bluuebunny/crossref_metadata_2025 huggingface dataset:
An investigation of the fixation and staining of lipids by a combination of malachite green or other triphenylmethane dyes with glutaraldehyde
Authors: Lawton June R.
Published: August 2011

Mixtures of the monocationic triphenylmethane dyes, malachite green or crystal violet, with glutaraldehyde, retained and stained phospholipid droplets in chloroplasts of leaves of Lolium multiflorum Lam. These dyes also stained trilinolenin; phosphatidic acid dioleoyl, dipalmitoyl; phosphatidylcholine dioleoyl, dilinoleoyl; and phosphatidylethanolamine dioleoyl on filter paper models. In this model system the dipalmitoyl derivatives of phosphatidylcholine and phosphatidylethanolamine did not stain well, if at all. Washing of the dyed samples with 0.5 M sodium chloride solution did not remove the colour, suggesting that the interaction is unlikely to be purely ionic. Except with trilinolenin, the colour and possibly the lipid samples were removed from the filter paper model system on washing with 100% ethyl alcohol.

Other triphenylmethane dyes (methyl green, light green and fast green FCF) did not retain phospholipid droplets in tissue. Fast green did, however, stain phospholipids in the model system. Two quinone‐imine dyes, neutral red and toluidine blue O, while staining phospholipids in the model system did not retain droplets on the chloroplasts but did assist in the retention and staining of cell membranes.

The basis of the reaction between lipid and dye is discussed in relation to the structural formulae of the dyes and model lipids. It is possible that there is an interaction between the hydrophobic fatty acid ester side chains of the lipid and the dyes. Neither the phosphate nor the polyhydric alcohol moieties of the lipid seem to be essential for staining or retention of lipid.
Imaging of submicron objects with the light microscope
Authors: Delgado Reynolds M. , Fink Michael J. , Brown R. Malcolm
Published: August 2011

Using conventional optics coupled with image processing, several submicron objects have been studied with light microscopy. These include polystyrene beads (88, 264 and 557 nm), frustules from the diatom Pleurosigma angulatum and the T‐4 bacteriophage, either attached to its host, Escherichia coli, or free in the medium. The best results were obtained from dark‐field and phase contrast optics. Digital image processing with the use of colour look‐up tables in real time greatly promoted precise focusing of the objects. Selective discrimination of image histogram grey values allowed for selection of a sharp contour boundary, thus more effectively delimiting the submicron objects. User interactive scaling of the diffraction limited boundaries greatly improved discrimination of the objects. While Abbe limits have not been surpassed by these techniques, the greater ‘apparent resolution’ could be attributable to a ‘better recognition’ of the submicron objects examined. Equivalence of images of polystyrene beads and bacteriophages was demonstrated with light and electron microscopy of the same field.
Optical sectioning in confocal fluorescent microscopes
Authors: Wilson T.
Published: August 2011

We discuss the three‐dimensional imaging properties of confocal fluorescence scanning optical microscopes with particular reference to their optical sectioning property. We consider the practically important cases of the use of finite‐sized circular and slit detectors and show how they effect the strength of the sectioning. We find, inter alia, that the performance is always better if the excitation and fluorescent wavelengths are close to each other, indeed as the fluorescent wavelength becomes very large the sectioning disappears altogether. We also compare the strength of the sectioning with that obtained in non‐fluorescent confocal microscopy as this has implications in immuno‐gold labelling studies.
A practical approach to the stereology of anisotropic structures
Authors: Beneš Viktor
Published: August 2011

Inversion formulae for the rose of directions of fibre (surface) processes are evaluated by means of a parametric model of the rose of intersections. The problems of statistical tests of isotropy, degree of anisotropy and stereological formulae are solved. A short‐cut sampling procedure for the three‐dimensional case is proposed and applied in a metallographic situation.
An improved method for collecting and staining microorganisms for enumeration by fluorescence light microscopy
Authors: Williamson F. A. , Palframan K. R.
Published: August 2011

A rapid, robust method for the enumeration of total and viable microorganisms is described. A method using specific stains for viable and total cells and fluorescence light microscopy on membrane filters had been previously developed, but was sub‐optimal in that some non‐specific staining of the filters occurred and the filters were not flat enough for automatic image analysis methods to be employed, because not all cells in a field of view were in focus simultaneously.

