bluuebunny/crossref_metadata_2025 huggingface dataset:
Contrast in high‐resolution scanning electron microscope images
Authors: Joy David C.
Published: August 2011

Current scanning electron microscopes, equipped with field emission guns and high‐performance immersion lenses, can achieve spatial resolutions of the order of 1 nm in both secondary and backscattered imaging modes over a wide range of operating energies. The generation and interpretation of images with nanometre‐scale resolution relies on a detailed knowledge, and application, of electron‐solid interactions. This paper develops the practical steps required to produce a high‐resolution image, and discusses the principles which govern image interpretation. Attention is focused primarily on materials which are low in atomic number and density, such as biological tissue, but the results apply after appropriate scaling of the physical parameters to most other materials.
High‐resolution scanning electron microscopy of frozen‐hydrated and freeze‐substituted kidney tissue
Authors: Herter Peter , Tresp Gregor , Hentschel Hartmut , Zierold Karl , Walther Paul
Published: August 2011

Inner surfaces and fracture faces of rabbit kidney tissue were investigated with high‐resolution scanning electron microscopy using two different cryopreparation techniques: (i) for the observation of fracture faces, cryofixed tissue was fractured and coated in a cryopreparation chamber dedicated to SEM, vacuum transferred onto a cold stage and observed in the frozen‐hydrated state; (ii) for the observation of inner surfaces of the nephron, water was removed after freezing and fracturing by freeze substitution and critical‐point drying of the tissue.

By both methods, macromolecular structures such as intramembranous particles on fracture faces and particles on inner surfaces were imaged. The latter method was used to investigate in more detail surface structures of cells in the cortical collecting duct. These studies revealed a heterogeneity of intercalated cells not described thus far.
Modelling and analysis of 3‐D arrangements of particles by point processes with examples of application to biological data obtained by confocal scanning light microscopy
Authors: König D. , Carvajal‐Gonzalez S. , Downs A. M. , Vassy J. , Rigaut P.
Published: August 2011

Within the concept of point processes, a review is presented of quantities which can be used in studies of three‐dimensional (3‐D) aggregates of particles. Suitable characteristics and estimators are given for both unmarked and marked point processes. To demonstrate the feasibility of such quantitative approaches, an application in histology, dealing with 3‐D arrangements of cell nuclei in rat liver, is described. Using a confocal scanning light microscope, 3‐D images are recorded and image analysis used to obtain the coordinates of the centroid, together with the volume and DNA content, of each cell nucleus. Examples of results are given, using both unmarked and marked point processes. In the latter case, cell type, nuclear volume and ploidy group are suitable marks.
Ultra‐rapid freezing by spraying/plunging: pre‐cooling in the cold gaseous layer
Authors: Chang Zhaohua , Baust John G.
Published: August 2011

A thermophysical model is established to analyse the influence of pre‐cooling of a biological specimen in the cold gas layer associated with spray‐freezing techniques. The basic principles governing the process of pre‐cooling are provided. It is concluded that pre‐cooling is one of the major limiting steps in attaining an overall ultra‐rapid cooling rate. Pre‐cooling has a substantial influence on the nature of the final frozen specimens. In order completely to avoid crystallization before entry into the liquid cryogen and maximize the overall cooling rate of the specimen, precautions should be taken to control the height of the gaseous layer and the size of the specimen. The probability of the specimen being frozen in the cold gaseous layer is reduced by increasing the entry speed. The effectiveness, however, becomes less marked at speeds greater than 10 m/s. In order to minimize the risk of misinterpreting the measured cooling rate, it is necessary to specify the pre‐cooling conditions. The pre‐cooling effect is much more evident in liquid helium than in cryogens such as propane, ethane, Freon 12, 13 and 22.
Surfaces of cryosections: is cryosectioning ‘cutting’ or ‘fracturing’?
Authors: Kirk R. Gary , Knoff Laura , Lee Ping
Published: August 2011

In order to determine if cryosectioning involves ‘fracturing’ or ‘cutting’ we examined the surfaces obtained in cryosectioning by a metal‐replicating procedure commonly used in freeze‐fracture microscopy. Platinum‐carbon replicas were made of the surfaces of both the sections and the complementary surfaces of the sample stubs from which the sections were cut. When samples of frozen red cells were sectioned at −120°C with large knife advancements (1 μm), the chips produced did not resemble sections. Membrane fracture faces, produced by splitting of the lipid bilayer, were found in electron micrographs of replicas of the sample stubs. This demonstrates that a cryomicrotome can be used to produce large intact replicas. When dull knives were used with small knife advancements, both smooth and fractured regions were found. The sections produced with dull knives had a snowflake appearance in the light microscope. When sharp knives were used with small advancements (0·1 μm), replicas of the surfaces were free of fracture faces and the sections had a cellophane‐like appearance in the light microscope. Therefore, in cryosectioning a different process other than ‘fracturing’ is responsible. This ‘cutting’ process may be micromelting of a superficial layer by the mechanism of melting‐point depression from the pressure exerted by the sharp edge of the knife.
Radiation damage in soft X‐ray microscopy of live mammalian cells
Authors: Shinohara Kunio , Ito Atsushi
Published: August 2011

