bluuebunny/crossref_metadata_2025 huggingface dataset:
Beryllium coating for biological X‐ray microanalysis
Authors: Marshall A. T. , Carde D.
Published: August 2011

Beryllium is ideal for coating biological specimens for light element X‐ray microanalysis at low temperature. It has higher electrical and thermal conductivity at 100 K and lower absorption of X‐rays of biological interest than carbon, aluminium or chromium. It produces no detectable characteristic X‐rays. When adequate precautions are taken beryllium is a valuable alternative to other coating materials. Because of its toxicity, however, it should not be used indiscriminately.
GRIDSS: an integrated suite of microcomputer programs for three‐dimensional graphical reconstruction from serial sections
Authors: Briarty L. G. , Jenkins P. H.
Published: August 2011

A program for serial section reconstruction using computer graphics on a microcomputer is described, which uses freely available non‐specific hardware and software. Individual section data are input from a digitizer pad, and their reconstructed composite image can be displayed as if viewed from any direction.
Serial sectioning and digitization of porous media for two‐ and three‐dimensional analysis and reconstruction
Authors: Yanuka M. , Dullien F. A. L. , Elrick D. E.
Published: August 2011

A procedure is proposed for serial sectioning of impregnated porous media, for photographing the sections under the microscope, and for digitizing the image using an IBM PC (XT) with a digitizing board.

The result is a three‐dimensional matrix containing the enhanced digitized image in a binary form of O's and I's which represent solid and pore spaces respectively.

The procedure was tested on three different porous materials: (1) a pack of glass beads, 170–350 μm; (2) Berca sandstone; and (3) Elora clay loam soil. Each of these was impregnated, forty‐six to eighty cross‐sections were prepared by close parallel polishing of 10–50 μm per section, and then the sections were photographed and digitized.

Examples of the real and digitized cross‐sectioned samples are given as well as an example of reconstructed images in a plane perpendicular to the plane of sampling.

It is proposed that the method of serial sectioning and automated digitization of porous media provides a powerful tool for further three‐dimensional geometrical and topological investigation of pore space to be used in models of fluid transport phenomena.
Influence of fixative osmolality on the morphometric determination of extracellular space in normal and reperfused ischaemic myocardium
Authors: Prickaerts J. P. , Wilson G. J. , Bayliss C. E. , Baffour R.
Published: August 2011

Morphometric determination of extracellular space in control and post‐ischaemic reperfused rabbit myocardium was evaluated using two fixatives differing in their composition and total osmolality. Measurement of control extracellular space in an isotonic fixative (294 mOsm/kg water) was 20.8% and in a hypertonic fixative (1816 mOsm/kg water) was 22.2%. These values were not statistically different. Ischaemic durations of 15, 30, 60 and 90 min, followed by an equivalent period of reperfusion, created significant increases in extracellular space. The size of the extracellular space determined by both fixatives was found to be the same. Total fixative osmolality does not appear to influence morphometric evaluation of the extracellular space in control tissue or in tissue damaged by ischaemia and reperfusion.
Recent advances and applications of high resolution electron microscopy
Authors: Hutchison J. L.
Published: August 2011

This review covers several broad areas: firstly, recent developments in HREM instrumentation, and then novel techniques for imaging are discussed, including some of the problems of image interpretation. Applications of HREM techniques to a wide range of materials problems are described and include solid state chemistry, ceramics, semiconductors, metals and natural diamonds.

The next generation of high resolution microscopes will operate in the 300–400 kV range, have low Cs objective lenses, and have sufficiently good vacuum to allow the combined use of CBED and EELS facilities with imaging in the sub‐2 Å range. Microprocessor control of instrumental parameters such as astigmatism, alignment and defocus are seen as an important way forward in achieving the optimum performance of these instruments.
Dynamical excitation of thermal diffuse scattering in crystals of rutile
Authors: Bursill L. A. , Rossouw C. J.
Published: August 2011

Observations of thermal diffuse scattering (TDS) for the [001] projection of rutile are presented. New phenomena include: intense diffuse scattering directed inwards from {200} reflections, a sharply‐defined square of continuous intensity having {200} positions as vertices, relatively sharp <110> streaking along radial and non‐radial directions, and a distinct deficit scattering region surrounding the transmitted beam. Both the angular dependence of the TDS with respect to crystal tilts and the temperature variation of TDS in the range —180 <T < + 150°C are described.

The observed features are interpreted using calculations of the site dependence, within the [001] projection of the rutile unit cell, of the electron current density and its strong dependence on crystal tilt. N‐beam dynamical interactions generate a sophisticated probe of the atomic sources of inelastic scattering within the unit cell, a detailed understanding of which is necessary also for further improvement of energy dispersive X‐ray and electron energy loss spectroscopic analysis.
The application of EDXS to the biological sciences
Authors: Hall T. A. , Gupta B. L.
Published: August 2011

The distribution of chemical elements in soft tissues may be faithfully preserved by very rapid freezing. Most often the material is then cryosectioned and the sections frozen‐dried prior to analysis, but direct analysis in the hydrated state is an established alternative.