A new membrane filter has recently become available: the Anopore† membrane was described by the manufacturers as having a number of properties which would overcome these limitations. These include inorganic construction (giving resistance to solvents), high porosity (giving high flow rates), low surface adsorption (giving low background staining) and inherent hydrophilicity (simplifying wetting with aqueous solutions).

Anopore membrane filters were found to produce very high contrast images of bacteria stained with ethidium bromide. Even with a relatively low power (magnification = 40) dry objective, these images could be easily thresholded for image analysis using only grey‐level information. The methods developed here are considered to be a suitable basis for a fully automated procedure for the enumeration of total microbial populations.
The simulation of HOLZ line positions in electron diffraction patterns: a first order dynamical correction
Authors: Bithell E. G. , Stobbs W. M.
Published: August 2011

The accuracy of the geometric model for the simulation of higher order Laue zone (HOLZ) line positions in electron diffraction patterns is discussed and a modification proposed in which a first order correction is made for the position of the first branch of the zero layer dispersion surface. The modification provides a physically more realistic picture of the scattering process, and the situations in which the neglect of these dynamical effects might lead to significant errors are assessed. The degree to which it is worthwhile to include the effects of increasing numbers of diffracted beams is also considered.
Fundamental stereological formulae based on isotropically orientated probes through fixed points with applications to particle analysis
Authors: Jensen E. B. , Gundersen H. J. G.
Published: August 2011

A new set of fundamental stereological formulae based on isotropically orientated probes through fixed points is derived. Volume, surface area and integrals of curvature can be estimated by this method. Some of the estimators require, however, local 3‐D information. The estimators can be applied locally to each of a sample of particles whereby moments of particle volume, particle surface area and particle integral of curvature can be estimated. The results are derived from a generalized version of the so‐called Blaschke‐Petkantschin formula.
Precision of area estimation: a numerical study
Authors: Matérn Bertil
Published: August 2011

After listing some general formulae for sampling in n‐dimensional space, the author considers the one‐dimensional case: the estimation of the length of a line segment by counting the number of points that happen to fall within the segment. If the points are equidistantly located, the variance of the estimate is a strictly periodic function of the length of the segment. This systematic sample has a higher efficiency than simple and stratified random samples of the same intensity.

With some modifications, the results carry over to the two‐dimensional case: the estimation of the area of a plane figure by counting the number of sample points falling inside the figure. However, the strict periodicity of the variance in the one‐dimensional systematic case is replaced by a ‘Zitterbewegung’. The magnitude of this oscillation is seen to be very different for figures of different shapes.

Some results are presented also for the estimation of areas by line transects, and for the estimation of volumes by aid of lattices of points in R3, and R4. Some comments are also given on the practical implications of the results for sampling in the plane.
A review of the determination of dislocation parameters using strong‐ and weak‐beam electron microscopy
Authors: Nikolaichick V. I. , Khodos I. I.
Published: August 2011

This paper reviews the results of the investigation of dislocation image characteristics as formed using strong‐ and weak‐beam transmission electron microscopy as well as the application of these data for the determination of the basic dislocation parameters: the value, direction and sign of the Burgers vector, and the dissociation width of dislocations split into Shockley partials. The relative advantages and limitations of strong‐ and weak‐beam techniques in studies of dislocation parameters are discussed. This is exemplified by an examination of the possibility of revealing dislocation dissociation with separation Δ 10 nm using strong diffracted beams. The influence of the anisotropy of the elastic moduli on the strong‐beam images of dissociated dislocations is also discussed. The effects of the column approximation on the calculated weak‐beam images of dissociated dislocations are analysed using the results of non‐column calculations. The statistical analysis of the measured dissociation values is also described, allowing the enhancement of the precision of the weak‐beam measurements and their sensitivity in revealing small variations in dissociation width. Finally, the differences in the form of weak‐beam images of dissociated dislocations and of ordinary and faulted dipoles are discussed.
Application of the disector method in the light microscopic quantification of type II pneumocytes in control and ozone‐exposed rats
Authors: Dormans J. A. M. A.
Published: August 2011

Alveolar type II cells in control and ozone‐exposed rat lungs were counted at the light microscopical level with the ‘disector method’. The type II cells were unequivocally marked by histochemical staining for alkaline phosphatase activity in 2 μm plastic sections. By this counting method, the mean number of type II cells per lung in control rats was of the same magnitude as those reported in the literature, using point counting methods. After exposure of rats to 1.6 mg ozone/m3 for 7 days, a 50% increase in the mean number of type II cells was observed.