When specimens are observed by soft X‐ray microscopy, they always absorb many photons, causing radiation damage at the imaged site. The problems of radiation damage were studied in view of the principle of image formation; absorption contrast, scattering (holography), or phase contrast. In all cases, photons with a wavelength of 1–10 nm interact with the specimen mainly through the photoelectric effect followed by the transfer of energy to the imaging site either directly (absorption imaging) or indirectly (holography or phase contrast). This absorbed energy will cause structural changes to the imaging site. From a review of the literature the absorbed dose is estimated to be as high as 107 Gy when the expected resolution of the specimen (1–10 thick) is 10 nm. This dose is far in excess of the amount required for cells to be able to survive when live mammalian cells are exposed. The levels of radiation effects were extrapolated to the estimated absorbed dose from the reported values for cell survival, chromosome aberrations, and DNA strand breaks with respect to observations on mammalian chromosomes. The extrapolated results show that some damage will occur in every 10 times 10‐nm (expected resolution) size unit. Although these studies focused only on the effects on mammalian chromosomes, the present results are more or less common phenomena in the observation of biological specimens. Hence, the results suggest that dynamic observations will be difficult. On the other hand, a time‐scale study of the effects of radiation on structural integrity suggests that single‐shot imaging with short‐pulsed (probably shorter than a few milliseconds) X‐rays may be appropriate for the observation of intact live biological specimens in the hydrated condition, before they have deteriorated.
The use of adhesive‐coated grids for the X‐ray microanalysis of dry‐cut sections in the TEM
Authors: Fritz Eberhard
Published: August 2011

Sections cut dry for the X‐ray microanalysis of diffusible elements were fixed to adhesive‐coated single fine‐bar grids. The drawbacks of folding grids normally used for this purpose can thus largely be avoided.
Quantitative analysis of electron energy‐loss spectra from ultrathin‐sectioned biological material
Authors: Sorber C. W. J. , Ketelaars G. A. M. , Gelsema E. S. , Jongkind J. F. , De Bruijn W. C.
Published: August 2011

Electron energy‐loss spectroscopy (EELS) has been used to determine elemental concentrations in biological specimens, consisting of ultrathin‐sectioned cells and tissues. Chelex100‐based Ca‐ and Fe Bio‐standards are used for elemental quantification to establish iron and calcium concentrations. These Bio‐standards, as well as the biological materials, are treated in a standard EM procedure such that ‘known’ and ‘unknown’ sites are located in one cross‐section.

Uncertainties and variabilities present in the equations for calculating the concentration in the ‘unknown’ site (determined by comparing simplex‐fitted EEL spectra from Bio‐standards with those from tissue) are outlined in two examples. Using an H+ Bio‐standard, the matrix composition of such biological cell material is analysed, leading to values which approach each other closely. Quantitative EELS, using Chelex100‐based Bio‐standards, is advocated.
A simplified preparation procedure of plant material for elemental analysis by ESI and EELS techniques
Authors: Lehmann H. , Kunz U. , Jacob A.
Published: August 2011

Living plant cells were embedded in the water‐soluble resin Nanoplast FB 101 and cut into ultrathin sections. Non‐vacuolated cells of algae and fungi showed a satisfactory preservation of fine structure. First results of elemental mapping by the ESI technique indicate that phosphorus and sulphur are neither extracted nor dislocated within the cells.

The authors are members of the ‘Study Group for Electron Microscopy’ at the Hannover School of Veterinary Medicine.
The glycosomes of trypanosomes: number and distribution as revealed by electron spectroscopic imaging and 3‐D reconstruction
Authors: Tetley L. , Vickerman K.
Published: August 2011

Computer‐aided 3‐D reconstruction of typanosomes from 0·35‐μm‐thick sections imaged on the Zeiss 902 electron microscope are being used to study the dynamics of cell organization. Segregation of glycolytic enzymes into glycosomes raises questions concerning the distribution and biogenesis of these organelles. Direct counts of glycosomes from Trypanosoma evansi indicate 30–40 per cell and for the closely related T. brucei, 65 per cell. These figures contrast with the estimates of others who have used model‐based morphometric methods to obtain a value of 230 per cell.
Ultrastructural aluminium detection in amphibian tissues by electron spectroscopic imaging and electron energy‐loss spectroscopy
Authors: Böhmer J. , Rahmann H.
Published: August 2011

Aluminium causes a variety of toxic effects in living organisms but very little is known about its uptake, pathways and locations of deposition. We have applied electron spectroscopic imaging (ESI) and electron energy‐loss spectroscopy (EELS) to locate aluminium at the ultrastructural level in amphibian larvae from acidic ponds. It is found diffusely bound or precipitated in cell organelles. The spatial resolution of aluminium detection is high. The elemental composition of small areas can be demonstrated by EELS. Three different fixation procedures give similar results. The two‐ and three‐window methods at the K‐ and L‐edges are compared.
Demonstration of calcium in dermal melanocytes of Xenopus laevis and Poecilia reticulata with electron energy‐loss spectroscopy and electron spectroscopic imaging
Authors: Müller Thomas , Bereiter‐Hahn Jürgen
Published: August 2011