For bulk specimens, the shape of the analysed volume is uncertain. But whichever current model is accepted, analytical spatial resolution must generally be limited to the order of 1 μm. Such specimens can be suitable for the specific analysis of cytoplasm, cell nuclei and large extracellular spaces but not for study on a finer scale.

Analytical spatial resolution in the range 200–500 nm is obtainable with sections cut ∼ 1 μm thick. In the frozen‐hydrated state, small extracellular spaces can be analysed but multiple scattering obscures intracellular detail in the STEM image. The irradiation required for an EDXS analysis, approximately 50 nanoCoulomb (50 nanoAmpere seconds), need not produce intolerable radiation damage when spread over an area 200 nm or more in diameter.

Finer structure, for example mitochondria and regions of rough or smooth endoplasmic reticulum, can be identified and analysed in frozen‐dried cryosections cut ∼ 100 nm thick. Recently such features have been visualized in 100 nm frozen‐hydrated sections where the water is vitreous. This opens the prospect of analysing material where elemental distributions have been preserved on a very fine scale, since one might avoid even the ionic shifts from aqueous solution to supramolecular structures which must occur on freeze‐drying. But radiation damage may be prohibitive when an irradiation of 50 nanoCoulomb is concentrated into a hydrated area less than 200 nm in diameter.
On the use of ionization cross sections in analytical electron microscopy
Authors: Williams D. B. , Newbury D. E. , Goldstein J. I. , Fiori C. E.
Published: August 2011

There are two approaches to the utilization of the ionization cross section, Q, for use in the determination of kA B factors for quantitative microanalysis in the analytical electron microscope. The first approach is to interpolate a value of Q from experimentally determined kA B factors at a fixed accelerating voltage (kV). The second approach uses a theoretical parameterization of Q generated by fitting the fundamental Bethe expression to selected experimental values of Q over a wide range of kV. This paper discusses the relative merits of the two approaches.
Determination of UTW kXSi factors for seven elements from oxygen to iron
Authors: Cliff G. , Maher D. M. , Joy D. C.
Published: August 2011

This study reports k factors for an ultra‐thin window (UTW) Si(Li) energy dispersive (ED) X‐ray detector which extends the limit for X‐ray microanalysis in the analytical electron microscope (AEM) to elements below Na in the periodic table. The oxygen k factors reported for the UTW ED detector suggest that quantitative thin specimen X‐ray analysis can be extended to oxygen.
Correcting electron energy loss spectra for artefacts introduced by a serial data collection system
Authors: Craven A. J. , Buggy T. W.
Published: August 2011

The large range of signal intensities found in electron energy loss spectroscopy causes difficulty when trying to record the spectrum accurately. This paper describes a system which records the data serially as simultaneous pulse and analogue signals without the need for changing the gain of the photomultiplier tube during the scan. The effect of deadtime and dark current on the recorded signals is discussed and procedures for correcting the data for these effects are described. At the same time, it is possible to correct the data for stray signal in the spectrometer and non‐linearity in the photomultiplier head‐amplifier at high output signal levels. In addition to its use for electron energy loss spectroscopy, this system is ideal for recording other signals which have a wide dynamic range and change rapidly from point to point, e.g. energy filtered diffraction patterns from thin amorphous films.
Beam interactions, contrast and resolution in the SEM
Authors: Joy David C.
Published: August 2011

Backscattered and secondary electrons are both used in the SEM for imaging purposes. The backscattered signal is the result of high angle elastic scattering events, while the secondary signal is the result of knock‐on inelastic collisions. The characteristic differences between images in the two modes arise from the details of the relevant interactions in the two cases. In order to examine this in a quantitative manner Monte Carlo electron trajectory simulation techniques have been used. Calculations of the ultimate resolution and depth of information of the secondary and backscattered images are presented, together with simulations of the edge brightness effect in high resolution secondary images and an analysis of the microanalytical application of atom number contrast in the backscattered mode.
A perimeter algorithm applied to lung pathology
Authors: Kemeny A. , Barres D. , Lafuma C. , Mignot J. , Robert A. M.
Published: August 2011

A new image analyser, the NS 2000, explores the image using 256 photodiodes and, according to an adjustable threshold, converts it into a set of digital points of logical level 0 or 1. The method of fitting squares of increasing size consists of checking the presence of a square of a given size and of a given logical value throughout the image studied and gives the areas of successive erosions of the image. Using the parameters provided by the device one can obtain the total boundary BA per unit area for the phase studied, which is proportional to the number of squares having points of the two logical levels. Errors in length measurement as a function of orientation and for isotropic structures are compared to other automatic perimeter algorithms and to manual square grid measurement.

