bluuebunny/crossref_metadata_2025 huggingface dataset:
The nucleator
Authors: Gundersen H. J. G.
Published: August 2011

A new, fundamental stereological principle is described which allows unbiased estimates of absolute structural quantities in arbitrarily shaped structures to be made from observations sampled in arbitrary points on independently isotropic probes. As an introduction to the principle, method(s) are described which lead to assumption‐free estimates of mean volume from the usual number‐distribution of particle sizes. Specifically, the estimation is unbiased when arbitrarily shaped particles are sampled with uniform probability using the disector or one of its many modifications. A special case, of interest when observations are restricted to sections, occurs when the particles are associated with some recognizable unit, like eukaryotic cells in biology. The estimation may then be carried out on a few sections of unknown thickness, or—under verifiable assumptions—on just one random section.
Quick sampling and perpendicular cryosectioning of cell monolayers for the X‐ray microanalysis of diffusible elements
Authors: Tvedt K. E. , Halgunset J. , Haugen O. A.
Published: August 2011

A quick sampling and preparation method for freezing of cell monolayers is described. The cells are grown on a large Formvar film supported by a frame of polystyrene. A polyvinylpyrrolidone (PVP) solution is applied to one side of the film forming a flat disc when frozen with a pair of pliers precooled in liquid nitrogen. The PVP solution provides the specimen with sufficient strength and may be used as an elemental standard for absolute quantification if salts of known concentrations are added. Manipulation of the cells prior to freezing is thus restricted to a minimum, which eliminates possible harmful treatments like scraping and centrifugation. The procedure is quickly performed, the freezing being completed within 30 s of the cells having been removed from the culture well. The analytical results reveal low and stable Na: K ratios. Our results confirm that cells in vitro are comparable to cells in vivo with respect to elemental composition.
Quantitative high resolution transmission electron microscopy: the need for energy filtering and the advantages of energy‐loss imaging
Authors: Stobbs W. M. , Saxton W. O.
Published: August 2011

Evidence is presented that inelastically scattered electrons contribute significant detail at the atomic level to high resolution images, particularly in high voltage instruments. The implications for quantitative image interpretation are shown to be serious and a case is made for incorporating facilities for energy‐filtered imaging in future high resolution electron microscopes.
Freeze‐fracture of 3T3 cells for high‐resolution scanning electron microscopy
Authors: Haggis G. H. , Pawley J. B.
Published: August 2011

Triton‐extracted, freeze‐fractured 3T3 cells have been examined in the Hitachi S‐900 field‐emission SEM, after light platinum coating, at low beam voltage to evaluate the performance of the microscope under these conditions. For unstained material fixed in glutaraldehyde alone, high‐resolution images can be obtained, at accelerating voltages of 1.5‐5kV, after rotary deposition of platinum to an average thickness of 1.5‐3nm. Comparisons are made between these results and those of studies by TEM of deep‐etch replicas of similar material previously published.
An interesting pair of identically distributed random tessellations, with vertices of Y‐type, which always cross orthogonally
Authors: MILES R. E.
Published: August 2011

It is shown that the skeletons of the polygons determined by a line process in the plane form two identically distributed tessellations I′, I″ with all vertices of Y‐type, such that wherever I′, I″ cross, they do so at a right angle. The characteristics N̄, B̄, Ā for I′ or I″ are derived, along with the joint distribution of the random angles, at a random vertex. The space‐filling three‐dimensional versions are also considered.
An alternate formulation of mean value for random geometric figures*
Authors: Vitale Richard A.
Published: August 2011

In contrast to the traditional technique of averaging functionals of a random geometric figure, it is possible to consider an averaging process which treats the figure as a whole. The result in probabilistic terminology is an expectation, which is itself a figure. Theoretical foundations and various instances are presented in an informal survey.
An extremal property of random flats
Authors: Mecke J.
Published: August 2011

This paper deals with anisotropic random two‐dimensional flats meeting a ball B in the four‐dimensional Euclidean space. The probability p that two independent, identically distributed random flats have a common point in B depends on the direction distribution of the random flats. The direction distributions maximizing p are determined. If the random flats are isotropic, p is not maximal, in contrast to the case of random hyperplanes (three‐dimensional flats). Related results for flat processes are explained.
Random packing and random tessellation in relation to the dimension of space
Authors: Tanemura Masaharu
Published: August 2011

The random packing of non‐overlapping objects and the random tessellation of space by objects are discussed with relation to the dimension of space. The random packing discussed here is random ‘sequential’ packing and random tessellation is defined in this paper as the Voronoi tessellation of Poisson point processes. The conjecture by Palásti is examined for homothetic packing of cubes by using the existing data up to four dimensions; the generalized Palásti conjecture for spheres is also examined from the results of Monte Carlo simulations for two‐ and three‐dimensional space. As for the random tessellation, Kiang's conjecture is examined using the data for two‐ and three‐dimensional space. The results of the study indicate that all of the conjectures are false. Some details are given of the simulation methods.
Some stereological and statistical consequences derived from Cartier's formula
Authors: Lantuéjoul Ch.
Published: August 2011