The subcellular distribution of calcium in dermal melanocytes of Xenopus laevis and Poecilia reticulata has been analysed. Using two cytochemical methods, phosphate precipitation and a combined oxalate‐pyroantimonate technique, electron energy‐loss spectroscopy and electron spectroscopic imaging have been applied for elemental analysis. Both precipitation techniques revealed a high calcium content in the melanosomes of both species. Calcium was also located in the vicinity of collagen fibrils and in the plasma membrane.
Electron spectroscopic diffraction and imaging of the early and mature stages of calcium phosphate formation in the epiphyseal growth plate
Authors: Barckhaus R. H. , Höhling H. J. , Fromm I. , Hirsch P. , Reimer L.
Published: August 2011

A review of different models of biomineralization in collagen‐rich hard tissues shows that further investigations of crystal formation are necessary. The electron spectroscopic diffraction (ESD) mode of operation of an energy‐filtering electron microscope offers the possibility of being able to avoid the background from inelastic scattering in selected‐area electron diffraction patterns. First experiments on the different stages of mineralization in the epiphyseal growth plate have only indicated the presence of apatite. The ESD mode can be complemented by the electron spectroscopic imaging mode and by elemental mapping of calcium.
A quantitative computer simulation of microscopic imaging
Authors: Goldstein D. J.
Published: August 2011

A versatile program is described for a BBC‐ or IBM‐compatible microcomputer, which uses a fast Fourier transform in calculating diffraction‐limited microscopic images. The defined ‘object’ can be a diffraction grating or a discrete specimen seen in cross‐section, of arbitrary interval (or width), transmittance and retardation. Many microscopical techniques can be simulated including bright‐field, phase contrast, central and peripheral dark‐field, schlieren, apodization, fluorescence, differential interference contrast and confocal scanning. Instrumental settings which can be varied include the aperture, focus and primary spherical aberration of the objective, the coherence and obliquity of the illumination, and in phase contrast the width, transmittance and retardation of the phase plate. The Fourier transform and various other algorithms used in the program are explained.
Study of potassium deficiency in cardiac muscle: quantitative X‐ray microanalysis and cryotechniques
Authors: Pogorelov A. , Allachverdov B. , Burovina I. , Mazay G. , Pogorelova V.
Published: August 2011

An imbalance of potassium in cardiac muscle causes an alteration of heart function. The distribution and concentration of potassium in rat papillary heart muscle was studied using cryofixation and X‐ray microanalysis. Freeze‐dried cryosections and sections of freeze‐dried, embedded tissue were analysed. Bulk frozen specimens were freeze‐dried either in a vacuum or by a new technique using liquid propane as a cryodehydration medium. These two methods of freeze‐drying were tested for elemental retention in other specimens, with comparable results. A potassium concentration of 120 mmol/l was measured in normal myocytes of cardiac papillary muscle compared to 80 mmol/l in myocytes of animals stressed by a temperature of 45°C for 1 h. The presumed physiological significance of the findings is discussed.
An improved analysis of experimental data from 125 I hot‐line autoradiographs: allowing for the effects of background grains
Authors: Aykroyd R. G. , Anderson C. W. , Junger E. , Williams M. A.
Published: August 2011

In this paper we suggest a new statistical method of correcting the results of hot‐line experiments for the effects of background sources and we use the new method to reassess the adequacy of three probability distributions proposed in the literature for image spread from line sources. The data are from sources labelled with 125I in semi‐thin resin sections 0·4‐0·8 μm in thickness. The new method reveals that two of the models describe the empirical distributions more closely than earlier analysis had suggested, and it confirms an increasing relationship between half distance of image spread and the thickness of the source. However, it also confirms that considerable ‘inter hot‐line’ experimental variation remains, even after background correction. This suggests that multiple experiments are needed to produce reliable estimates of half distance.
Structural characterization of air‐oxidized chromium particles by extended energy‐loss fine‐structure spectroscopy
Authors: Diociaiuti Marco , Paoletti Luigi
Published: August 2011

Extended energy‐loss fine‐structure spectroscopy (EXELFS) and electron energy‐loss spectroscopy (EELS) microanalysis have been used to study the structural and chemical modifications caused by interaction with the atmosphere of small, very reactive chromium (Cr) particles whose diameters range from 150 to 1300 Å.

Analysis of the data allows us to deduce that the particles consist of a Cr core with a body‐centred cubic structure and with a lattice parameter reduced by about 4% with respect to the bulk value by surface stresses (for the smallest diameter), surrounded by a Cr2O3 shell of about 15‐Å width.
Comparison between the conventional method and the simple desiccation method in porcine sperm processing for scanning electron microscopy
Authors: Bonet S. , Briz M.
Published: August 2011

Sperm quality has been studied using different microscopic methods. Light microscopy gives similar percentages of sperm typologies (mature, immature and aberrant) as scanning electron microscope (SEM) techniques using both normal (critical‐point drying) and simple desiccation techniques. Artefactual forms, which are easily recognizable, are not taken into account. The artefactual forms observed by the simple desiccation SEM method were spermatozoa with tails folded in a simple loop, and spermatozoa with short tails coiled at their extreme. The simple desiccation SEM method is a useful and rapid technique for the testing of porcine sperm quality.
Alkaline hydrolysis/methylation‐acetylation: a new technique for ultrastructural DNA cytochemistry
Authors: Romero J. B. , Ferrer J. M. , Tato A. , Tandler C. J. , Llorente A. R. , Del Castillo P. , Stockert J. C.
Published: August 2011