Using the parameters provided by the device for the quantitative evaluation of lesions of elastase induced emphysema in hamster lungs, the values obtained for internal surface area (ISA) of alveoli, are lower by about 40% in elastase treated hamsters when compared to normal controls. This difference is highly significant (P < 0.001) and the results obtained by the described method are in good agreement with those obtained by the classical manual procedure. Efficiency, defined as the precision of the estimate per unit measurement time on a given set of sample units (pictures in this paper), is given for both NS 2000 and manual procedures. The results obtained show comparable values but the automatic procedure includes a statistical treatment and it is significantly faster on histological sections than on micrographs, which was not the case with the manual method.
Improved anticontaminator for cryo‐electron microscopy with a Philips EM 400
Authors: Homo Jean‐Claude , Booy Frank , Labouesse Patrick , Lepault Jean , Dubochet Jacques
Published: August 2011

Electron microscopy of frozen‐hydrated specimens is frequently impaired by atmospheric humidity, introduced together with the cold specimen and subsequently condensing on it. This effect can be prevented in the Philips 400 electron microscope operating with the cold stage PW6591 by an improved anticontaminator described in this note. The retractable anticontaminator is formed by two adjustable liquid‐nitrogen cooled blades placed just above and below the specimen. It does not interfere with any other feature of the microscope.
On estimating freezing times during tissue rapid freezing
Authors: Jones G. J.
Published: August 2011

For the study of morphological changes that are associated with fast physiological processes, it is important to know the times at which the surface regions of specimens are frozen during rapid freezing. A simple physical model has been used to estimate the freezing times and the cooling rates at 10 μm depths in specimens. The calculations indicate that cooling rates in excess of 4 × 104 K s−1 are associated with freezing times of less than 0.5 ms. Using the same model, experimental measurements of freezing times at much larger depths have been extrapolated to a depth of 10 μm, the times obtained are 0.1‐0.6 ms for freezing by rapid immersion in cryogenic liquids, and 0.1 ms or less for freezing on a metal block. It is concluded that the delay time between contact with a cryogenic source and specimen freezing is less than 0.5 ms. The uncertainty in the time of freezing may be larger than this, because of an uncertainty of about ± 0.5 ms in determining the exact time of contact and, for freeze fracture studies, because of an uncertainty of up to 0.5 ms due to imprecision in the depth of fracture. At the same time it is estimated that the time during which freezing takes place may be as high as 250 μs, which can be taken as an upper limit for the resolution time for rapid freezing.
A simplified procedure for correction of errors in scanning stage absorbance photometry
Authors: de Vries Uwe , Schindler Joachim F.
Published: August 2011

Measurements with a scanning stage cytophotometer are subject to systematic errors. Residual distributional error, diffraction error and glare are due to physical limitations in measuring diaphragms and step width. These errors mainly depend on the ratio measuring stop/object size, mean local absorbance and shape of the measuring object. Small dense objects give rise to increasing inaccuracy. These errors were minimized by a set of easily handled equations, making use of the area and mean local absorbance only. These equations are easily incorporated in scanning programmes. The usefulness of this correction was demonstrated in Feulgen‐stained preparations.
A comparative study of softeners and catalyst systems upon dimensional changes and sectioning quality of glycol methacrylate sections
Authors: Gerrits P. O. , van Leeuwen M. B. M.
Published: August 2011

It is shown that softeners and temperature have an important influence upon the stretching parameters of glycol methacrylate sections. Also it is demonstrated that the catalyst system used may significantly interfere with section stretching.
The influence of tissue handling before fixation on the morphology of the chick blastoderm
Authors: Vanroelen Ch. , Vakaet L.
Published: August 2011

The morphology of cells and tissues of chick blastoderms handled before fixation in different ways, was compared. Blastoderms were fixed in ovo or after mounting on a glass ring (New, 1955). Differences in the morphology were observed.
Liquid propane jet‐freezing, freeze‐drying and rotary replication of the cytoskeleton and plasma membrane associated structures in human monocytes
Authors: Espevik Terje , Elgsaeter Arnljot
Published: August 2011

We have studied the cytoskeleton and plasma membrane associated structures of human monocytes using liquid propane jet‐freezing, freeze‐drying and rotary replication. We find that the cytoskeleton of these cells consists mainly of actin filaments. The actin filaments were identified by their ability to bind myosin sub‐fragment S1. A large fraction of these actin filaments radiates from dense cytoskeletal foci. The presence of such actin filament rich foci also in unfrozen cells was demonstrated using immunofluorescence light microscopic techniques. In addition to coated pits a network of thin fibrils (diameter 4–5 nm) were observed on the protoplasmic side of the plasma membrane facing the substrate. These results indicate that liquid propane jet‐freezing may represent a simple, inexpensive alternative to impact freezing at liquid helium temperature for obtaining the high cooling rate necessary for successful freeze‐drying and rotary replication of cytoskeletal structures.
Extraction of lipids during freeze‐substitution of Acholeplasma laidlawii‐cells for electron microscopy
Authors: Weibull C. , Villiger W. , Carlemalm E.
Published: August 2011