Let X and Y be two random variables with distribution functions Fx and FY, respectively. If Cartier's formula E[Y|X]=X holds, then Fx is necessarily less dispersed than FY. In this paper, the main consequences of Cartier's formula are derived. Some examples where such a formula holds are also given, namely in stereology (comparison of various estimators of Minkowsky's functionals) and in geostatistics (the change of support problem).
The control of temperature during polymerization of Lowicryl K4M: there is a low‐temperature embedding method
Authors: Glauert Audrey M. , Young Robert D.
Published: August 2011

Methods are described for controlling the temperature of Lowicryl K4M in flat embeddings and in capsules during polymerization under ultra‐violet (UV) irradiation in an Agar UVF 35 low‐temperature cabinet. Aluminium blocks or an ethanol bath are used as heat ‘sinks’. Consequently the question posed by Ashford et al. (1986) — “Is there a low‐temperature embedding method?” — is answered in the affirmative. This control of temperature is particularly important when using gelatin capsules since a temperature rise of as little as 2 K results in uneven polymerization.
3‐D reconstruction from optical sections of the traps of the nematophagous fungus Arthrobotrys oligospora
Authors: Wharton D. A. , Murray D. S.
Published: August 2011

A technique is described for the 3‐D reconstruction from serial optical sections of the traps and invasive hyphae of the nematophagous fungus Arthrobotrys oligospora, using differential interference contrast optics and 3‐D acquisition and reconstruction programs on an Apple Macintosh microcomputer. The technique preserves the true morphology of the trap, enables its complex arrangement to be correctly interpreted, is non‐destructive and uses relatively inexpensive hardware.
Conditions required for detection of specimen‐specific SE‐I secondary electrons in an analytical SEM
Authors: Apkarian Robert P.
Published: August 2011

An analytical SEM equipped with an above‐the‐lens detector, an in‐the‐lens specimen stage and a high brightness LaB6 emitter was used to produce a specimen‐specific, secondary electron‐I (SE‐I) signal for recording edge brightness contrast with high intensity on small particles at high magnification (200,000). The SE‐I edge brightness contrast produced from 20–40 nm colloidal gold on silicon wafers was useful for estimating instrument resolution since the edge brightness is the sum of the SE‐I signal range (≌ 1 nm) and the beam diameter. LaB6 crystal saturation and gun conditions were determined in order to minimize the probe diameter at the first cross‐over position. Ferritin particles also on the silicon wafers were imaged by adjustments of the gun bias voltage conditions. Establishment of these conditions was useful for high resolution SEM studies of appropriately coated bulk biological specimens.
The effect of aberrations on the axial response of confocal imaging systems
Authors: Wilson T. , Carlini A. R.
Published: August 2011

A confocal scanning microscope permits three‐dimensional imaging of a volume specimen. The role of lens aberrations on the axial response of the system is considered both experimentally and theoretically. It is found that primary coma, for example, plays no part in the axial response from a perfect reflector. The role of detector size is also considered and compared with theory. In general, the effect of aberrations is increased as the size of the detector is increased.
Freeze‐substitution of plant tissues with a new medium containing dimethoxypropane*
Authors: Kaeser Wulf
Published: August 2011

Meristematic cells from root tips of Dahlia variabilis (L.) and highly vacuolated storage parenchyma cells of Helianthus tuberosus (L.) were fixed by the high‐pressure freezing technique and freeze‐substituted. A new water‐free medium for freeze‐substitution was developed to obtain better preservation of the ultrastructure of plant tissues. Adding dimethoxypropane (DMP) to the substitution medium gave two positive results: (1) water could be withdrawn from the samples without an exchange of the medium and without adding drying agents or working in a nitrogen atmosphere, and (2) the ultrastructural preservation of the substituted tissues was better than in a methanol medium and at least as good as in tissues fixed chemically at room temperature.
A simplification of the ‘step’ method for estimating mean section thickness
Authors: Evans S. M. , Howard V.
Published: August 2011

An inexpensive and efficient method for estimating mean section thickness based on an estimate of the sectioning depth is presented. This value is required for the estimation of particle number using the disector.
Energy filtered STEM imaging of thick biological sections
Authors: Colliex C. , Mory C. , Olins A. L. , Olins D. E. , Tencé M.
Published: August 2011

Energy filtered imaging of thick biological specimens was analysed using a dedicated STEM fitted with an energy loss spectrometer and interfaced with a sophisticated data collection setup. All images were digital, thus permitting a quantitative analysis of the data. We also present a mathematical explanation of the data, which is useful in pradicting the quality of thick specimen images formed with energy filtered electrons.

