualized as a round granule.
The characterization of microstructures in the atom probe
Authors: Hetherington M. G. , Miller M. K.
Published: August 2011

The atom probe can be used to characterize fine‐scale microstructures. The results presented in this and a subsequent paper attempt to develop a methodology for interpreting information concerning 3‐D structures from the sequence of ions detected in the atom probe. Probability theory (Bernoulli trials and Markov processes) is used to build appropriate mathematical descriptions of different types of microstructure.
Estimation of section thickness and quantification of iron standards with EELS
Authors: Door R. , Frösch D. , Martin R.
Published: August 2011

The possibilities of using electron energy‐loss spectroscopy (EELS) for quantification of elemental concentrations in ultrathin sections are examined. Dynabeads, which are polystyrene beads with a known iron content, are proposed as internal iron standards. The quantity of an element present depends on the thickness of the specimen. A prerequisite for estimation of absolute section thicknesses with EELS is the knowledge of the mean free path λ of electrons in the specimen. This factor is determined for the embedding resins Epon and Nanoplast by comparing EELS data with directly observed thicknesses in re‐embedded sections. Dynabeads were found to include iron in a homogeneous distribution and to be stable in the electron beam.
Improved preservation of phospholipid‐rich multilamellar bodies in conventionally embedded mammalian lung tissue—an electron spectroscopic study
Authors: Fehrenbach H. , Richter J. , Schnabel Ph. A.
Published: August 2011

Different conventional methods of tissue processing were studied to determine the extent to which phospholipid‐rich multilamellar bodies of pulmonary alveolar epithelial type II cells of the pig were preserved. Prolonged treatment with half‐saturated aqueous uranyl acetate yielded excellent results on the stabilization of the multilamellar substructure, irrespective of whether glutaraldehyde‐paraformaldehyde or glutaraldehydetannic acid was used as a primary fixative. The lamellar periodicities were observed to be 5·5–6·1 nm.

Differences in the phosphorus distribution among several types of lipid bodies of alveolar epithelial type II cells were studied by means of electron spectroscopic imaging and electron energy‐loss spectroscopy. Multilamellar bodies gave phosphorus signals which were significantly higher than those obtained from granular regions of composite bodies, whereas homogeneous bodies gave phosphorus signals which were even lower than those obtained from mitochondria, endoplasmic reticulum membranes or ribosomes.
EXELFS analysis of amorphous and crystalline silicon carbide
Authors: Martin J. M. , Mansot J. L.
Published: August 2011

Core‐shell ionization edges from thin specimens of cubic silicon carbide (c‐SiC) are studied by means of electron energy‐loss spectroscopy (EELS) in a transmission electron microscope (TEM). Special attention is paid to the investigation of extended energy‐loss fine structures (EXELFS) to study local order effects around silicon atoms as a function of disorder in SiC.

Two forms of SiC were used: c‐SiC for the crystalline form, and friction‐induced amorphous silicon carbide (a‐SiC) for the disordered form.

EXELFS are generated by the elastic backscattering of outgoing electron waves by neighbouring atoms. Results show that both Si‐K and Si‐L edges contain structural information. EXELFS at the Si‐K edge can be used to calculate the radial distribution function (RDF) in the same way as extended X‐ray absorption fine structures (EXAFS). EXELFS at the Si‐L edge can be isolated by digital filtering. The high signal‐to‐noise ratio at the Si‐L edge permits the effect of larger interatomic distances to be observed, and thus the EXELFS technique is of promise for studying the effect of disorder in Si‐based ceramic materials with a high spatial resolution (nanometre scale).
Electron spectroscopic imaging and X‐ray microanalysis of acrylic fibres
Authors: Carboni V. , Lanzillotta F. , Carpanese G. F. , Mariani P. , Barbatelli G. , Sbarbati A. , Cinti S.
Published: August 2011