Cells of the bacterium Acholeplasma laidlawii were rapidly frozen against a copper block cooled by liquid helium. The frozen cells were transferred to liquid nitrogen and subsequently to acetone or methanol at 183 K. After 24 h the cells were infiltrated at 203 K with a non‐polar methacrylate resin of the same type as Lowicryl HM20. The resin was cured at the same temperature. Acetone extracted approximately 5% of the lipid content of the cells, methanol 15–45% and the resin only negligible amounts. Similar results were obtained with A. laidlawii‐ghosts. The cells appeared well preserved when examined in the electron microscope.
Descriptors of flatness and roughness
Authors: Serra J.
Published: August 2011

The two notions of flatness and roughness refer to local properties of the profiles and of the surfaces. They are defined in two‐ and three‐dimensional spaces. Flatness is the ratio of the measure of the surface divided by its projection, and roughness the average of the square of the mean curvature of the surface per unit area. Whatever the number of dimensions of the space is, both parameters are accessible from vertical plane sections. The problem of their digital version is discussed, and algorithms are given. The approach also applies for grey tone functions, in R2 and in R3. Finally the results are extended to the n‐dimensional case.
Measurement of cellular elastic properties by acoustic microscopy
Authors: Hildebrand J. A. , Rugar D.
Published: August 2011

The acoustic microscope is used to investigate the elastic properties of living biological cells. A quantitative model is developed relating acoustic microscope image contrast to cellular elastic properties. Cytoplasmic acoustic attentuation is measured by focusing the acoustic microscope on the surface of the underlying substrate. Cytoplasmic acoustic impedance is measured by focusing the acoustic microscope on the top surface of the cell. The model allows the acoustic microscope to give quantitative information about cellular elasticity on a subcellular scale.
Specimen holder design for X‐ray microanalysis of thin films in the TEM: reduction of spurious X‐rays
Authors: Ryan K. G. , Flower N. E. , Presland M. R.
Published: August 2011

Spurious Cu and Fe peaks were observed during energy dispersive X‐ray spectroscopy of thin resin sections or of thin gold films in a Philips EM400T. Since our field of interest lies in the distribution of Cu in thin sections, experiments were aimed at the elimination of this peak. Replacement of the low background specimen holder with one fashioned from a small strip of Al foil established that the source of the Cu was the Cu alloy of the specimen holder. Tests showed that the incident beam was well collimated and that the fluorescence of the holder was due to X‐rays scattered from the analysed volume.

We have made a new low background specimen holder from an Al alloy (6351 T6) containing 1% Si, 1% Mg, 0·7% Mn and 0·4% Cu. Although the tensile strength of this material is only two‐thirds that of the Cu alloy, it is still sufficient for a mechanically robust holder. The spurious CuKα peak was not observed in the X‐ray spectrum when analyses were performed with this holder and when the beryllium top plate on the holder was doubled in thickness, no other peaks were introduced to the spectrum. The importance of eliminating spurious peaks is discussed.
A clearing method for the observation of lateral root primordia
Authors: Casero V. J. , Lloret P. G. , Vidal M. R. , Abadía‐Fenoll F.
Published: August 2011

A clearing method is described to show the adventitious root tissues of Allium cepa by transparency. This method allows the lateral root primordia to be observed in situ, and enables them to be dissected out more easily. The histological sections show that both the tissues of the adventitious and lateral root primordia are perfectly preserved so that it is possible to carry out morphological observations.
Electron phase‐contrast imaging of melamine‐embedded, unstained horse‐spleen ferritin
Authors: Westphal C. , Bachhuber K. , Frösch D.
Published: August 2011

A pellet of aldehyde‐fixed horse‐spleen ferritin was embedded in a water‐soluble melamine resin for ultrathin sectioning. Overfocused electron micrographs of individual molecules show the four‐lobed iron‐core and the peripheral protein subunits in great detail. They confirm earlier X‐ray diffraction data of hydrated horse‐spleen apoferritin (Hoare et al., Nature, 255, 653, 1975), which postulate a molecule with twenty‐four protein subunits occupying octahedral lattice sites around the iron‐core.
An improved detection system for electrical microcharacterization in a scanning electron microscope
Authors: Lesniak M. , Unvala B. A. , Holt D. B.
Published: August 2011

An improved, modular detection system for conductive mode, charge collection (CC) microscopy and electrical microcharacterization in a scanning electron microscope is reported. It makes possible the recording of both the short‐circuit current (EBIC) and the open‐circuit voltage (EBIV) generated by the barrier or the bulk electron voltaic effect even in the presence of biases up to 700 V. The constant‐current β‐conductivity voltage signal and the constant‐voltage β‐conductivity current signal can also both be recorded.

The design of the system emphasizes noise minimization, a wide range of operating characteristics all independently controllable, quantitative monitoring and simple signal processing. An interface to a microcomputer is provided for more detailed quantitative analyses of the signals. The system has been calibrated so the accuracy of the measurements is known.