It is known that increasing specimen thickness leads to a decrease of the zero energy loss intensity and an increase in higher (multiply scattered) energy loss electrons. We show that contrast decreases gradually with increased energy loss but, most important, the signal to noise ratio is maximal at an energy loss position slightly below the intensity maximum. This is the optimal position for imaging thick specimens. Moreover our studies confirm that the following parameters have similar effects on the energy loss spectra: (1) increased thickness (t); (2) higher average Z number elements (or lower mean free path Ai); and (3) lower primary voltage (V0).
Examination of mucosal surfaces by scanning electron microscopy before and after removal of debris
Authors: Dickson G. R. , Mckenna S. , Mccoll K. , Carr K. E.
Published: August 2011

A rapid method is described whereby gastrointestinal biopsy specimen surfaces can be examined by scanning electron microscopy with overlying tract contents (debris) intact, and also re‐examined after cleaning to determine the structure of the underlying mucosal surface. The conductive coating of gold is removed using mercury, a non‐wetting agent. The specimen surface is suitably cleaned of debris after a brief ultrasonication in absolute ethanol which mixes with the transitional fluid (CO2) used for critical point drying.
Phospholipid, Nature's own slide and cover slip for cryo‐electron microscopy
Authors: Frederik P. M. , Stuart M. C. A. , Bomans P. H. H. , Busing W. M.
Published: August 2011

Thin films of surface‐active compounds, with or without particulate material, can be obtained by immersing and withdrawing a bare specimen grid from a solution/suspension of the compound. Immediately after withdrawing the grid, thinning of the film starts. Thinning is initially powered by gravity and capillary forces and will proceed in thin films (< 100 nm) driven by intermolecular forces until the London‐van der Waals attractive forces come to an equilibrium with electrostatic repulsion of similarly charged surfaces of the film. With small unilamellar vesicles prepared from the phospholipid dimyristoyl phosphatidyl choline (DMPC) the draining behaviour of these films was studied by cryo‐electron microscopy. Small unilamellar vesicles were observed within the film as well as the coalescence of these vesicles into sheets (‘leaky’ membrane fusion). Sheets dominate the images when films are allowed to drain for longer periods (>3min).

Thin films were formed on grids from catalase crystals suspended in a DMPC suspension and vitrified by cooling. High‐resolution information was obtained by electron diffraction at low temperature and under low‐dose conditions from catalase crystals surrounded by small vesicles as well as from catalase crystals surrounded by sheets of DMPC. In the latter case the water content drops from 99% (DMPC in small vesicles) to less than 30% (DMPC in sheets) during draining. Ferritin was added to a DMPC suspension and thin films were prepared and vitrified. After prolonged draining ferritin molecules were deposited in layers with a stepwise increase in thickness. Draining of thin films has thus a dehydrating effect as well as a sorting and ordering effect. These effects must be considered when using surface‐active compounds at air‐water interfaces as a slide and cover slip for electron microscopy.
Inverse problems
Authors: Coleman Rodney
Published: August 2011

Positron emission tomography involves constructing an image of brain tissue from gamma rays counted at detectors surrounding the head. This is an inverse problem: how to measure a phenomenon from data taken from a derived distribution. We have noise and a loss of high frequency signal, both of which contribute to ill‐conditioning. A brief and informal review of the mathematical and statistical methods available for handling inverse problems which are linear or linearizable is given.
Analysis of heterogeneous fluorescence photobleaching by video kinetics imaging: The method of cumulants
Authors: Koppel Dennis E. , Carlson Curt , Smilowitz Henry
Published: August 2011

The method of cumulants has been applied to digital video fluorescence microscopy. The method is used to reconstruct the distribution of fluorescent molecules before the initiation of fluorescence photobleaching, and to characterize heterogeneous photobleaching by imaging one or more of the cumulants of the bleaching decay rate. Using the pipelined pixel processor of the image analysis system for the bulk of the calculations, rather than the general‐purpose host‐computer CPU, the video kinetics imaging can be performed in near real‐time. The method is applied to chick embryo myotubes labelled with fluorescein‐conjugated α‐bungarotoxin. The pre‐bleach fluorescence distribution is derived, and the image of fluorescein fluorescence is separated from glutaraldehyde‐induced autofluorescence on the basis of the spatially resolved average photobleaching decay rate.
Aluminium binding to chromatin DNA as revealed by formation of fluorescent complexes with 8‐hydroxyquinoline and other ligands
Authors: Llorente A. R. , Castillo P. Del , Stockert J. C.
Published: August 2011

After aluminium mordanting of chicken blood smears, several chelating dyes and reagents were tested in order to detect the metal binding to chromatin DNA by fluorescence microscopy. 8‐Hydroxyquinoline induced the most selective fluorescence reaction, which was abolished after DNase or trichloroacetic acid treatment. Morin, purpurin and nuclear fast red showed an intense nuclear fluorescence, but lower selectivity. Fluorescence was also observed in chromatin treated with morin or cur‐cumin without mordanting. The simplicity and microscopical characteristics of the Al/8‐hydroxyquinoline fluorescence reaction make it very suitable for cytological studies of chromatin.
The development of biological preparative techniques for light microscopy, 1839–1989
Authors: Bracegirdle Brian
Published: August 2011

The development of techniques of preparing biological material for microscopical observation is summarized, with much greater emphasis being given to the introduction and establishment of the various techniques, largely in the nineteenth century, than to their perfection and extremely widespread use in the twentieth century. The development of the microtome as an instrument of the greatest importance is summarized and an assessment is made of the impact of preparative techniques on the development of the medical sciences.
TEM in materials science—past, present and future
Authors: Hirsch P. B.
Published: August 2011