The use of the disector method at the light microscopical level offers some advantages above a quantification at the electron microscopical level. The procedure is less time‐consuming, larger areas can be screened, two parallel countings can be performed in one set of sections and there is no need for an exact knowledge about the diameter of the measured particle.
Spatial orientation of arterial sections determined from aligned vascular smooth muscle
Authors: Finlay H. M. , Whittaker P. , Hicks J. G. , Taylor C. P. S. , Park Y. W. , Canham P. B.
Published: August 2011

The alignment of the arterial axis can be used as the required reference for three‐dimensional (3‐D) measurements of structures within the artery wall. Our hypothesis is that this alignment reference may be derived mathematically if one uses medial smooth muscle, a tissue component we have found to have a circumferential organization in human cerebral and coronary arteries. We tested this hypothesis for angles of sectioning up to 60°, using arteries fixed at normal distending pressure. These segments of artery were sectioned at precisely measured angles, using a specially designed mitre box, and the sections were stained to enhance birefringence of the smooth muscle. Arteries from eleven autopsies were obtained from the heart and brain, and measurements of 3‐D orientation on medial smooth muscle were made with a polarizing light microscope equipped with a four‐axis Universal stage. By comparing the cutting angle, derived from our mathematically obtained values, to the predetermined cutting angle, we deduce that we can determine the obliqueness of cut relative to the arterial axis to within 4.6°. The application that is important to us, and possibly to other laboratories, is that the necessary direction reference for an artery is completely contained within a single tissue section.
Triumphs and conflicts in microscopy
Authors: Hartley W. G.
Published: August 2011

The controversies surrounding the Abbe theory and its final acceptance are discussed. The practical problems of lens design and manufacture, the rise and fall of the ultraviolet microscope and reflecting microscope and the development of fluorescence microscopy are briefly surveyed. In particular, the conflicts which arose between Fellows of singular or opposing views—E. M. Nelson, A. E. Conrady, J. W. Gordon—are presented as illustrative of the failure to recognize the nature of the point at issue.
The electron microscope: The British contribution
Authors: Mulvey Tom
Published: August 2011

The unpredictable development of the electron microscope is reviewed in the context of the 150th Anniversary of the Royal Microscopical Society. Abbe convinced himself that an electron microscope could never be constructed. Later, J. J. Thomson established the minute corpuscular nature of the electron and Rutherford realized that such beams can reveal the nature of the atom; his student H. G. Moseley invented electron probe analysis, while looking for X‐ray spectra, but the technique did not seem very practical. G. P. Thomson invented the electron diffraction camera, but it was of limited use without an electron lens. In 1931, Knoll and Ruska built a two‐stage TEM with magnetic lenses and understood where Abbe went wrong. Although Ernst Ruska surpassed the resolving power of the light microscope in 1933, the first commercial TEM was manufactured in the U.K., leading to many further commercial developments. The realization of a digital computer with a stored program, now indispensible in microscopy, was also achieved first in the U.K. Gabor's electron beam holography, invented in Rugby in 1949, was not a challenge to the conventional TEM at first, but recently atomic resolution holography has been achieved in Abbe's native country, using a digital computer to process the hologram, ironically making use of the very principles laid down by Abbe in the last century for the light microscope.
Measuring through the microscope: Development and evolution of stereological methods
Authors: Weibel Ewald R.
Published: August 2011

Obtaining, by means of microscopy, meaningful measurements pertaining to spatial structures requires methods which allow three‐dimensional quantitative information to be derived from the reduced information available on the two‐dimensional flat sections of the structure. The most powerful methods to that effect are those of stereology which are based on mathematical principles. This paper reviews the early invention of these methods, which sought to solve practical problems, and their further evolution as more rigorous mathematical foundations were developed. It is demonstrated that stereological methods are essentially sampling methods and that newer trends provide new and sound solutions to old and elusive problems, such as anisotropy or particle number and size.
Contrast definition and contour detection for logarithmic images
Authors: Jourlin Michel , Pinoli Jean‐Charles , Zeboudj Rachid
Published: August 2011

Logarithmic images, such as images obtained by transmitted light or those produced by the human visual system, differ from linear images. Their processing and analysis require consequently specific laws and structures. The latter have been developed in the concept of a logarithmic image processing (LIP) model (Jourlin & Pinoli, 1987, 1988; Pinoli, 1987a). This model permits the introduction of a well‐justified contrast definition: from a physical point of view, it is closely linked with logarithmic images and from a mathematical point of view, it is set up in an algebraic structure.