Acrylic fibres are synthetic fibres produced by extruding viscous solutions of acrylonitrile co‐polymers. A spin finish is applied during the fibre‐forming process. In this work the structure of acrylic fibres has been correlated with spin finish distribution by a combined application of transmission electron microscopy (TEM), electron spectroscopic imaging (ESI) and X‐ray microanalysis (XRMA). Many irregularly shaped microcavities at the periphery of the fibres were detected by TEM. XRMA revealed that potassium and phosphorus are the elements most often found inside the spin finish material. ESI revealed that phosphorus is constantly present in all the microcavities. Therefore it seems likely that a preferential distribution of the spin finish is found inside the microcavities, just at the periphery of the fibre. This work is also an example of how both ESI and XRMA are powerful tools in morphological studies.
Stereology for pores in wheat bread: statistical analyses for the Boolean model by serial sections
Authors: Bindrich Ute , Stoyan Dietrich
Published: August 2011

A simple approximative stereological method is proposed for obtaining the fundamental parameters of a three‐dimensional Boolean model. It uses serial sections for the determination of N+V, the specific convexity number. This procedure was applied to two different examinations of bread, which can be very well described by Boolean models.
A review of methods for the production and use of monoclonal antibodies to study zoosporic plant pathogens
Authors: Hardham A. R. , Gubler F. , Duniec J. , Elliott J.
Published: August 2011

For many species of Oomycetes, the infection of host plants is initiated by motile biflagellate zoospores. In recent years, studies of these zoospores have utilized monoclonal antibodies directed towards a variety of zoospore components. Although only two species of fungi, Phytophthora cinnamomi and Pythium aphanidermatum, have been used for immunization and initial screening, the reactions of these antibodies with over thirty species of fungi in the Peronosporales or Saprolegniales have now been determined. The present paper reviews the methods employed to produce the monoclonal antibodies and to use them to study the biology of the zoospores and the infection process.

The lack of a cell wall means that fixation protocols for zoospores for immunization and screening must be chosen carefully so that cell‐surface or intracellular sites will be accessible to the antibodies. The inclusion of glutaraldehyde in the fixative helps keep the zoospore plasma membrane as intact as possible, and screening with cells fixed in the presence of glutaraldehyde selects for antibodies that bind to the surface of the zoospores. Five different patterns of labelling to the zoospore surface have been found. Other antibodies bind with three distinct patterns to the surface of zoospores and/or cysts.

The use of formaldehyde alone in the fixative solution allows fragmentation of the plasma membrane and the exposure of intracellular components. In P. cinnamomi attempts to obtain antibodies directed against intracellular antigens were hampered by the presence of an immunologically dominant component that is stored in small vesicles in the zoospore cortex and secreted onto the surface of cysts. This problem was resolved by immunotolerizing mice neonatally before proceeding with immunization 2 months later. Antibodies directed towards a number of novel sites were obtained in this way.

Monoclonal antibodies generated by these methods have been used to identify taxonomically specific spore components, to locate surface molecules that might be responsible for the induction of zoospore encystment and to characterize molecules involved in spore adhesion to potential hosts.
Freeze‐substitution of scleractinian coral for confocal scanning laser microscopy and X‐ray microanalysis
Authors: Marshall A. T. , Wright O. P.
Published: August 2011