The progress in transmission electron microscopy as applied in materials science is reviewed briefly, from the era of replica techniques in the 1940s and 1950s, through the development of the diffraction contrast technique in the late 1950s and 1960s, to the present day when instrumental resolution is sufficient to obtain structure images of a wide variety of crystalline solids. One of the most important advances has been the development of combined imaging and microanalytical techniques in a single instrument. The versatility of the TEM technique with atomic resolution and microanalytical facilities, the variety of signals and contrast effects available, and its universal application, establishes it as an essential tool in materials science for the foreseeable future.
Does light microscopy have a future?
Authors: Tanke Hans J.
Published: August 2011

The application of light microscopy in medicine and cell biology has been significantly influenced by both the availability of specific biological reagents such as monoclonal antibodies and nucleic acid probes, as well as by the enormous progress in microelectronics and computer technology. It is likely that specific reagents for a variety of cellular macromolecules will become available on a large scale in the coming years. Moreover, methods using both sensitive detection devices such as charge‐coupled device (CCD) cameras and sophisticated image processing exist to quantify this information at the single molecule level in morphologically intact cells.

This paper describes the impact of these two factors on the light microscope of the future, with special emphasis on fluorescence. It defines the improvements that still are required to solve some of the challenging problems such as the quantification of unique genes and their products in intact cells, the quantification of DNA adducts and the detection of rare mutant cells or circulating tumour cells.
Mathematical morphology and convolutions
Authors: Mazille J. E.
Published: August 2011

The transforms of mathematical morphology may be generated by convolutions followed by thresholdings. Furthermore, the convolution‐thresholding pair may generalize mathematical morphology in three directions thanks to adjustments of the thresholding level, weighting of the coefficients of the convolution matrix and iterations. This leads to a wide set of non‐linear operators and structuring elements, including powerful directional transforms and an accurate iterative approach of a circular structuring element of any size and thus to a good approximation of the Euclidean distance.
Shape characterization of particles via generalized Fourier analysis
Authors: Réti Tamás , Czinege Imre
Published: August 2011

A general method for shape characterization of two‐dimensional (2‐D) microscopical particles is presented. The proposed procedure based on Fourier analysis techniques can be regarded as a generalization and further development of methods described in the literature. The geometry of the particles is characterized by a set of shape functions which is invariant under translation, rotation and dilation. Shape descriptors generated from the Fourier coefficients are used for shape evaluation. Similarity between particles has been evaluated using an appropriately defined distance measure. The method is demonstrated by an application using a collection of 2‐D objects.
Boolean random functions
Authors: Serra J.
Published: August 2011

A Boolean function f in Rn is the supremum of upper semi‐continuous random functions f'i which are almost surely positive, bounded with compact support and centred at the Poisson points (i). They generalize to functions of classical Boolean model for sets. The Boolean function f may be studied via its subgraph, i.e. as a random set in Rn x R. The key notion is then the functional Q(Bt), i.e. the probability that a compact set Bt centred at altitude t misses the subgraph of f. The general expression of Q(Bt) is given, and followed by a series of important derivations (volumes, gradients, numbers of summits, etc). Theorems of structure are given: they concern the properties of infinite divisibility for the sup, and domains of attraction for Boolean functions. The last sections are devoted to the study of two particular Boolean functions; emphasis is put on the stereological implications of the approach. A critical example illustrates the theory.
An interactive image analysis system for mean particle volume estimation using stereological principles
Authors: Moss M. C. , Browne M. A. , Howard C. V. , Joyner D. J.
Published: August 2011

A semi‐automatic image analysis system based on the stereological approach of point‐sampled intercepts for mean volume estimation is described. The method used requires no assumptions about particle geometries and is thus general in its application. To ease implementation in this application, the assumption of particle convexity is made, making the measurement of intercepts easier for the user.

The system was tested with computer‐generated sample sections to determine the accuracy and precision of the estimates obtained. The results were found to be within 10% of expected values in all cases. When tested with ‘real’ samples a typical result gave a coefficient of variation of 9% from eight independent estimates of one sample.
High‐resolution low‐temperature scanning electron microscopy for observing intracellular structures of quick frozen biological specimens
Authors: Inoué Takao , Koike Hirotami
Published: August 2011