The applications presented at the end of this paper concern image preprocessing and segmentation. In particular, in the case of microscopic images, the proposed method of segmentation gives good results with transmitted light (thin foils in biology or transmitted electronic microscopy). However, images obtained by reflected light microscopy are not within the scope of this model.
Microcomputer system for ion microscopy digital imaging and processing
Authors: Olivo J. C. , Kahn E. , Halpern S. , Briancon C. , Fragu P. , DiPaola R.
Published: August 2011

Analytical ion microscopy is a powerful tool for biological tissue analysis as it allows direct chemical distribution imaging, even at low element concentrations. A microcomputer based digital imaging system achieving acquisition at low light level is presented. It includes a high sensitivity video camera connected to a specialized image processor subsystem. Acquired images consist of 512 times 512 pixels with 8 bits accuracy. Real‐time image processing software has been implemented so that image processing may be performed on‐line. Image processing software allows off‐line image manipulation and correlation for biological interpretation of elemental mapping images. System capabilities are illustrated by a study of stable and radio iodine mapping in rat thyroid tissue.
A method for measuring the various constituents of the human hair follicle
Authors: Corcuff P. , Roguet R. , Kermici M.
Published: August 2011

Hair follicles from scalp biopsies (temporal and parietal regions) were isolated by microdissection. This technique allows preservation of the whole structure of the follicle in its fibrous sheath, or isolation of certain elements: bulb and dermal papilla.

Each follicle is examined by transmission optical microscopy and its image is digitized into sixty‐four grey levels by an image analyser. Follicle images are memorized on a hard disc, then recalled individually for measurement.

The image analysis consists of thresholding, interactive selecting, then measurement of the following elements: diameter of the hair follicle, volume of the bulb, height of the keratogenous zone, mean diameter of the hair and size of the dermal papilla. These parameters were related to a clinical classification (terminal, dystrophic, vellus). This morphometric study constitutes an objective approach which is different from, but complementary, to the classic trichogram (telogen/anagen).
The use of local entropy measures in edge detection for cytological image analysis
Authors: Barba Joseph , Jeanty Henrick , Fenster Paul , Gil Joan
Published: August 2011

Accurate edge detection is a fundamental problem in the areas of image processing and pattern recognition/classification. The lack of effective edge detection methods has slowed the application of image processing to many areas, in particular diagnostic cytology, and is a major factor in the lack of acceptance of image processing in service orientated pathology. In this paper, we present a two‐step procedure which detects edges. Since most images are corrupted by noise and often contain artefacts, the first step is to clean up the image. Our approach is to use a median filter to reduce noise and background artefacts. The second operation is to locate image pixels which are ‘information rich’ by using local statistics. This step locates the regions of the image most likely to contain edges. The application of a threshold can then pin‐point those pixels forming the edge of structures of interest. The procedure has been tested on routine cytologic specimens.
Countercurrent plunge cooling: A new approach to increase reproducibility in the quick freezing of biological tissue
Authors: Murray P. W. Le R. , Robards A. W. , Waites P. R.
Published: August 2011

Conditions fulfilling all the accepted criteria for optimum cryoquenching are realized for the first time in a plunge freezing device. Samples are plunged down through a smoothly rising cryogen stream that is maintained within ± 0.25 K of a preselected temperature. Dry nitrogen gas at ambient temperature removes evaporated cryogen molecules from the plunging path immediately above the liquid coolant to prevent pre‐cooling of the sample. Symmetrical instrument design and conditions of flow ensure symmetrical freezing under optimum conditions in a totally repeatable fashion. The device has been used with halocarbons, propane and ethane. It provides consistent cooling rates comparable with the best so far obtained by any other plunging method.
Correction for specimen movement after acquisition of element‐specific electron microprobe images
Authors: Lamvik M. K. , Ingram P. , Menon R. G. , Beese L. S. , Davilla S. D. , LeFurgey A.
Published: August 2011