A new technique for the visualization of DNA‐containing structures in electron microscopy is described. Samples of glutaraldehyde‐fixed bone marrow from rats were subjected to alkaline hydrolysis to remove RNA and the phosphate of phospho‐proteins, followed by a combined blockage of protein carboxyl and amino groups through methylation‐acetylation. After uranyl acetate staining of epoxy‐embedded ultrathin sections, chromatin from all cell types showed a highly selective and intense electron opacity. Staining methods for DNA were also positive in semithin sections. This simple procedure could be very useful in ultrastructural cytochemistry of DNA and chromatin.
Electron probe X‐ray microanalysis of intracellular element concentrations in cryosections in the presence of changes in cell volume
Authors: Bostrom T. E. , Field M. J. , Györy A. Z. , Dyne M. , Cockayne D. J. H.
Published: August 2011

The interpretation of element concentration data for X‐ray microanalyses of biological tissues, which are subjected to some experimental treatment, can be complicated by changes in cell volume and total cell dry matter induced by the treatment. We have examined the manner in which such changes would affect the values measured in frozen‐dried cryosections of soft tissues, and how they may be taken into account in the interpretation of the results. The element content (mass per unit dry weight) measured by the peak‐to‐continuum or Hall method is independent of changes in cell volume, but is sensitive to a change in the local dry mass. Conversely, intracellular concentrations in terms of mass per unit volume, as determined by the peripheral or internal standard technique, are dependent on volume changes but independent of dry mass. The estimated dry weight fraction is affected by changes in both volume and dry mass. The results obtained from both quantification methods can therefore provide information on the combination of changes in cellular element levels, volume and total dry mass that may occur following the experimental treatment.

In a study of the late effect of the drug cisplatin on electrolyte concentrations in kidney proximal tubules, both quantification methods have been used to obtain wet weight and dry weight concentrations. By applying the above considerations, the analytical results have been interpreted as a combination of changes in element levels and a shrinkage of the tubule cells. Cell shrinkage was confirmed by morphometric analysis of tubular cross‐sections.
A device for freeze‐substituting a large number of samples under controlled conditions
Authors: Marshall A. T. , Kent M.
Published: August 2011

An inexpensive device for reproducible freeze‐substitution is described. The equipment consists of a reciprocating shaker, holding vials of substitution fluid, mounted in the gas phase of a large liquid nitrogen refrigerator. The shaker temperature is controlled by a programable microprocessor.
Advances in ALCHEMI analysis
Authors: Turner P. S. , White T. J. , O'Connor A. J. , Rossouw C. J.
Published: August 2011

The ALCHEMI method for locating the sites of foreign atoms within crystals is known to be sensitive to the delocalized emission of X‐rays. This can result in large errors in some cases through differences in delocalization for different excitations or by error amplification in the ratio method of analysis. An alternative approach to the analysis of ALCHEMI data, using multivariate statistical analysis, is extended to the case of multiple impurities. Initial results from zone‐axis channelling experiments for a Yb‐doped zirconolite (CaZrTi2O7) are shown to confirm the improved accuracy of this method, especially for axial orientations. Data were collected using a 400‐keV analytical electron microscope fitted with an intrinsic Ge X‐ray detector. The potential advantages for ALCHEMI analysis of Ge detectors are considered.
Electron microscopy in synthetic polymer membrane research
Authors: Kim K. J. , Dickson M. R. , Fane A. G. , Fell C. J. D.
Published: August 2011

The use of electron microscopy (EM) to study microporous membranes is described. Application of EM to polymer membranes can be classified into two major areas: elucidation of membrane formation mechanisms (the effect of preparative parameters on morphology of the final membrane) and physical characterization of membranes.

Conventional transmission and scanning electron microscopes have been used successfully to clarify details of membrane organization (structure) and formation mechanisms as well as supplying some information on the pores. However, neither transmission electron microscopy nor scanning electron microscopy could resolve the fine surface morphology of ultrafiltration membranes due to instrumentation limitations. The recent development of field‐emission scanning electron microscopy has opened new opportunities in membrane research.
Estimating the length of a bounded curve in three dimensions using total vertical projections
Authors: Cruz‐Orive Luis M. , Howard* C. Vyvyan
Published: August 2011

A new method is proposed to estimate the total length of a bounded, isolated linear feature in three dimensions from ‘total vertical projections’, obtained by rotating the curve about a fixed axis (arbitrarily called ‘vertical’) and projecting it onto a fixed vertical plane. No sections are required. Properly stained and embedded neuron dendrites, mycelial trees, fluorescent cytoskeletal filaments within a cell, etc., are candidate specimens for the method, especially in combination with the new devices for non‐invasive three‐dimensional microscopy. It is necessary that the specimen curve is rigid (i.e. of constant shape), that its length density is not too high (so that overlapping effects are not important) and that the embedding medium is fairly transparent. Given these requirements, the method can be very accurate and convenient to use, as is exemplified here.
An evanescent‐field optical microscope
Authors: van Hulst N. F. , de Boer N. P. , Bölger B.
Published: August 2011

The classic diffraction limit of resolution in optical microscopy (∼γ/2) can be overcome by detecting the diffracted field of a submicrometre‐size probe in its near field. The present stage of this so‐called scanning near‐field optical microscopy (SNOM) is reviewed.