The potential of this detection system is illustrated by means of a number of applications.
Design and application of a freeze‐fracture stage with a cleaver device for Balzers ‘freeze‐etching’ apparatus
Authors: Stolinski C. , Landmann L. , Abt H.
Published: August 2011

A fracturing mechanism in the form of a double‐wedged cleaver, used previously on other freeze‐fracture machines, has been incorporated in the Balzers BAF 400 D apparatus. The design of the stage, with the cleaver, is explained and its performance and advantages are outlined. By minimizing mechanical damage to the exposed surface of the frozen specimen and reducing contamination by effective shrouding, an improvement in the quality of the replicas is achieved. The suitability of this mechanism for the fracturing of tough tissues such as skin is also outlined.
Stereological estimation of average cell volume in monolayer culture by combined light and electron microscopy
Authors: Halgunset Jostein
Published: August 2011

In order to convert stereological ratio estimates into ‘absolute’ values (‘per cell’ values) the average cell volume must be estimated. The present paper describes a stereological method based on well‐known point counting procedures for the estimation of average cell volumes in monolayer cultures fixed in situ. This method involves estimation of the average attachment area per cell by light microscopy combined with estimation of the attachment membrane surface density by electron microscopy. There is no need for any assumption as to cellular or nuclear shape.

The method has been tested on an established cell line, NHIK 3025, and shows a good accuracy. It has also been used to analyse the volume changes that take place in human monocytes during monolayer culture, demonstrating a 28‐fold increase of the average cell volume over 10 days.
An improved approach to the analysis of autoradiographs containing isolated sources of simple shape: Method, theoretical basis and reference data
Authors: Downs A. M. , Williams M. A.
Published: August 2011

There are circumstances in which, by assuming that a high proportion of the radioactivity in a biological specimen is associated with certain well‐dispersed structures of simple shape, a relatively simple and effective objective analysis can be carried out. The usefulness of this type of restricted analysis has previously been severely limited by the requirement that all the profiles of interest should be of roughly the same size, which is rarely the case. A procedure is proposed which, by appropriately combining image spread functions for profiles of the same shape but of various sizes, allows realistic hypothesis testing in such circumstances. Details for the practical application of the method are offered and criteria for assessing the feasibility of applying the method in particular circumstances are discussed.

The necessary backup information to the method is supplied in the form of the derivation of the fundamental image spread function, the introduction of a coherent notation and reference data quantitatively describing the image spread around certain extended sources of simple shape.
New classification methods of branching patterns
Authors: van Pelt J. , Verwer R. W. H.
Published: August 2011

Binary branching patterns are described in this paper by their topological structure. The statistical properties of these structures appear to be highly dependent on their growth patterns. As such, observed trees may be used for testing growth models. This paper describes three options in the construction of frequency distributions of topological parameters and their corresponding probability distributions, arising from the terminal and segmental growth models. The construction of these distributions makes the analysis of observed tree structures possible in those experimental conditions where small numbers of observations or differing sizes of trees would form serious obstacles to alternative analytical procedures.
Low voltage scanning electron microscopy
Authors: Pawley James
Published: August 2011

The scanning electron microscope (SEM) is usually operated with a beam voltage, V0, in the range of 10–30 kV, even though many early workers had suggested the use of lower voltages to increase topographic contrast and to reduce specimen charging and beam damage. The chief reason for this contradiction is poor instrumental performance when V0=1–3 kV, The problems include low source brightness, greater defocusing due to chromatic aberration greater sensitivity to stray fields, and difficulty in collecting the secondary electron signal. Responding to the needs of the semiconductor industry, which uses low V0 to reduce beam damage, considerable efforts have been made to overcome these problems. The resulting equipment has greatly improved performance at low kV and substantially removes the practical deterrents to operation in this mode. This paper reviews the advantages of low voltage operation, recent progress in instrumentation and describes a prototype instrument designed and built for optimum performance at 1 kV. Other limitations to high resolution topographic imaging such as surface contamination, the de‐localized nature of the inelastic scattering event and radiation damage are also discussed.
Locational stereology performed within sampling fields in rat aortic wall, a polarized tissue
Authors: Guyton John R.
Published: August 2011

One‐dimensional structural localization on a scale approximating the sizes of the structures themselves is accomplished for polarized tissue by a new microcomputer‐based stereological method The tissue of interest is the inner rat aortic wall, viewed by transmission electron microscopy. Particular attention is given to the interface between intima and media. Locational analysis of smooth muscle cells, elastin, collagen and other structures near this interface can be performed. The key to accomplishing the analysis within a reasonable time rests with a microcomputer program which simultaneously records structure codes from the microcomputer keyboard and X, Y‐coordinates from an electronic digitizing board. The stereological solution to this problem is applicable to a wide range of situations with polarized geometries.
A methodological basis for SEM autoradiography: biosynthesis and radioligand binding
Authors: Cheng G. , Hodges G. M. , Trejdosiewicz L. K.
Published: August 2011

A method is described for scanning electron microscope (SEM) autoradiography whereby preservation of high resolution cell surface details is retained together with degelatination of the emulsion without gross loss or redistribution of silver grains. This method should provide a convenient medium‐sized marker for SEM (using secondary, backscattered electron and X‐ray imaging) topographic studies of biosynthesized molecules, and of cell surface receptors and antigens, using indirect or direct labelling procedures with radio‐labelled ligands.
Polyethylene glycol embedding of plant tissues for transmission electron microscopy
Authors: Hawes C. R. , Horne J. C.
Published: August 2011