Because a long time is generally required to generate X‐ray maps of specific elements by electron beam methods, images are subject to a loss of resolution due to stage movement. Methods have been previously described for correcting stage drift during exposure by sensing the drift and deflecting the beam to follow the stage; but these methods require modifications of the equipment. When the drift is not excessive, it is possible to correct a series of images after the exposure series is finished. Here we demonstrate two methods for correcting the drift, one based on manual assignment of specimen position and one on the use of cross‐correlation functions to determine objectively the misalignment of images in the series. The success of the methods is illustrated in calcium‐specific images of a bone section that show the collagen periodicity after drift correction.
The relative intensities of electron diffraction maxima in mixed rare‐earth oxides
Authors: Schaffer G. B. , Loretto M. H. , Smallman R. E.
Published: August 2011

The relative intensities of electron diffraction maxima from yttrium aluminium perovskite were analysed in terms of the atomic scattering contribution to the structure factors and in terms of double diffraction. It has been shown that double diffraction influences the visible intensity between diffraction spots in electron diffraction patterns to a greater extent than does the magnitude of the structure factor. A direct consequence of this is that the intensity of diffraction maxima can increase when a specimen is tilted off an exact zone axis.
Ultrathin sectioning for electron microscopy: The distilled water in the knife trough may extract phosphatase reaction products from the sections
Authors: Rostgaard J. , Qvortrup K.
Published: August 2011

During an electron microscopical study of the localization of the nucleoside diphos‐phatase IDPase in Reissner's membrane of the inner ear, it was discovered that the distilled water in the knife trough produced an annoying artefact. It dissolved all the lead phosphate reaction product from the sections, and thus converted a positive phosphatase reactivity to a false negative one. The water in the knife trough had a pH of approximately 5.4.

Calculations showed that this is an expected acidity, if CO2 in the air equilibrates with distilled water, and that there is 200,000 times more acid in the trough than necessary to dissolve all the reaction product from a ribbon of ultrathin sections. Experiments showed that the artefact could be avoided by adjusting the pH to neutrality with dilute ammonia.
A new rapid method of air‐drying for scanning electron microscopy using tetramethylsilane
Authors: Dey Sudip , Baul T. S. Basu , Roy B. , Dey D.
Published: August 2011

A new rapid air‐drying technique which gives results comparable to critical point‐drying is described for scanning electron microscopy, using a trematode parasite, Homalogaster paloniae, as a test specimen.
Fast Monte Carlo method for simulating electron scattering in solids
Authors: Czyżewski Zbigniew , Joy David C.
Published: August 2011

A simplified Monte Carlo approach to the simulation of the scattering process of penetrating electrons in solids is presented. The Mott scattering cross‐section and simulated angular distributions for plural scattering at low energies are used to describe the angular scattering of electrons. The Mott cross‐section is more accurate than the Rutherford one and provides a longer mean free path for low electron energies. This method is particularly applicable for microcomputers.
Artefacts arising during preparation of hydrated paper pulp samples for low‐temperature SEM
Authors: Moss P. A. , Howard R. C. , Sheffield E.
Published: August 2011

Beating, a pulp treatment widely used in the paper industry, causes disruption of cell wall layers and fibrillation. Previous studies of the effects of beating on fibre morphology have used conventional methods of specimen preparation, with all the attendant problems of shrinkage and distortion during dehydration. Low‐temperature scanning electron microscopy (LTSEM) therefore seemed to offer an ideal method for examining fully hydrated wood pulp fibres. Cryofixation of pulp followed by sublimation of superficial ice, however, is shown to generate artefacts indistinguishable from structures present in the samples. Fibrillar and membranous structures were generated in LTSEM‐prepared sugar solutions; their presence in pulp samples was therefore attributed to the dissolved carbohydrates inherent in pulp suspensions. Since artefact and fact are currently impossible to distinguish in LTSEM‐prepared pulp samples, it seems that the technique should be applied to wet paper or pulp samples with considerable circumspection.
Three‐dimensional imaging of neurons by confocal fluorescence microscopy
Authors: Carlsson K. , Wallén P. , Brodin L.
Published: August 2011