Intracellular structures of rapidly frozen biological tissues were observed in 3‐D under a low‐temperature scanning electron microscope using a newly developed side‐entry type cryo‐holder. The present low‐temperature SEM is simple, easy to operate and effective for observing biological materials at high magnification. Biological tissues (the pancreas, small intestine, brown adipose tissue and Harderian gland) freshly removed from the mouse were immediately frozen in liquid propane cooled with liquid nitrogen, and their surfaces were manually fractured using a precooled razor blade in liquid nitrogen before introducing the cryo‐holder into the SEM. When intracellular structures were revealed after appropriate sublimation, the specimens were coated with gold using a metal evaporator fitted to the side of the microscope column at one of the specimen chamber ports. The cryo‐holder was connected to a copper braid coming from a liquid nitrogen reservoir to maintain a low temperature. Using this method, intracellular structures such as the mitochondria and endoplasmic reticulum were demonstrated at high magnifications. Ribosomal granules were discerned on the rough endoplasmic reticulum of the pancreatic acinar cells. Granular substances, presumably elementary particles, were also recognized on the mitochondrial cristae of the brown adipose tissue. The method was particularly effective for studying the 3‐D configuration of lipid droplets which had been difficult to preserve by chemical fixation.
A microscope fluorimeter using multiple‐wavelength excitation for ultrasensitive single‐cell emission spectrometry
Authors: Nobiling R. , Bührle C. P.
Published: August 2011

Fluorimetric measurements with Ca2+‐chelating or pH‐sensitive dyes at the level of individual small cells (diameter < 10 μm) require special modifications of and attachments to conventional light microscopes: (1) a subassembly for multiple‐wavelength excitation by rapidly interchanging narrow‐band interference filters in the excitation light path permits quasi‐continuous dual‐ or triple‐wavelength determinations of the signals as well as ratio‐forming; (2) a gated, photon‐counting multiplier tube is mandatory for ultrasensitive light measurements. Data processing is done digitally and accomplished by circuitry using standard logic elements, for example ECL, TTL and CMOS counters and gates.

We also present examples of application to some typical problems in the field of cell biology: (A) distribution of Ca2+ in isolated, living cells; (B) Ca2+‐transients in single cells and tissue explants exposed to selected stimuli.

Outlines are given for converting conventional light microscopes to ultrasensitive photon‐counting analysing instruments which may then be modified according to the experimental requirements and the type of microscope available. In addition, we determined whether calibration techniques used in macroscopical work, i.e. with conventional spectrofluorimeters, also produced meaningful results when applied to microspectrofluorimetry of very small cells that yield only low count rates.
Serial sectioning, electron microscopy, and three‐dimensional reconstruction of fine nerve fibres and other extended objects
Authors: Heppelmann B. , Meßlinger K. , Schmidt R. F.
Published: August 2011

Two different techniques for three‐dimensional reconstructions of nerve fibres or other extended fine tissue structures were developed to study the afferent innervation of the cat's knee‐joint capsule.

The re‐embedding technique starts with series of semithin sections for light microscopic reconstructions. In a second step, selected semithin sections can be re‐embedded for ultramicrotomy to examine ultrastructural details. This method offers the possibility to investigate fine structures over a distance of several hundred micrometres without any loss of ultrastructural information.

The serial section‐ESI technique is based on the electron spectroscopic imaging of semithin sections. Small tissue blocks are cut into series of either semithin or alternate semi‐ and ultrathin sections which can be directly used for a complete ultrastructural investigation.

Finally, true‐to‐scale three‐dimensional reconstructions are performed by graphical techniques or computer‐aided methods.
Accurate composition determination of Be‐Ti alloys by electron energy loss spectroscopy
Authors: Liu D. R. , Williams D. B.
Published: August 2011

Accurate quantification of the Be content in Be‐Ti alloys on a submicrometre scale can be accomplished with electron energy loss spectroscopy in an analytical electron microscope. The three major steps required to ensure the accuracy of the numerical results are analysed. The first step is the choice of the specimen thickness which should be such that the influence of the specimen surface effects can be ignored yet thin enough so that deconvolution of the spectra is unnecessary. The second step is the background extrapolation under the ionization edge of interest. In this study, a direct least‐squares fit with a progressive weighting is used to avoid the drawbacks of the conventional linear least‐squares fit. The third step is the calibration of the partial ionization cross‐section ratio with the use of a standard specimen. Without this calibration step, the error in the final microanalysis result could be excessive, as demonstrated. With all these precautions taken into consideration, we are able to show that the intermetallic phase TiBe12 exhibits a great deviation from its nominal stoichiometry.
An X‐ray projection microscope with field emission gun
Authors: Kolařík V. , Svoboda V.
Published: August 2011

The application of a field emission gun in an X‐ray projection microscope is described. In the microscope, a tungsten cathode activated by zirconium and oxygen in combination with the magnetic one‐lens imaging system was used. In order to obtain a sufficiently small‐sized X‐ray source, a Cu‐Be‐Al sandwich target was applied.
Back‐scatter electron images at 200 kV of subsurface structures in composite materials
Authors: Fourie J. T.
Published: August 2011

Back‐scatter electron images of subsurface structures of higher atomic number than the matrix are obtained at accelerating voltages of 50, 100, 150 and 200 kV. Similar back‐scatter images of structures on the bottom surface of single crystal foils of copper and mica ranging in thickness from 1 to 4 μm are compared with STEM images. It is concluded that at 200 kV a reasonable resolution of structures down to 1 μm subsurface is possible.
Size distribution of granular materials from unthresholded images
Authors: Michelland S. , Schiborr B. , Coster M. , Mordike B. L. , Chermant J. L.
Published: August 2011