Freeze‐substitution in combination with confocal scanning laser microscopy provides a unique means by which the microstructure of undecalcified, and chemically untreated, polyps of scleractinian corals can be examined free of preparation artefacts. Mucocytes and intercellular spaces are particularly well preserved and the relationships of cell layers to each other and to the skeleton are undisturbed. Freeze‐substitution also permits X‐ray microanalysis of bulk samples and thin sections, a procedure which has hitherto been impossible to carry out on corals except on fixed tissue samples. Analyses indicated a high retention of Na+ and Cl− in spaces thought to be filled with fluid similar in composition to sea water. This increases confidence in freeze‐substitution as a means of retaining diffusible ions. Zooxanthellae contained metal/Ca ratios within the range of those previously reported for extracted zooxanthellae. It is shown that the mucocytes contain very high concentrations of S, K, Ca and Sr which are specifically localized in mucous granules. The concentrations differ between, and are characteristic of, different epithelial cell layers. Remarkably good correspondence was obtained between the two entirely different X‐ray analytical methods. This is the first time such a comparision between methods has been made. It is suggested that the uptake of transepithelially transported Ca2+ and Sr2+ by mucocytes may be a means of regulating the deposition of these ions in the skeleton.
Immunoelectron microscopic localization of elastic tissue components in archival tissue samples
Authors: Fanning Joseph C. , White Jacinta F. , Polewski Roman , Cleary Edward G.
Published: August 2011

Tissue samples that have been stored for many years, in different media and under a variety of conditions, have been examined by modern techniques of immunoelectron microscopy, using antibodies against elastic tissue components. A range of post‐embedding restorative procedures has been identified, which will allow reliable immunolocalization of antibodies against the elastic tissue component of such specimens. These methods have been applied successfully to autopsy‐derived material, fixed in buffered formaldehyde, to archival material stored frozen at −70 or −20·C, to specimens fixed for electron microscopy and stored for many years in buffer, and even to archival material from formaldehyde‐fixed, paraffin‐embedded blocks, reprocessed for electron microscopic examination.

The successful restorative methods included pre‐treatment of the sections with 6***M guanidine hydrochloride, or 1M Tris/saline, each containing 100 ***mM dithiothreitol (a reducing agent) followed by alkylation with 220 mM iodoacetamide. The application of these techniques allowed reliable study of elastic tissue antibody distributions in archival tissues that could not be obtained again, as well as comparative studies with tissues processed many years previously.
Preliminary characterization of an intrinsic germanium detector on a 400‐keV microscope
Authors: White T. J. , Cousens D. R. , Auchterlonie G. J.
Published: August 2011

An evaluation was made of an intrinsic germanium X‐ray detector fitted to a 400‐keV electron microscope. Its characteristics as a function of accelerating voltage, pulse processor settings and count rate were investigated. Comparisons are drawn between intrinsic Ge and Si(Li) detectors at both an experimental and theoretical level. The software requirements for the successful collection of spectra are discussed.
Cryosectioning of plant material frozen at high pressure
Authors: Michel M. , Hillmann T. , Müller M.
Published: August 2011

Low‐temperature electron microscopy combined with electron diffraction allows determination of the state of structural preservation as well as that of the solidified cellular water in frozen‐hydrated sections. Apple leaves frozen at high pressure and cryosectioned with glass knives covered with a very thin layer of frozen water exhibit a minimum of artefacts and are thin enough for structural analysis. The Zeiss 902 EM energy‐filtering microscope helps to improve the poor contrast of frozen‐hydrated sections by imaging in the zero‐loss mode. All sections analysed showed the electron diffraction pattern of vitreous water. This is ideal: the character of the liquid phase is preserved. A very high yield of good cryosections is achieved because the samples are vitrified and the friction on the glass knife surface is reduced by a thin ice layer.
A model for cryosectioning based on the morphology of vitrified ultrathin sections
Authors: Richter Karsten , Gnägi Helmut , Dubochet Jacques
Published: August 2011

Electron microscopy of vitrified ultrathin sections allows cell ultrastructure to be studied in the hydrated state. Sectioning of the frozen material is, however, a limiting step, since the cutting forces cause severe mechanical deformation. In order to address this problem, we have investigated the surface of cryosections. It is shown that cryosections have two fundamentally different surfaces. One surface is rough, deformed by cutting‐induced deformation lines which are orientated perpendicular to the cutting direction. The other surface, in comparison, is not affected by those deformation lines. Except for knife marks it is smooth. In order to explain the observations, the following model is proposed. The rough relief corresponds to the former block face. Its roughness originates from material that is squeezed out of the section plane when the section is compressed in the cutting direction and bent away from the specimen block. The smooth section surface is the surface in contact with the knife during the sectioning. This contact keeps the surface smooth while imprinting the knife marks.
Application of high‐resolution scanning electron microscopy to biological macromolecules
Authors: Nakadera Takashi , Mitsushima Akira , Tanaka Keiichi
Published: August 2011