The study of neuronal architecture by means of confocal laser microscopy is described. Optical serial sectioning has been performed on whole‐mount specimens, and the resulting stacks of digitally recorded images have been processed with the help of a computer. Specimen preparation is described, as well as the instrument and its performance. It is shown that the limits in photometric quality are set by photon quantum noise. As both light absorption and scattering was low in the studied specimens, the maximum scanning depth was limited mainly by the working distance of the objectives. Compared with traditional methods, confocal microscopy in combination with digital image processing has the following advantages: (1) a truly three‐dimensional (3‐D) reconstruction is obtained, (2) the specimen remains intact, (3) a higher resolution can be obtained, (4) the process is automated and less time‐consuming and (5) various kinds of data processing are possible.
Applications of confocal microscopy in industrial solid materials: some examples and a first evaluation
Authors: Knoester A. , Brakenhoff G. J.
Published: August 2011

We report here the first results of the application of confocal scanning laser microscopy (CSLM) for the study of the microstructure of solid industrial materials. Glass‐fibre‐reinforced composites, heterogeneous and conductive polymers, homogeneous as well as heterogeneous catalyst (precursor) specimens and soils were examined. We conclude that both the fluorescence and reflection modes of CSLM can yield valuable information. In particular, the optical sectioning capability of CSLM appears to be of great value as it enables one to access the 3‐D organization of the specimen without the need for a difficult and time‐consuming specimen preparation procedure. However, local obscuration may be an important factor in confocal image formation, limiting the penetration capabilities of the technique for industrial materials.
Standards for the application of X‐ray microanalysis to biological specimens
Authors: Warley Alice
Published: August 2011

A review on the subject of compounds used as standards for biological X‐ray microanalysis is presented. The general approach used for standardization has been to use standards which resemble the specimen closely in composition. Thus, standards based on proteins have been used for analysis of quench‐frozen cryosectioned specimens, whereas standards based on embedding resins have been used for resin‐embedded material. The properties of, and problems associated with, each type of standard are recognized and have been well documented. The choice and analysis of standard should not be a drawback to fully quantitative analysis of biological material. Attention is drawn to the fact that the problems associated with any quantification procedure need to be kept in mind when analysis of standards is undertaken.
Estimation of structural anisotropy based on volume orientation. A new concept
Authors: Odgaard A. , Jensen E. B. , Gundersen H. J. G.
Published: August 2011

The quantification of anisotropy—its main direction and the degree of dispersion around it—is desirable in numerous research fields dealing with physical structures. Conventional methods are based on the orientation of interface elements. The results of these methods do not always agree with perceived anisotropy, and anisotropic structures do not necessarily turn out to be ‘anisotropic’ using these methods. In the present paper, we propose an alternative to curve and surface orientation, namely volume orientation. Using trabecular bone as an example of a two‐phase anisotropic structure, the new concept is studied in some detail. In particular, a parametric method of estimating volume orientation from sections is presented and discussed.
Fluorescence discrimination in a reflecting environment
Authors: Galassi L.
Published: August 2011

A method to discriminate fluorescence from reflected or scattered light in epifluorescence microscopy is described. The method is based on the fact that the intensity of fluorescence measured with a long pass (LP) filter placed along the emerging beam (i.e. as a barrier filter) is higher than that measured with the same filter placed ahead of the objective (i.e. along the incident beam). On the other hand, when specific precautions are taken, the intensity of reflected or scattered light is identical to within a small difference. Within the margins of error of this small difference, it can be concluded that when the light intensity I measured with an LP filter along the emerging beam is higher than the intensity I' measured with the same filter along the incident beam, a fluorescent emission is present and the fraction of fluorescent light in the emerging beam is at least (I — I')/I.
Fuzzy sets‐based classification of electron microscopy images of biological macromolecules with an application to ribosomal particles
Authors: Carazo J. M. , Rivera F. F. , Zapata E. L. , Radermacher M. , Frank J.
Published: August 2011

Pattern recognition methods based on the theory of fuzzy sets are tested for their ability to classify electron microscopy images of biological specimens. The concept of fuzzy sets was chosen for its ability to represent classes of objects that are vaguely described from the measured data. A number of partitional clustering algorithms and an extensive set of cluster‐validity functionals (some already reported and some newly developed) have been applied to a test‐data set and to two real‐data sets of images. One of the real‐data sets corresponded to images of the Escherichia coli 50S ribosomal subunits depleted of proteins L7/L12 and the other set to images of the E. coli 70S monosome in the range of overlap views. These two latter sets had been previously studied by another clustering methodology. The new results obtained by the application of fuzzy clustering techniques will be compared to those previously obtained and some conclusions about the consistency of these classifications will be drawn from this comparison.
A quantitative interference light microscope study of human first trimester chorionic villi
Authors: Ockleford C. D.
Published: August 2011

Using a Jamin‐Lebedeff‐type interference microscope an analysis of frozen sections of human first trimester chorionic villi reveals regional differences down to subcellular resolution. The evidence indicates compositional differences between villus cell types and shows that the syncytiotrophoblast is differentiated into at least three layers, one of which corresponds positionally to the previously described syncytioskeletal layer.