The use of high molecular weight polyethylene glycol as an embedding medium for the production of resin‐free sections of plant cells is described. Specimens can be observed at a range of accelerating voltages, are high in contrast and require no heavy metal staining. All the major organelles appear intact and a three‐dimensional network of filaments in the cytoplasm and nucleoplasm is reported.
Measurement of the width of thin, cylindrical, transparent objects by phase contrast light microscopy6
Authors: Knight Peter , Parsons Nick
Published: August 2011

Measurement of the width of a thin, cylindrical, transparent object by phase contrast light microscopy has been frustrated by the absence of an established relationship between the true width of the object and its apparent width in the phase contrast image. We have solved this problem by devising a simple method by which individual glass fibres may be measured using both phase contrast light microscopy and electron microscopy. Using this method we have constructed calibration curves relating the diameter measured by phase contrast microscopy to the real diameter of the fibres. These curves are linear in the range 0.10‐2.5 μm real diameter, with slopes close to unity and intercepts of about 0.2 μm. Thus widths of such objects are overestimated. The precise value of the intercept is related to the overall numerical aperture of the optical system. Each calibration curve permits the true width of a cylindrical object to be estimated by phase contrast microscopy with an accuracy of better than ±0.05 μm. We have found that greater precision is obtained by taking measurements of light micrographs subjectively using a microcomparator rather than objectively using a microdensitometer.
Weighted least squares estimation of background in EELS imaging
Authors: Pun Thierry , Ellis James R. , Eden Murray
Published: August 2011

In quantitative Electron Energy Loss Spectrometry, a weighted least squares estimation should theoretically be used to estimate the background law below core edge energy, since the variances of the data vary. However, it is found that proper weighting makes the above edge signal‐to‐noise ratio decrease rather than increase. This result is discussed, and the influence of the bias introduced by the logarithmic transformation of the data is quantified.
Automated recognition of atypical nuclei in breast cancer cytology specimens by iterative image transformations
Authors: Cornelisse C. J. , van Driel‐Kulker A. M. J. , Meyer F. , Ploem J. S.
Published: August 2011

In order to develop an objective grading system for nuclear atypia in breast cancer, an image analysis technique has been applied for the automated recognition of enlarged and hyperchromatic nuclei in cytology specimens. The image segmentation algorithm, based on the ‘top hat’ image transformation developed in mathematical morphology, is implemented on the LEYTAS automated microscope system. The performance of the segmentation algorithm has been evaluated for fifty malignant and eighty‐five benign breast lesions by visual inspection of the displayed ‘flagged’ objects. The mean number of flagged objects per 1600 image fields for breast cancers was 887 (range 0–7920) of which 87% consisted of single, atypical nuclei. For benign lesions the mean number was 30 (range 0–307) of which 20% were single nuclei. By adaptation of the ‘top hat’ parameter values, a more extreme subpopulation of atypical nuclei could be discriminated. The large interspecimen variation in the breast cancer results was related to differences in DNA content distribution and mean nuclear area, determined independently with scanning cytophotometry, and to some extent with the histological type.
Systematic inquiry in tests of negative/positive replica combinations for SEM
Authors: Bromage Timothy G.
Published: August 2011

High resolution replica materials are routinely used in scanning electron microscopy. A systematic evaluating procedure for replica combinations is proposed which details fourteen points to be recorded. These points include quantitative and qualitative information useful when assessing a replica combination for a particular research problem. A case study employing one silicone‐based impression material and one epoxy resin is performed as an example of the procedure.
Digital mapping of metal‐bearing amoebocytes in tissue sections of the Pacific oyster by scanning microphotometry
Authors: Lytton D. G. , Eastgate A. R. , Ashbolt N. J.
Published: August 2011

Automated scanning microphotometry provides an objective assessment of the distribution of metal‐bearing (MB) amoebocytes in various tissues of the Pacific oyster, Crassostrea gigas. Silver‐staining of these cells in paraffin‐embedded sections gave good histochemical enhancement for effective photometric discrimination. Metal‐associated pixel fractions (MAPF) for 0.41 mm2 tissue‐fields were calculated from the results of microphotometric scans, giving an estimate of the metal content and an expression of the population density of MB cells. Gut‐associated MB cells in oysters obtained from two geographically‐separate metalliferous estuaries had similar distribution patterns, with MAPF values that correlated with metal concentrations in visceral mass determined by atomic absorption spectrophotometry (AAS). The metal content of other tissues varied by two orders of magnitude, the gill having the highest MAPF values. Intermediate MAPF values were found for digestive diverticula and the lowest values in the gonad. There was little difference between gut‐associated MB cell populations in oysters from the two collection sites, despite differences in size, glycogen content and gonadal maturation. This non‐destructive method of tissue microanalysis may provide a better way of assessing tissue metal‐load in individual organisms, and one that is independent of physiological variables.
A microcomputer based digital imaging system for ion microanalysis
Authors: Bryan S. R. , Woodward W. S. , Griffis D. P. , Linton R. W.
Published: August 2011