The development of ultrahigh‐resolution scanning electron microscopes (SEMs) has made the observation of biological macromolecules feasible, but adequate preparation methods have not yet been established. Although it has been possible to observe some molecules after they have been spread on a carbon substrate, this method has not proved suitable for other molecules which exhibit lower contrast, or are more susceptible to damage by the electron beam. In this study we have applied heavy‐metal impregnation methods using phosphotungstic acid, uranyl acetate, or osmium tetroxide mordanted by tannic acid. In addition, contamination due to the electron beam was reduced by improving the vacuum in the specimen chamber, and by the use of a heated specimen stage. Using these measures, haemocyanin, ferritin, apoferritin, thyro‐globulin and immunoglobulin M were successfully imaged. Ultrahigh‐resolution SEM seems likely to become an important means for studying the morphology of biological macromolecules.
A quantitative analysis of transparency in the human sclera and cornea using Fourier methods
Authors: Vaezy Shahram , Clark John I.
Published: August 2011

Cellular microstructure observed by scanning transmission electron microscopy (STEM) was analysed using Fourier methods. Fourier components of the density fluctuations in the sclera and the cornea of the human eye were quantified. The results show that the Fourier components responsible for the opacity of the sclera have sizes of the order of the wavelength of visible light. In the sclera, approximately 54% of the spectral energy of the density fluctuation falls in the range of 200–1100 nm (scattering range). In the cornea, approximately 24% of the total spectral energy falls in this range. The predominant Fourier components of the density fluctuations in the opaque sclera are approximately 300 nm in wavelength, whereas those of the transparent cornea are approximately 80 nm in wavelength. This method will be useful in quantitative analysis of microstructural changes accompanying biological phenomena such as normal development of transparency in the human lens, and abnormal loss of transparency during cataract formation.
The influence of specimen refractive index, detector signal integration, and non‐uniform scan speed on the imaging properties in confocal microscopy
Authors: Carlsson K.
Published: August 2011

Refraction of light in a specimen volume may cause aberrations that influence the imaging properties in confocal microscopy. In this paper the influence on three‐dimensional resolution and geometry is experimentally investigated for a uniform specimen volume. It is found that the depth resolution is more severely affected than the lateral resolution. This is unfortunate, because even under ideal conditions the depth resolution is lower than the lateral resolution. Lateral image geometry is little affected by the specimen refractive index, whereas the depth scale can be considerably elongated or compressed.

The influence of a finite detector integration time is also considered. This can give a noticeable, but not particularly severe effect on the image resolution in the line‐scan direction. Because the integration time can be accurately controlled, a shorter integration time can be used when maximum resolution is essential, albeit at the price of a higher noise level.

In scanning fluorescence microscopy a non‐uniform scan speed may give large variations in bleaching over the specimen surface. Experiments illustrate how serious such non‐uniform bleaching effects can be when a specimen area is repeatedly scanned, for example when recording optical serial sections.
Absorption and scattering correction in fluorescence confocal microscopy
Authors: Visser T. D. , Groen F. C. A. , Brakenhoff G. J.
Published: August 2011