A new method of obtaining information on the size distribution directly from grey‐tone images (from SEM for example) is proposed and discussed. Good agreement was obtained using this method and with the classical semi‐automatic method using a digitizing table.
Characterization of coals by automated optical image analysis 1. Vitrinite reflectance
Authors: Unsworth J. F. , Gough H.
Published: August 2011

An increasing awareness of the importance of petrographic characterization of coals to efficient selection of coals for pulverized coal combustion and metallurgical coking, has highlighted the need to improve slow and subjective optical microscopic procedures. Automated image‐analysis procedures to measure vitrinite random reflectance are examined here in some detail, giving particular attention to inertinite‐rich coals. It is shown by consideration of intra‐ and inter‐particle reflectance variance that a given accuracy for vitrinite mean random reflectance (Rv) can be achieved by selection of an appropriate surface sampling procedure. The repeatability of Rv for single coals is similar to that for manual microscopy, but the reproducibility, as established by an international interchange exercise, is not yet good enough to specify a standard procedure. In some coals even vitrinite sub‐macerals can be distinguished; however, caution is required when extending this method to vitrinite reflectance distributions of blends containing different rank coals or of heat‐altered coals.
Characterization of coals by automated optical image analysis 2. Inertinite reflectance
Authors: Unsworth J. F. , Gough H.
Published: August 2011

It is now well established that the combustion and carbonization behaviour of the coal maceral group inertinite is correlated with its reflectance. Inertinite reflectance studies have shown that, whereas accurate measurement by conventional optical microscopy is a prohibitively time‐consuming operation, image analysis techniques are limited by their inability to distinguish vitrinite and inertinite with the same grey level. Nevertheless, automated inertinite reflectance measurement has been used to demonstrate a potential quantitative distinction between fusinite and semifusinite. Furthermore, the proportions of vitrinite and inertinite in a coal sample can be determined by image analysis as accurately as by conventional methods.

Reflectance distributions including all components within a coal have also been investigated; this could provide a way of characterizing coals, in particular inertinite‐rich ones, without the need for maceral identification. This approach is almost free of subjective interpretation; however, there are some limitations due to erroneous measurements at inter‐maceral boundaries. The reproducibility of this procedure between different laboratories is sufficiently encouraging to consider its use in a quality control environment.
Computer‐based image analysis of cartilage differentiation in embryonic limb bud micromass cultures
Authors: Garrison James C. , Peterson Paul , Uyeki Edwin M.
Published: August 2011

A computer imaging system was used to analyse the effects of benzamide (BAM), sodium butyrate and dimethyl sulphoxide (DMSO) on chondrogenesis in culture. Embryonic chick limb bud cells were used as a source of undifferentiated mesenchyme, and cultures were prepared using a micromass culture technique. The degree of chondrogenesis is directly related to the amount of Alcian blue staining. Standard assays include either manual tally of individual cartilage nodules, which is both tedious and time‐consuming, or extracting bound dye for spectrophotometric analysis, which obscures individual variations because it requires pooled samples. In the present study, low‐magnification images of individual micromass colonies were converted into derived images based on their percentage transmittance relative to the background. These derived images were analysed for relative degree of chondrogenesis, using area and integrated optical density (IOD) as descriptors. While both types of data were useful for ranking the degree of chondrogenesis in culture, IOD was the preferred descriptor because of its accuracy over a wide range of threshold values. The area and IOD data both demonstrate that BAM produces marked enhancement of chondrogenesis in culture, sodium butyrate causes marked inhibition and DMSO produces mixed results (enhancement at the high dose, inhibition at the low dose). While the present study demonstrates the usefulness of computer‐aided microscopy for analysis of low‐magnification images, the same descriptors (area and IOD) should be useful in quantifying data from a variety of objects (cells, nuclei, etc.) which can be stained in a selected, quantitative fashion.
Immunohistochemical staining of bromodeoxyuridine‐labelled cells in the mouse embryo
Authors: Cottell David C. , Gilmartin Linda , Bannigan John G.
Published: August 2011

The aim of this study was to compare two immunohistochemical methods, avidin‐biotin peroxidase and immunogold silver staining (IGSS), in the detection of 5‐bromo‐deoxyuridine incorporation in mouse embryo tissues. In addition, two fixation schedules, formal‐saline and a mixture of formaldehyde and glutaraldehyde (4F1G), and two embedding procedures, paraffin wax and the acrylic resin L.R. White, were also compared.

Pregnant mice were injected with 600mg per kilogram body weight on days 10 or 15 (plug day = day 1) of gestation and the embryos recovered 2 h after treatment and fixed in formal‐saline or 4F1G. Fixed material was then processed into wax blocks or L. R. White resin.