An evanescent‐field optical microscope (EFOM) is presented in which the near‐field regime is provided by the exponentially decaying evanescent field caused by total internal reflection at a refractive‐index transition. A sample placed in this field causes a spatial variation of the evanescent field which is characteristic for the dielectric and topographic properties of the sample. The evanescent field is frustrated by a dielectric probe and thus converted into a radiative field.

In our case the probe consists either of an etched optical fibre or of a highly sharpened diamond tip. The probe is scanned over the sample surface with nanometre precision using a piezo‐electric positioner. The distance between probe and sample is controlled by a feedback on the detected optical signal. The resolution of the microscope is determined by both the gradient of the evanescent field and the sharpness of the tip. Details of the experimental set‐up are discussed.

The coupling of the evanescent field to the submicrometre probe as a function of probe‐sample distance, angle of incidence and polarization has been characterized quantitatively. The observed coupling is generally in agreement with presented theoretical calculations.

Microscopy has been performed on a regular latex sphere structure, which clearly demonstrates the capacity of the evanescent‐field optical microscope for nanometre‐scale optical imaging. Resolution is typically 100 nm laterally and 10 nm vertically.

The technique is promising for biological applications, especially if combined with optical spectroscopy.
The point‐spread function of a confocal microscope: its measurement and use in deconvolution of 3‐D data
Authors: Shaw Peter J. , Rawlins David J.
Published: August 2011

We have measured the point‐spread function (PSF) for an MRC‐500 confocal scanning laser microscope using subresolution fluorescent beads. PSFs were measured for two lenses of high numerical aperture—the Zeiss plan‐neofluar 63 × water immersion and Leitz plan‐apo 63 × oil immersion—at three different sizes of the confocal detector aperture. The measured PSFs are fairly symmetrical, both radially and axially. In particular there is considerably less axial asymmetry than has been demonstrated in measurements of conventional (non‐confocal) PSFs. Measurements of the peak width at half‐maximum peak height for the minimum detector aperture gave approximately 0·23 and 0·8 μm for the radial and axial resolution respectively (4·6 and 15·9 in dimensionless optical units). This increased to 0·38 and 1·5 μm (7·5 and 29·8 in dimensionless units) for the largest detector aperture examined. The resulting optical transfer functions (OTFs) were used in an iterative, constrained deconvolution procedure to process three‐dimensional confocal data sets from a biological specimen—pea root cells labelled in situ with a fluorescent probe to ribosomal genes. The deconvolution significantly improved the clarity and contrast of the data. Furthermore, the loss in resolution produced by increasing the size of the detector aperture could be restored by the deconvolution procedure. Therefore for many biological specimens which are only weakly fluorescent it may be preferable to open the detector aperture to increase the strength of the detected signal, and thus the signal‐to‐noise ratio, and then to restore the resolution by deconvolution.
Digital three‐dimensional visualization of cellular organelles studied by medium‐ and high‐voltage electron microscopy
Authors: Beorchia A. , Ploton D. , Menager M. , Lebonvallet S. , Thiry M. , Bonnet N.
Published: August 2011

Tilted thick sections (one‐half to several micrometres) of biological specimens observed with medium‐ to high‐voltage electron microscopes are extremely useful for the study of the three‐dimensional (3‐D) structure of organelles.

If high resolution in 3‐D visualization and 3‐D reconstruction is needed, many images corresponding to various angles of rotation and tilt must be recorded. This necessitates very time‐consuming work—including eventual photographic processing—before good positioning of the object is defined.

We have developed software which permits very rapid and precise determination of the tilt‐axis, the registration of tilted views, 3‐D measurements and 3‐D visualization. Images are digitized either from negative films or directly with a camera fitted to the microscope. The application of the software is performed in minutes and allows for a rapid check of the quality of the tilt‐series and of the features of interest of the object.

Application of the software to the study of the 3‐D structure of active components of the nucleolus stained with silver is shown.
SEM electron channelling patterns as a technique for the characterization of ion‐implantation damage
Authors: Page Trevor F. , McHargue Carl J. , White C. W.
Published: August 2011

Electron channelling patterns (ECPs) formed in back‐scattered images in the scanning electron microscope (SEM) have been used occasionally to confirm surface amorphization during ion implantation. In order to place such observations on a more quantitative basis, the study reported here has explored the variation of ECP appearance with both specimen damage levels (and thus subsurface structures) and SEM accelerating voltage (i.e. sampled depth).

Polished and annealed (0001) single crystal sapphire discs were implanted to various damage levels up to both subsurface and full surface amorphization. Damage levels were measured independently by Rutherford back‐scattering (RBS). Selected‐area ECPs were obtained in a Jeol‐840 electron microscope operating over the range 5–40 kV in 5‐kV steps.