A microcomputer based digital imaging system was developed for a Cameca IMS‐3f ion microscope permitting real‐time digital acquisition of secondary ion images. Image signal‐to‐noise enhancement results from random noise reduction by real‐time ensemble averaging and from a reduction of pattern noise in the charge injection device (CID) array by subtraction of blank frames. Acquired images comprise 244times248 pixel arrays with 8‐bit intensity resolution. Images are displayed on a colour monitor in a grey scale or pseudo‐colour using one of four programmable lookup tables. Image processing software permits off‐line ion image enhancement and manipulation as well as multitechnique digital image correlations. System capability is illustrated by a biological example involving digital imaging studies of Al distribution in osteomalacic bone tissue, including correlative light microscopy and ion microanalysis.
Differences in apparent elemental composition of tissues and cells using a fully quantitative X‐ray microanalysis system
Authors: Kendall Marion D. , Warley Alice , Morris I. W.
Published: August 2011

The elemental composition of tissues and cells in suspension was studied using fully quantitative X‐ray microanalysis. Quantitation was achieved using a commercially available program with reference to gelatin standards. Results showed that cells isolated in suspension differed in composition from those in the corresponding tissue. Elevated concentrations of Na, P, S and Cl were found in the cells in suspension. Concentrations of K and Mg were affected to a lesser extent and concentrations of Fe, Zn and Ca were unaffected by the suspension procedures.
Inaccuracies with the Hall technique due to continuum variation in the analytical microscope
Authors: Warner R. R. , Myers M. C. , Taylor D. A.
Published: August 2011

We have identified a source of extraneous (non‐sample) continuum in analytical microscopes with high‐angle EDS detectors. The extraneous continuum is generated by specimen‐scattered electrons but is not associated with a characteristic peak. This peakless continuum varies in magnitude with position across a grid and can cause large errors in quantitation with the Hall technique. The use of a modified specimen holder and an objective lens with a widened exit bore in the lower pole piece reduces the peakless continuum to acceptable levels in our instrument. Peripheral standards attached to the specimen can further reduce the impact of this extraneous continuum. Analytical microscopes with horizontal EDS detectors can also have serious problems with continuum variation across a grid, although the effect is likely due to X‐ray absorption by the specimen holder rather than extraneous continuum.
Effect of glutaraldehyde on the osmolarity of the buffer vehicle
Authors: Coetzee J. , van der Merwe C. F.
Published: August 2011

The osmolarity of four different buffers (Na‐cacodylate, PIPES, Na‐K‐phosphate and Na‐Na‐phosphate) at different pH‐values and molarities was measured. On the addition of glutaraldehyde to these buffers, an increase in osmolarity was observed. This increase was more than could be explained by the numerical addition of the osmolar values of the glutaraldehyde and of the buffer. The increase is due to a higher buffer dissociation constant. On the addition of 5% glutaraldehyde to a 0.05 M buffer, the increase in buffer osmolarity may be as much as 107 mOsm. Graphs are given to allow the determination of K‐values for these four buffers at different molarities and pH‐values. The effect of different glutaraldehyde concentrations on these K‐values is presented graphically. The time‐dependent increase in osmolarity of glutaraldehyde solutions in water is described.
A procedure for increasing the contrast of biological specimens in edge‐projection TEM
Authors: Panitz J. A. , Bear D. G.
Published: August 2011

Rotary shadowing has been used to increase the image contrast of biological specimens during edge‐projection imaging in the transmission electron microscope (TEM). In this imaging mode, biological specimens are adsorbed from aqueous solution onto a highly curved substrate and observed in a direction parallel to its surface. High contrast TEM images are obtained at 200 kV when a 1–3 nm layer of tungsten is thermally evaporated onto the substrate at an angle of about 9°. Individual adsorbates are clearly delineated by the smooth, continuous, and fine‐grained tungsten layer that surrounds them. TEM images obtained with this technique can provide a unique view of biological adsorbates on metal, insulator or semiconductor substrates.
The ‘bi‐corpuscle’ stereological problem: estimation of distributions of distances between paired spheres, from measurements in slabs showing overprojection and truncation, and its application to binucleated cells
Authors: Rigaut Jean Paul
Published: August 2011

A non‐parametric stereological method for the estimation of distributions of distances (d) between paired spheres of the same size, from the distances between their profiles observed in sections, is presented. The model accounts for sphere overprojection and truncation effects.

Using the mean value of d (d̄), it is then possible, with simple formulae, to compute the probabilities of the co‐observation of two profiles from paired spheres with distance d between their centres, when at least one profile is observed.

A useful application is the estimation of true binucleated cell frequencies from observations on tissue sections. An example for rat liver diploid cells is shown.