In three‐dimensional (3‐D) fluorescence images produced by a confocal scanning laser microscope (CSLM), the contribution of the deeper layers is attenuated due to absorption and scattering of both the excitation and the fluorescence light. Because of these effects a quantitative analysis of the images is not always possible without restoration. Both scattering and absorption are governed by an exponential decay law. Using only one (space‐dependent) extinction coefficient, the total attenuation process can be described. Given the extinction coefficient we calculate within a non‐uniform object the relative intensity of the excitation light at its deeper layers. We also give a method to estimate the extinction coefficients which are required to restore 3‐D images. An implementation of such a restoration filter is discussed and an example of a successful restoration is given.
Working with the confocal scanning UV‐laser microscope: specific DNA localization at high sensitivity and multiple‐parameter fluorescence
Authors: Montag Markus , Kukulies Jörg , Jörgens Reinhard , Gundlach Heinz , Trendelenburg Michael F. , Spring Herbert
Published: August 2011

The use of fast‐staining DNA‐specific dyes such as DAPI or Hoechst 33342/33258 has been a major problem for confocal scanning laser microscopy (CSLM) studies of intranuclear chromatin organization. Moreover, the availability of a confocal ultraviolet scanning laser microscope configuration, which allows an excitation at wavelengths of 364 nm as well as 488, 514 and 543 nm, is a prerequisite for single as well as multiple fluorescence parameter studies, especially if these studies are concerned with the precise localization of intranuclear signals.

Here we report the characteristics and application of a CSLM, which was adapted for UV‐excitation and therefore enables comparison of the spatial distribution of several types of signals within one preparation. In addition to multiple‐parameter studies, we have also investigated the sensitivity of the system with regard to the identification of the double‐stranded DNA of lampbrush chromosome loops in germinal vesicles of amphibian oocytes.
Accumulation of fluorescent non‐cationic probes in mitochondria of cultured cells: observations, a proposed mechanism, and some implications
Authors: Rashid Fiza , Horobin Richard W.
Published: August 2011

Cultured rat fibroblasts were exposed to millimolar concentrations of forty‐four non‐cationic fluorescent probes, of very varied physico‐chemical properties. Mitochondrial staining occurred with nineteen of these probes, nine of which were nominally anionic and ten nominally non‐ionic. All nineteen were in fact lipophilic weak acids. Using structural parameters these could be specified numerically as follows: electric charge ≤ 0; log P(less‐ionized form) < 0; and pKa ∼ 7. In addition to these structural variables, dye concentration and the time of exposure of cells to probes were significant factors for the staining of mitochondria. Accumulation of these compounds can be understood in terms of ion‐trapping of hydrophilic salts of lipophilic weak acids, due to the internal pH of respiring mitochondria being higher than the cytosolic pH. As a case example of the application of this approach, the mode of action of many inhibitors of mitochondrial anabolism is discussed in terms of the mechanisms introduced here.
Reflection electron imaging and spectroscopy studies of aluminium metal reduction at α‐aluminium oxide (0,1,2) surfaces
Authors: Wang Z. L.
Published: August 2011

Cleaved α‐aluminium oxide (0,1,2) surfaces are studied using combined techniques of scanning reflection electron microscopy and reflection electron energy‐loss spectroscopy. An α‐aluminium oxide (0,1,2) surface can terminate with a layer of either oxygen or Al atoms depending on where the bonds break. Aluminium metal can be reduced only from the domains initially terminated with oxygen by an intensive electron beam. The interpretation of this damage effect is based on the desorption process of oxygen atoms after Auger decay. The resistance to beam damage of the domains initially terminated with Al is found to be due to the protection of the surface adsorbed amorphous oxygen layer.
Images of 16S ribosomal RNA by scanning tunnelling microscopy
Authors: Flamion Pierre‐Jacques , Cachia Claire , Schreiber Jean‐Pierre , David Thierry , Lesniewska Eric , Goudonnet Jean‐Pierre
Published: August 2011