Progressive ECP degradation—in terms of high‐order line disappearance—was observed with increasing dose, culminating in total pattern loss when full surface amorphization occurred. However, ECP information could still be obtained from the damaged near‐surface material even when a subsurface amorphous layer was present, thus demonstrating the shallow retrieval depth of information from the ECP technique. Indeed, because the spatial distribution of damage from ion implantation is both calculable and measurable, these experiments have also allowed us, for the first time, to explore and demonstrate the shallow sample depths from which the majority of ECP contrast originates (< 150 nm in sapphire at an accelerating voltage of 35 kV), even when the beam penetration is considerable by comparison (∼ 5 μm). Furthermore, the way in which this sampled depth varies with SEM accelerating voltage is both demonstrated and shown to be a powerful diagnostic technique for studying the distribution of near‐surface structural damage.
Determination of the Cnl parameter in the Bethe formula for the ionization cross‐section by the use of Cliff‐Lorimer KAB factors obtained at different accelerating voltages in a TEM
Authors: Gauvin R. , L'Espérance G.
Published: August 2011

There is a clear advantage in using theoretical KAB factors to perform quantitative microanalysis, because they do not have to be experimentally determined. To compute KAB factors, we need a very good parameterization of ionization cross‐sections. If the bnl parameter in the Bethe formula for the ionization cross‐section is not a function of the element (A or B), then only the Cnl parameter is required to compute KAB factors. In this paper we present a new method to measure Cnl based on the ratio of KAB factors obtained at different accelerating voltages.
A modified Jelley refractometer
Authors: Langford Stephen A.
Published: August 2011

Light beams are read directly (not as virtual images) against a scale erected about 74 cm in front of the prism, with a modified Jelley refractometer. Liquid temperatures are controlled by thermistors placed at each end of the prism trough, which is enclosed in an aluminium block.

The scale is calibrated against the refractive index (RI) of distilled water, seven (sic) RI estimates for six Cargille liquids related to six glass standards, and the label RIs of ninety‐eight other Cargille liquids.

The calibrated refractometer is used accurately and precisely to calibrate ninety‐six Cargille liquids, over the inclusive 1ċ400–1ċ800 (25ċ0°C, 589ċ3 nm, RI unit) label range, at liquid temperatures of 19–49°C and illuminating wavelengths of 435ċ8–694ċ3 nm.
Axial resolution of confocal microscopes with parallel‐beam detection
Authors: Shao Zhifeng , Baumann Otto , Somlyo Andrew P.
Published: August 2011

We present a simple theory for the evaluation of the axial resolution of a confocal scanning microscope with parallel‐beam detection. The results demonstrate that, in certain cases, the collection efficiency is low compared with a conventional confocal microscope, but the axial resolution may be further improved.
Electron energy‐loss spectroscopy analysis of adriamycin‐plasma membrane interaction
Authors: Diociaiuti M. , Molinari A. , Calcabrini A. , Arancia G.
Published: August 2011

Our previous studies on the mechanism of cytotoxic action of the anti‐tumour drug adriamycin (ADR) indicated that this anthracyclinic antibiotic strongly modified the molecular architecture of the plasma membrane of human erythrocytes, presumably becoming incorporated within both lipid layers. In order to verify this hypothesis, electron energy‐loss spectroscopy (EELS) has been used to compare the P content in control and ADR‐treated erythrocyte ghosts. EELS measurements allowed us to reveal a significant reduction in the P/C ratio in erythrocyte ghosts after ADR treatment. This finding seems to reflect a phospholipid ‘dilution’ produced by the incorporation of the drug molecules in the membrane layers. A structural model of the ADR‐membrane interaction is proposed.
Quantitative theory of ideal phase‐contrast microscopy, taking object width into account
Authors: Goldstein D. J.
Published: August 2011

In ‘ideal’ phase‐contrast microscopy all the direct light and none of the diffracted light is influenced by the phase plate in the back focal plane of the objective. Contrary to almost all previous work, it appears that the intensity of an ideal phase‐contrast image is affected not only by the transmittance and retardation of the object and of the phase plate, but also by the width of the specimen (or total width of multiple specimens) relative to the microscopic field. Equations and computer code are presented with which the intensity of such images can be calculated. Previously published equations are special cases, and implicitly or explictly assume either that the object is of negligible width, or occupies precisely half the microscopic field.

The absolute brightness of an image in ideal central dark‐field microscopy is a function of the object retardation, but the intensity of the image relative to the background is a function only of the width of the object(s) relative to the field.