After sectioning, the antigen‐antibody reaction was visualized using either the avidin‐biotin peroxidase or IGSS methods. All methods tested gave a well‐contrasted, highly specific reaction, but IGSS in combination with 4F1G and L. R. White gave a clearer and more sensitive signal than other combinations.
The ‘corpuscle’ stereological problem—re‐evaluation using slab fragment volumes and application to the correction of DNA histograms from sections of spherical nuclei
Authors: Rigaut J. P. , Persoz A.
Published: August 2011

A new category of stereological size distribution unfolding models is introduced. It is based on the use of the volumes of particle slab fragments, in addition to their profile dimensions. When spheres are cut by a slab of known (constant) thickness, an estimation of discrete sphere sizes from section data is then possible, as only one parent sphere solution exists for any slab fragment, given the latter's projection size and volume. The unfolding algorithm consists in sequentially testing a set of equations: only one of the solutions satisfies various constraints on bounds. A precise determination of the section thickness is required. Truncation parameters, instead of being troublesome inputs as in classical unfolding models, become valuable outputs.

This model offers the first stereologically valid solution to the important problem of correcting DNA‐amount histograms obtained from sectioned spherical nuclei. Under the (biologically reasonable) assumption that the nuclear volume is proportional to the DNA amount, it is possible to estimate the DNA concentration and, subsequently, compute discrete slab fragment volumes from corresponding DNA values.

An application to Feulgen‐stained rat liver sections is shown. Measurements of hepatocytic nuclear‐profile areas and integrated optical densities are obtained by automated image analysis (IBAS), and the model is used to unfold the section‐obtained DNA histogram. A maximum likelihood fitting of the final distribution with chi functions allows a parametric estimation of ploidy frequencies.

This model can only be used for acceptably spherically shaped particles.
Neuronal imaging with colloidal gold
Authors: Pol Anthony N. van den
Published: August 2011

Colloidal gold is easily prepared, and readily adsorbs to a number of immunoreagents and other proteins for a wide variety of uses for neuronal visualization. Gold probes serve a role as immunolabels for both light and electron microscopy. As an ultrastructural immunocytochemical marker for detection of proteins, peptides or amino acids, gold can be used for immunostaining thick or thin sections prior to embedding, or for immunostaining ultrathin sections after embedding tissue in conventional or unusual embedding matrices. By virtue of its particulate nature, gold as an immunolabel facilitates a semi‐quantitative analysis of relative antigen densities on ultrathin sections. Various combinations of different size gold particles or dual immunolabelling with enzymatic immunolabels together with colloidal gold or silver‐intensified gold serve well for ultrastructural immunocytochemical localization of two antigens in the same tissue section. Colloidal gold can be detected with light microscopy, transmission and scanning electron microscopy, and with confocal laser microscopy. Silver intensification allows detection of gold at both the light and electron microscope level, and increases the sensitivity of immunogold procedures. Colloidal gold is useful as a tracer for physiological studies of transport and internalization in neurons in vivo and in vitro; computer‐assisted video imaging techniques allow detection and tracking of single gold particles in living cells.
Optimizing sampling designs for volume measurements of components of human brain using a stereological method
Authors: Regeur L. , Pakkenberg B.
Published: August 2011

Measurements of the cerebral cortical volume used to be very laborious, due to the 3‐D complexity of the gyral pattern. Using stereological methods, which allow the quantification of 3‐D structures from measurements on 2‐D cross‐sections, the difficulties have been overcome.

In thirty formalin‐fixed normal human brains the total volumes were measured by saline displacement. The brains were serially sliced in coronal sections and the fractional areas of the cortex, white matter, central grey structures and ventricles were determined by point‐counting. Using Cavaliéri's principle the volumes of these structures were calculated. The average cortical fixed volume was 549 ml (SD + 107) corresponding to 54% of the total volume of the hemispheres. The coefficient of error of the cortical volume determinations was 2.6%. The efficiency of the design and the possibilities for optimizing the design are discussed. This point‐counting method was preferred to the use of an automatic image analyser, being precise, easy to handle and not interfering with further tissue processing for histological preparation.
Three‐dimensional image restoration and super‐resolution in fluorescence confocal microscopy
Authors: Bertero M. , Boccacci P. , Brakenhoff G. J. , Malfanti F. , van der Voort H. T. M.
Published: August 2011

Confocal scanning laser microscopy (CSLM) provides optical sectioning of a fluorescent sample and improved resolution with respect to conventional optical microscopy. As a result, three‐dimensional (3‐D) imaging of biological objects becomes possible. A difficulty is that the lateral resolution is better than the axial resolution and, thus, the microscope provides orientation‐dependent images.

However, a theoretical investigation of the process of image formation in CSLM shows that it must be possible to improve the resolution obtained in practice. We present two methods for achieving such a result in the case of 3‐D fluorescent objects.

The first method applies to conventional CSLM, where the image is detected only on the optical axis for any scanning position. Since the resulting 3‐D image is the convolution of the object with the impulse‐response function of the instrument, the problem of image restoration is a deconvolution problem and is affected by numerical instability. A short introduction to the linear methods developed for obtaining stable solutions of these problems (the so‐called regularization theory of ill‐posed problems) is given and an application to a real image is discussed.