We report the use of scanning tunnelling microscopy (STM) to study surface topographies of complex nucleic acid structures. From low‐resolution STM images of uncoated 16S ribosomal RNA, we demonstrate the possibility of determining several objective parameters (molecular mass and radius of gyration) in order to characterize and identify the molecules observed. These parameters were compared with values obtained by other physical methods and the radius of gyration was found to be the most reliable. At high resolution, it was possible to measure the main dimensions of selected V‐form particles more precisely than with electron microscopy. Images of the more compact form have been also obtained that show different domains in the macromolecular structure.
Application of scanning electron microscopy (SEM) and microbead techniques to study the localization of p24 and p18 antigens of HIV‐1 on the surface of HIV‐1‐infected H9‐lymphocytes
Authors: Dennin Reinhard H. , Beyer Axel
Published: August 2011

Immunofluorescence staining techniques at present, when applied to follow the expression of HIV‐1‐specific antigens on infected cells, only give the information that the antigens detected are localized in the outer region of the membrane of the infected cell. We therefore set up a procedure using magnetic polystyrol particles coated with antibodies specific for the HIV‐1 antigens under study, in combination with scanning electron microscopy. We were able to demonstrate that p24 and p18 structural antigens are clearly expressed on the surface of HIV‐1‐infected H9 lymphocytes. This means that there was no steric hindrance for structures of cell‐like size specific for HIV‐1 antigens to interact with their target antigens. Other antigens may be hidden in membrane structures and are therefore inaccessible, for example, to the beads used here, which were of a similar size to antigen‐specific cells in vivo. The results of this model system must be seen with respect to the interaction of antigen‐specific cell‐mediated immunity with full antibody‐dependent cellular cytotoxicity, or without cytotoxic T lymphocytes, the mediator function of antibodies.
Visualization of ultraviolet microbeam irradiation sites with uranyl acetate
Authors: Czaban B. Barbara , Forer Arthur
Published: August 2011

A method which enables visualization of the position of the focused ultraviolet (UV) light (260–300 nm) in a UV microbeam apparatus is described. The UV light is focused onto uranyl acetate which fluoresces. The fluorescence is of low intensity, but can be visualized using a TV‐camera sensitive to low light levels. This is a simple, quick, and objective way to locate the position of the focused UV light in UV microbeam.
High‐pressure scanning electron microscopy of insulating materials: A new approach
Authors: FARLEY A. N. , SHAH J. S.
Published: August 2011

A new SEM technique for imaging uncoated non‐conducting specimens at high beam voltages is described which employs a high‐pressure environment and an electric field to achieve charge neutralization. During imaging, the specimen surface is kept at a stable low voltage, near earth potential, by directing a flow of positive gas ions at the specimen surface under the action of an electric bias field at a pressure of about 200 Pa. In this way charge neutrality is continuously maintained to obtain micrographs free of charging artefacts. Images are formed by specimen current detection containing both secondary electron and backscattered electron signal information. Micrographs of geological, ceramic, and semiconductor materials obtained with this method are presented. The technique is also useful for the SEM examination of histological sections of biological specimens without any further preparation.

A simple theory for the charge neutralization process is described. It is based on the interaction of the primary and emissive signal components with the surrounding gas medium and the resulting neutralizing currents. Further micrographs are presented to illustrate the pressure dependence of the charge neutralization process in two glass specimens which show clearly identifiable charging artefacts in conventional microscopy.
A simple ethanol‐based freeze‐substitution technique for marine invertebrate embryos which allows retention of antigenicity
Authors: Campbell Stephen S. , Crawford Bruce J. , Reimer Corinne L.
Published: August 2011