The equations give results for ideal phase‐contrast microscopy identical with those of a computer program simulating microscopic imaging. The program can in addition take into account non‐ideal factors including a finite width of phase plate, finite objective aperture, deviations from best focus, glare, primary spherical aberration and obliquity of the coherent illumination.
The experimental effect of detector size on confocal lateral resolution
Authors: Glass Monty , Dabbs Tim
Published: August 2011

The lateral resolution of a scanning‐spot microscope has been measured as the product of the pin‐hole size and lens numerical aperture of the detector approaches the confocal limit. The resolution follows an earlier prediction, improving approximately 30% in going from that of a conventional scanning‐spot or broad‐viewing‐area microscope to that of a fully confocal microscope. The discrepancy between the data and the theoretical curve can be attributed to lens apodization arising from the use of multi‐element thick lenses and a non‐ideal, truncated Gaussian beam profile.
Microscopy of cell cultures through thin plastic films
Authors: Moroz Pavel E.
Published: August 2011

Microscopy of cells growing in vessels containing plastic films as a substrate or as a transparent window is facilitated by a contact cap on the objective or the contact objectives for intravital microscopy. When applied to microscopical examination of living cells through a thin film, the cap considerably improves the conditions of observation with high‐power dry objectives and makes it possible to use water‐ and oil‐immersion objectives.
A device to ease grid coating for EM autoradiography
Authors: Riboni Laura , Núñez‐Durán Harol
Published: August 2011

A modification to the standard device used to hold grids during the process of coating with emulsion for EM autoradiography is described. The device has a plastic base with six bronze poles attached. Each pole has a groove machined along its side to the top, and a shallow depression at the end of the groove. Instructions for the construction of the poles and for use of the device are given. The innovative design allows the user to feel the top of the pole, and thus simplifies selection of the coated grids. As grids are less exposed to safe light than in the standard procedure, the background of silver grains decreases; also the chance of missing a grid decreases.
Freeze‐substitution techniques for preparing nematodes for scanning electron microscopy
Authors: Wharton D. A.
Published: August 2011

The effect of different substitution times, temperatures and the incorporation of fixatives on the preservation of three species of nematode for scanning electron microscopy by freeze substitution with methanol, followed by critical point drying, is investigated. Hammerschmidtiella diesingi adults and Trichostrongylus colubriformis infective juveniles were successfully preserved using methanol at 253 K as the substitution medium. Preservation deteriorated with long substitution times, suggesting the extraction of material and that substitution times should be kept as brief as possible. Panagrolaimus davidi was not successfully preserved using pure methanol, but preservation was improved by using fixatives in the substitution medium, the best results being obtained with 1% OsO4/3% glutaraldehyde in methanol. A substitution temperature of 193 K did not give any improvement in preservation. The differences in the quality of preservation between the three species may be due to the relative ability of the cuticle to withstand collapse during critical point drying. Chemical fixation using cold fixative resulted in the retention of a natural posture but poor preservation, whereas hot fixatives resulted in good preservation but the loss of a natural posture. Freeze substitution in methanol may prove useful in the preparation of specimens possessing cuticles or cell walls which have sufficient strength to withstand the drying process (e.g. arthropods, plants, fungi, nematodes). More delicate specimens may require the incorporation of fixatives into the substitution medium or conventional fixation.
Image registration and distortion correction in ion microscopy
Authors: Olivo J. C. , Kahn E. , Halpern S. , Fragu P.
Published: August 2011

We present a method whereby the geometric registration of a series of ion microscopic images is performed by applying a two‐step procedure. After applying a global linear transformation that corrects for geometric differences, a non‐linear elastic transform is used in order to match local properties and structures of the images. Transformation parameters are computed on the basis of shape‐specific points in the images. Distortion correction is achieved by relating ion images to the optical image of the same field and by using the two‐step algorithm to register the images. This methodology is evaluated on synthetic misaligned objects and on thyroid tissue images.
Real‐time quantitative elemental analysis and mapping: microchemical imaging in cell physiology
Authors: LeFurgey A. , Davilla S. D. , Kopf D. A. , Sommer J. R. , Ingram P.
Published: August 2011

Recent advances in widely available microcomputers have made the acquisition and processing of digital quantitative X‐ray maps of one to several cells readily feasible. Here we describe a system which uses a graphics‐based microcomputer to acquire spectrally filtered X‐ray elemental image maps that are fitted to standards, to display the image in real time, and to correct the post‐acquisition image map with regard to specimen drift. Both high‐resolution quantitative energy‐dispersive X‐ray images of freeze‐dried cyrosections and low‐dose quantitative bright‐field images of frozen‐hydrated sections can be acquired to obtain element and water content from the same intracellular regions. The software programs developed, together with the associated hardware, also allow static probe acquisition of data from selected cell regions with spectral processing and quantification performed on‐line in real time. In addition, the unified design of the software program provides for off‐line processing and analysing by several investigators at microcomputers remote from the microscope. The overall experimental strategy employs computer‐aided imaging, combined with static probes, as an essential interactive tool of investigation for biological analysis. This type of microchemical microscopy facilitates studies in cell physiology and pathophysiology which focus on mechanisms of ionic (elemental) compartmentation, i.e. structure‐function correlation at cellular and subcellular levels; it allows investigation of intracellular concentration gradients, of the heterogeneity of cell responses to stimuli, of certain fast physiological events in vivo at ultrastructural resolution, and of events occurring with low incidence or involving cell‐to‐cell interactions.
Cryopreservation evaluated with mitochondrial and Z line ultrastructure in striated muscle
Authors: Dalen H. , Scheie P. , Nassar R. , High T. , Scherer B. , Taylor I. , Wallace N. R. , Sommer J. R.
Published: August 2011