The sphere distribution must be studied beforehand and, when the size variation is unacceptably large, implying that the assumption of a constant sphere radius is unreasonable, the main limitation of the model is met.
Correction coefficients for the size distribution of photographically recorded spherical particles
Authors: Horálek Vratislav , Coleman Rodney
Published: August 2011

Spheres which are part of a spatial process are photographed. The relationship is derived between the sizes of the images and the actual sphere size distribution. Non‐orthogonal projection given by the linear optics of a pinhole camera is assumed. The depth of field is brought into the calculation.
Digital image tiles: a method for the processing of large sections
Authors: Silage Dennis A. , Gil Joan
Published: August 2011

Segmentation of large areas of light microscopic slides into N by N fields, and each of these fields into M digital image tiles, allows the scanning, storage and digital processing of large images. Any of the original N2 fields or composites of M adjacent tiles can be recalled to the video display for analysis. Developed procedures for use on a microscope equipped with a precision scanning stage allow registration of the image coordinates (X‐Y) for any original or composite field and the alignment of one of these fields along the depth (Z) axis by means of external, machined fiducial marks in serial sections. To facilitate work whenever unavoidable, we have incorporated methods for digital image panning and zooming (changes of magnification) and discuss their use and implications.
New progress in X‐ray microscopy
Authors: Cosslett V. E.
Published: August 2011

Interest has recently revived in microscopy at X‐ray wavelengths, stimulated in part by work for microelectronic lithography. The use of synchrotron radiation and other intense sources of X‐rays, the development of plastic foils in place of photo‐emulsions for recording and the fabrication of Fresnel zone lenses for focusing X‐ray beams have all played a part. Some progress has already been made in improving the resolution and contrast of this method of microscopy, in the wavelength range 2–4 nm. The limitation imposed by radiation damage in biological specimens still has to be explored.
X‐ray microscopy: experimental results with the Göttingen X‐ray microscope at the electron storage ring BESSY in Berlin
Authors: Schmahl G. , Niemann B. , Rudolph D. , Guttmann P. , Sarafis V.
Published: August 2011

An X‐ray microscope and X‐ray microscopy experiments with biological specimens are described. The experiments have been performed with a resolution of about 0.05 μm using the synchrotron radiation of the electron storage ring BESSY.
Soft X‐ray contact microscopy with nanosecond exposure times
Authors: Rosser R. J. , Baldwin K. G. , Feder R. , Bassett D. , Coles A. , Eason R. W.
Published: August 2011

High resolution (better than 20 nm) contact micrographs have been produced with exposure times of about a nanosecond. The illuminating source was a short‐lived carbon plasma produced by focusing a single short (∼1 ns) 100 J pulse from the Vulcan laser at the Rutherford Appleton Laboratory (RAL) to a 300 μm spot on a graphite target. This plasma emits strongly in the soft X‐ray region, particularly at the CVI (3.37 nm) and CV (4.03 nm) lines. The specimens were behind a 100 nm thick Si3N4 window, at atmospheric pressure in an environmental cell. The images of diatoms recorded on X‐ray resist showed features down to the limit of resolution of the SEM used to view the developed resist, which was about 20 nm.
Theoretical calculation of layer geometry in rotary shadowed models of membrane‐associated particles
Authors: Landmann L. , Roth J.
Published: August 2011

The geometry of the evaporated metal layer on models of membrane‐associated particles has been calculated theoretically and the respective density profiles have been estimated. The models were a hollow cylinder, a hemisphere and a rotary ellipsoid substituting the freeze‐fracture appearance of gap junction connexons and globular integral membrane proteins respectively. These calculations may be used as a help for choosing an optimal compromise between shadowing angle and layer thickness. They furthermore provide a basis for assaying the extent of shadowing artefacts such as decoration and lead to a more accurate interpretation of rotary shadowed structures.
An improved Senarmont retardation analysis method
Authors: Laughlin R. G. , Marrer A. M. , Marcott C. , Munyon R. L.
Published: August 2011

The phase difference between two plane polarized light rays whose electric field vectors (planes of polarization) are at right angles is often determined by Senarmont analysis. In this analysis, a quarter‐wave plate whose vibration axes lie at an azimuth of ±45° to the planes of polarization of the two rays is inserted. The rotation of the analyser that leads to extinction is then determined by manual adjustment. This classical method has been significantly improved by, instead, measuring light intensities at thirty‐two evenly spaced analyser angles which span 180° of analyser rotation. An unbiased estimate of the phase difference may then be extracted with high accuracy by analysis as a designed experiment.

Of the several numerical analysis methods investigated, harmonic analysis is particularly attractive for routine use—especially with small computers. Iterative non‐linear least‐squares analysis on a mainframe computer was taken as the standard of numerical accuracy, against which direct algebraic solutions on a personal computer were evaluated. An ‘accuracy’ statistic (which measures the goodness‐of‐fit to the theoretical cosine function) has proven invaluable in assessing the validity of actual data.

Data of exceptional quality are produced by the Leitz Jamin–Lebedeff optical system using intensities measured with a photodiode detector placed in the camera tube of the microscope. These data will support analysis to better than 0.0001° of analyser position, or <6×10−7 periods. Since alignment of the optics with comparable accuracy is impossible, a method for accurately quantitating the contribution to the observed retardation that originates from within the optics, by performing the analysis on an air sample, has been devised.