A simple technique has been developed for flash freezing and freeze substituting small (0.5 mm) marine embryos, which effectively preserves both cellular and extracellular components, using inexpensive equipment that is readily available in most laboratories. To achieve this, embryos of the starfish, Pisaster ochraceus, were isolated on copper freeze‐fracture EM grids. The embryos were then rapidly frozen by plunging the grids into a supercooled liquid cryogen, and stored in liquid nitrogen. Freeze substitution was carried out by placing the specimens in sealed vials containing anhydrous ethanol at −90°C for 4–5 days. Following substitution, the specimens were passively warmed to −20°C over 2 h and then to room temperature over a further 2 h. They were then embedded in either JB4 for light microscopy or Epon or LR White resins for transmission electron microscopy. Four different liquid cryogens, Freon 12, ethane, propane, and nitrogen slush, were tested. Freezing in propane, the best cryogen of the four, gave good preservation of the embryonic cells but poor preservation of the extracellular matrix (ECM). To overcome this, the embryos were exposed to four cryoprotective agents, dimethylsulphoxide, glycerol, ethylene glycol and propylene glycol prior to freezing, and the results were assessed. The experiments demonstrated that good preservation of both cells and ECM could be achieved by adding 15% propylene glycol in sea water to the embryos prior to freezing in propane. Material preserved in this manner not only gave excellent morphological results, but the antigenicity of both native antigens of ECM components and antibodies to which the animals had been exposed in vivo were retained. The application of this technique to other tissues and embryos should prove useful in many future studies.
Surface areas of basolateral membranes in renal distal tubules estimated by vertical sections
Authors: Mathiasen Flemming Ø. , Gundersen Hans Jøorgen G. , Maunsbach Arvid B. , Skriver Elisabeth
Published: August 2011

The surface area of the Na, K‐ATPase‐rich basolateral membranes in the medullary thick ascending limb of the distal tubule in the rabbit kidney was determined stereologically using the method of ‘vertical sections’, whereby unbiased surface estimates are obtained by imposing a cycloid test‐lattice on micrographs of ultrathin sections cut roughly longitudinal to the tubular axis.

The unbiased estimate of the surface area of basolateral membranes per tubule length in the thick ascending limb was 1.45 × 106 μm2/mm. The results are compared with previous surface area measurements in this segment of the tubule and discussed with respect to the contributions from all sampling levels to the real biological variation. An optimized sampling scheme with a roughly fourfold reduction in workload is suggested.
An approach based on two‐dimensional graph theory for structural cluster detection and its histopathological application
Authors: Kayser K. , Sandau K. , Paul J. , Weisse G.
Published: August 2011

An approach based on graph theory is described for detecting clusters of cells in tissue specimens (two‐dimensional space). With a set of discrete basic elements (cell nuclei) having several measurable features (area, surface, main and minor axis of best‐fitting ellipses) a graph is defined as having attributes associated with edges. Different minimum spanning trees (MSTs) can be constructed using different weight functions on the attributes (attributed MST). Analysis of the MST and of an attributed MST by use of a decomposition function allows detection of image areas with similar local properties. These clusters, which are then clusters of the tree, describe, for example, partial growth in different directions in a case of a human fibrosarcoma assuming that tumour cell nuclei are homogeneous with respect to their configuration and size. The model allows the separation of clusters of tumour cells growing in different directions and the approximation of the different growth angles. This decomposition also allows us to create new (higher) orders of structure (cluster tree).
The significance of 3‐D transfer functions in confocal scanning microscopy
Authors: Sheppard C. J. R. , Gu Min
Published: August 2011

The three‐dimensional (3‐D) transfer function is a useful concept for describing image formation in confocal scanning microscopy. From it we can derive the corresponding 2‐D transfer function for in‐focus imaging. In confocal transmission this can be derived analytically. The 1‐D transfer function for on‐axis imaging, which can be expressed in an analytical form even for confocal fluorescence with differing wavelengths of excitation and fluorescence, can be derived from the 3‐D transfer function. The 2‐D transfer function for in‐focus imaging in confocal fluorescence microscopy with a finite‐sized detector is also presented, which is shown to exhibit sign changes and can therefore result in reversals of image contrast.
The double disector: unbiased stereological estimation of the number of particles inside other particles
Authors: Marcussen Niels
Published: August 2011