Single, intact, frog skeletal muscle fibres and whole frog hearts were quick‐frozen on a polished, liquid‐He‐cooled copper block and examined in the electron microscope after freeze‐substitution and freeze‐fracture. In both kinds of striated muscle, collapse of the peripheral and intracristal membrane spaces in mitochondria was found to increase with increasing distance from the point of first impact (PFI) of the muscle cells on the cold copper block. The changes correlated with a previously described gradient of Z line and A band cryodamage occurring with distance from the PFI. The findings in thin sections from freeze‐substituted preparations were confirmed by freeze‐fracture preparations. It is concluded that, since the mitochondrial membrane changes are concurrent with, and follow the same spatial distribution of, other manifest cryoarte‐facts, the cryoartefactual nature of the mitochondrial changes must be excluded before functional significance is attributed to them. The collapse of mitochondrial membrane spaces as a sensitive indicator of quality of cryopreservation may apply to non‐muscle cells as well.
An improved cryofixation method: cryoquenching of small tissue blocks during microwave irradiation
Authors: Hanyu Yoshiro , Ichikawa Michinori , Matsumoto Gen
Published: August 2011

The metal contact method of rapid freezing is greatly improved by irradiating the specimen with microwaves at 2.45 GHz for a short period of time (50 ms), while pushing the specimen onto the surface of the copper block cooled by liquid N2. The microwave irradiation, together with two technical improvements (a light‐mass plunger and a recently developed β‐gel shock absorber) for preventing bounce, produces a good freezing zone for squid retina, with high reproducibility for each experimental trial, extending from the contact surface to a depth of about 15 μm, which is comparable to the depth obtained by the metal contact method using liquid He in the absence of microwave irradiation. A good freezing zone was also experimentally demonstrated in specimens of rat liver and heart muscle. Microwave irradiation does not have appreciable effects on the ultrastructure of squid retina. The mechanism underlying the improvement in the rapid freezing under the microwave irradiation is discussed.
The effects of glycol methacrylate as a dehydrating agent on the dimensional changes of liver tissue
Authors: Gerrits Peter O. , Horobin Richard W. , Stokroos Ietse
Published: August 2011

The dimensional changes of liver sections during the course of processing with glycol methacrylate (GMA) or with ethanol are described. Tissue processing with ethanol served as a control. During prolonged processing steps (24 h each), linear shrinkage of tissue specimens dehydrated with GMA at room temperature was 13.2%. Subsequent infiltration with GMA resulted in trivial swelling, and polymerization in slight shrinkage (2.3%). In comparison, processing with cold GMA resulted in shrinkage during dehydration (about 10.8%), a slight swelling in pure GMA, followed by shrinkage during polymerization (2.2%). Short routine processing schedules resulted in similar shrinkage/swelling patterns, although precise values differed slightly. In all experiments, ethanolic dehydration resulted in smaller dimensional tissue changes than did GMA dehydration.

The dimensional changes of tissue sections during stretching on water, mounting and drying compensated for the major part of the shrinkage manifested during processing.
Non‐raster isotropic scanning for analytical instruments
Authors: Sasov A.
Published: August 2011

A new type of image‐forming, non‐raster isotropic scanning is presented. Isotropic scanning may be used in electron microscopy, for TV systems, image processing equipment, etc. With this approach it is possible to obtain less distorted images than with raster scanning, and more reliable quantitative characteristics in the image processing systems.
Digital filters to restore information from fast scanning tunnelling microscopy images
Authors: Aguilar M. , Anguiano E. , Diaspro A. , Pancorbo M.
Published: August 2011

A transfer function—similar to that used in optical cases to correct blurring effects due to the circular aperture of the system—is presented here to restore scanning tunnelling microscopy (STM) images. Due to the conical geometry of the tip‐sample system, we have established an analogy between the process of image formation in STM and in optical systems. The transfer function utilized, similar to that calculated by Stokseth, allows us to differentiate between the blurring effects introduced along the x and y axes. These effects are different due, mainly, to the different velocities achieved along the x and y directions. Furthermore we have measured the β parameter that characterizes the classical 1/fβ noise present in STM data, demonstrating its independence from experimental conditions. A Wiener filter is utilized to restore the images using the previous assumptions given for the transfer function and noise effects.
The quantification of fluorescent emission from biological samples using analysis of polarization
Authors: Entwistle A. , Noble M.
Published: August 2011

The quantification of fluorescent emission from biological specimens can only be carried out in cellular regions where the relationship between fluorophore concentration and fluorescent emission is linear. Using a confocal scanning laser microscope, we show that quantification of fluorescent emission from biological samples labelled with fluorescein and fluorescein analogues mounted in a viscous medium can be readily achieved. Where the distribution of fluorophore is highly localized, for example in cells labelled for immunofluorescence analysis, we demonstrate that analysis of fluorescence depolarization can identify regions in which fluorophore concentration exceeds the range in which the relationship to fluorescent emission is linear. We also demonstrate that, under the conditions examined, depth‐dependent effects, fading and quenching are either small enough to be ignored or can be corrected for mathematically when quantifying fluorescent emission.