The second method applies to a new version of CSLM proposed in recent years. In such a case the full image must be measured by a suitable array of detectors. For each scanning position the data are not single numbers but vectors. Then, in order to recover the object, one must solve a Fredholm integral equation of the first kind. A method for the solution of this equation is presented and the possibility of achieving super‐resolution is demonstrated. More precisely, we show that it is possible to improve by about a factor of 2 the resolution of conventional CSLM both in the lateral and axial directions.
Fluorescence in the tandem scanning microscope
Authors: Boyde A. , Jones S. J. , Taylor M. L. , Wolfe L. A. , Watson T. F.
Published: August 2011

Our studies have shown that the fluorescence mode can be used to good effect in both tandem scanning microscopes (TSM: direct view confocal microscopes) as well as confocal scanning laser microscopes (CSLM). Applications are presented which show that the two great advantages of TSM are real‐time viewing and real colour, which allow faster use and interpretation. CSLM are complementary, not competitive, being currently more sophisticated for low‐level fluorescence work. This is equally possible with available TSM, but requires further development using CCD cameras and image‐processing systems.
The application of real‐time confocal microscopy to the study of high‐speed dental‐bur‐tooth‐cutting interactions
Authors: Watson Timothy F.
Published: August 2011

A tandem scanning reflected light microscope (TSM) has been used to study the cutting interactions of dental burs with enamel. Confocal microscopy produces high‐resolution images of subsurface structures in semi‐transparent specimens such as teeth. The TSM has a real‐time imaging ability which allows the visualization of the high‐speed failure of a substrate which is being machined. This paper describes the criteria for successful imaging and their implementation in the design of a stage for controlling these cutting interactions. Examples of the results achieved are given and further applications for this technique in the field of biomaterials suggested.
Probing DNA structure and function with a multiwavelength fluorescence confocal laser microscope
Authors: Arndt‐Jovin Donna J. , Robert‐Nicoud Michel , Jovin Thomas M.
Published: August 2011

Three levels of organization in DNA structure in the interphase cell nucleus are assessed by confocal laser scanning microscopy: (i) the conformational state of the double helix; (ii) the distribution of eu‐ and heterochromatin; and (iii) the localization of replication complexes throughout S phase. Multi‐parameter measurements were carried out in each optical section using two laser sources and combined stereoscopic reconstructions were used to assess the co‐localization of nuclear components.

DNA is highly polymorphic and can adopt a variety of different helical conformations as well as unusual structures (curved, cruciform, multi‐stranded). We have assessed by laser scanning microscopy the presence of left‐handed Z‐DNA in polytene chromosomes of Diptera as well as the spatio‐temporal distribution of Z‐DNA binding proteins in whole‐mount Drosophila embryos and ovaries.

We have determined the 3‐D distribution of replication sites relative to heterochromatin regions, nucleoli and nuclear membrane by using short pulses of BrdU incorporation in synchronized mouse and human fibroblasts. Replication sites were visualized with a monoclonal anti‐BrdU antibody combined with DNA fluorescent staining and antibody labelling of nuclear lamin. The implications of dynamic DNA movement and structural rearrangement to the organization of the nucleus in domains are discussed.
Fluorescent in‐situ hybridization to detect cellular RNA by flow cytometry and confocal microscopy†
Authors: Bauman Jan G. J. , Bayer Jan A. , van Dekken Herman
Published: August 2011

A fluorescent in‐situ hybridization procedure was developed which is suitable for detection of specific cellular RNA in cells fixed in suspension. The procedure was originally developed for analysis of single‐cell suspensions by flow cytometry. The resulting fluorescent cells proved to have their 3‐D morphology prefectly preserved. The spatial distribution of specific ribosomal RNA and messenger RNA could then be analysed by confocal microscopy of individual cells. In the hybridization procedure, biotinylated single‐stranded RNA probes were used that were produced by transcription from cloned DNA fragments. Detection of poly‐(A)+ RNA was performed with a poly‐biotin‐d(U)‐tailed oligo‐d(T) probe. Bound probe was detected using streptavidin–fluorescein isothiocyanate. For flow cytometry, nuclear DNA was counterstained with di‐amidine‐2‐phenyl indol. Multi‐parameter flow cytometry was used to quantify the fluorescence intensity, i.e. the hybridization signal, of thousands of cells. Confocal scanning microscopy on individual cells revealed the intracellular distribution of the target RNA. The distribution of ribosomal RNA and poly‐(A)+ RNA mRNA in mouse bone marrow and the human leukaemia cell line HL60 cells was investigated. Ribosomal RNA was confined to the cytoplasm of the cells, although in many cells the nucleoli could also be distinguished. Poly‐(A)+ RNA in HL60 cells was found both in the cytoplasm and nucleus. A granular cytoplasmic distribution was seen in part of these cells.
Localization of ribosomal and telomeric DNA sequences in intact plant nuclei by in‐situ hybridization and three‐dimensional optical microscopy
Authors: Rawlins D. J. , Shaw P. J.
Published: August 2011

We have combined the use of three‐dimensional (3‐D) fluorescence microscopy and computer image processing of images with in‐situ hybridization to analyse the 3‐D organization of interphase nuclei in plants. In sections of root tips of Pisum sativum, using cDNA probes, we have shown that telomeres are arranged around the nuclear periphery and that the ribosomal genes in this species appear to exist in discrete, 3‐D domains.