A double‐disector method for obtaining estimates of the number of particles inside other particles, illustrated by an estimation of the average cell number in a glomerulus, has been developed. The method is suitable for paraffin‐embedded tissue because it does not require knowledge about the section thickness. Most importantly, the estimate is absolutely unaffected by tissue shrinkage. The average number of cells in a human glomerulus is 2850 whereas in rats it is 580.
Geostatistical and morphological methods applied to three‐dimensional microscopy
Authors: Conan V. , Gesbert S. , Howard C.V. , Jeulin D. , Meyer F. , Renard D.
Published: August 2011

Three‐dimensional (3‐D) images of osteocyte lacunae were examined on a confocal microscope. Both geostatistical and morphological processing techniques were used to improve and to analyse them. By a geostatistical approach, this study aims at improving 3‐D confocal images before any further image processing. Optimized linear filters, which take account of the second‐order statistics and the 3‐D structure of the data, allow for the removal of imperfections such as noise and/or blur due to the axial convolution, and interpolate voxels on a face‐centred cubic grid from an initial cubic grid. An application of this technique to 3‐D biological images is demonstrated. In a second step, a 3‐D binary image is digitized and cleaned with 3‐D morphological filters. The standard 3‐D measurements cannot be applied in this case, since all osteocytes cut the border of the field. For this reason a 3‐D Boolean model has been adjusted, from which it is possible to derive all useful information on the repartition and the morphology of the osteocytes.
Interpretation of electron microscope autoradiographs: correction for cross‐fire when membrane components are highly labelled
Authors: Hejblum Gilles , Mary Jean‐Yves , Morice Vincent , Aurengo Andre
Published: August 2011

The interpretation of electron microscope autoradiographs concerns the estimate of the relative concentration of a radiolabelled molecule in subcellular compartments. Because of the radiation spread, an autoradiographic grain underlying a subcellular compartment cannot be directly assigned to the latter. Observed data must therefore be corrected for cross‐fire (CF). We studied the efficiency of several methods designed for the CF correction when membrane components possess a high concentration of labelling, resulting in a high proportion of CF. Grains generated from simulated sources were subjected to the CF method, to an original method based on the simulated annealing optimization algorithm, and to the expectation‐maximization algorithm. The expectation‐maximization algorithm appears to be clearly superior to the two other methods. Nevertheless, the variances of the estimates were higher than expected. Since no analytical expression of the estimates is available, an objective comparison of the labelling in different compartments is not possible with a standard test. Consequently, the interpretation of autoradiographs in electron microscopy is still an open problem.
Observation of rhombic facets on platinum wire after heating in air using scanning tunnelling microscopy
Authors: Hoffmann‐Millack Britta , Roberts Clive J. , Steer William S.
Published: August 2011

Evidence is provided for the formation of surface faceting due to heating in air at 1160 K. For the first time diamond‐shaped facets with angles of 60 and 120° have been observed on the surface of a sample of ultrapure platinum wire in air using scanning tunnelling microscopy. The maximum extension of the facets is about 1000 Å.
Methodology for spectrum evaluation in quantitative electron energy‐loss spectrometry using the Zeiss CEM902
Authors: van Puymbroeck J. , Jacob W. , van Espen P.
Published: August 2011

The spectral energy‐loss intensities acquired with the Zeiss CEM902 do not follow a Poisson law. The intensities are normally distributed and their variances are non‐uniform. When fitting the inverse power law after logarithmic transformation, it is expected that the power law coefficients and their uncertainties will be biased. Due to the particular dependence of the variance on the intensity which is observed in our experiments, it appears that no bias is introduced when using unweighted least‐squares fitting after log‐log transformation. Because the sample variances are estimated with low degrees of freedom, the residual variance of the fit is proposed as a criterion rather than the X2 test to evaluate the optimum width of the fitting window. The optimal width for the integration window can be determined as the window where the signal‐to‐noise ratio is at a maximum. Critical limits for qualitative and quantitative analysis are proposed which are based on the variance of the net intensity. They can be used to test the analytical significance of the signals.