Quantitative measurements have been made of specimen thickness, refractive index and dry mass of regions in the tissue.

Local differences in syncytiotrophoblast have been noted with respect to the content of a population of organelles with distinctive optical properties. These may correspond to stored forms of steroid hormone or their precursors.
Theoretical prediction of the electron backscattering coefficient for multilayer structures
Authors: August Hans‐Jürgen , Wernisch Johann
Published: August 2011

Based on existing formulae for the backscattering coefficient for coated samples, i.e. for samples consisting of one film on a massive homogeneous substrate, a theory for the backscattering coefficient for multilayer structures is derived in a clear mathematical way without using approximations. The resulting expression is simple and ready to be applied in scanning electron microscopy or other related analytical techniques.
The total number of neurons in the human neocortex unbiasedly estimated using optical disectors
Authors: Braendgaard H. , Evans S. M. , Howard C. V. , Gundersen H. J. G.
Published: August 2011

An efficient method is presented for obtaining, in under 4h, an unbiased estimate of the total number of neurons in the human neocortex, with a coefficient of error on the estimate of ∼ 5%. The novel sampling scheme used in this study is unbiased and was designed so that only a small amount of neocortical grey matter had to be removed. Hence, the majority of the cerebral grey matter and all the internal grey matter was left intact for further resampling and analysis. Each cerebral hemisphere was subdivided into the four major neocortical regions, sliced coronally at 7‐mm intervals and the volume of the neocortex determined using Cavalieri's principle. Uniform sampling of neocortex was performed in the hemisphere followed by regional subsampling with a varying sampling fraction being taken from each region. Neuronal density estimates were made in thick plastic sections using optical disectors. Shrinkage estimates were made in parallel with the number estimates and found to be negligible. The total number of neocortical neurons in the right hemisphere of five normal 80‐year‐old men was found to be 13·7 × 109 with an inter‐individual coefficient of variation of 12%.
Estimating mean particle volume and number from random sections by sampling profile boundaries
Authors: Gittes Frederick
Published: August 2011

We describe some new shape‐independent stereological estimates of particle mean volume and surface area. Finding volumes or surface areas of cell nuclei, from electron micrographs of random thin sections, is a central problem of biological stereology. The well‐known point‐sampled intercept (PSI) method samples profile interiors to find the volume‐weighted mean volume. This can be used in place of the true mean volume, but to do so introduces bias when volumes vary a great deal, as they do in fixed specimens. Jensen and Gundersen quite recently extended the PSI estimator to provide particle surface area, with no bias in the case of uniform surface areas. Here we extend the PSI volume estimator in a different way, sampling profile boundaries rather than their interiors. We obtain a boundary‐sampled intercept (BSI) volume estimator, simpler than the PSI surface area estimator, but also unbiased for uniform surface areas. Both of these estimators are attractive, for example, in measuring and counting cell nuclei, where membrane surface area varies less than volume. Furthermore, they have no shape bias whatsoever. This paper also examines the general relationship between boundary‐ and area‐sampled estimates, and we clarify the formal connection between our volume estimator and the PSI surface area estimator. We also calculate and compare their theoretical efficiencies.
Estimating the total number of lymphatic valves in infant lungs with the fractionator
Authors: Ogbuihi Sylvester , Cruz‐Orive Luis M.
Published: August 2011

The fractionator is illustrated by means of a biomedical example involving the estimation of the number of lymphatic valves in lungs of infants who had died from sudden infant death syndrome (SIDS) and other known causes.

The method is unbiased irrespective of tissue deformations and it does not require external information such as section thickness. An upper bound of the coefficient of error of the estimate of the number of valves within one lung was 6.5%, despite the fact that the number of valves counted per lung at the last stage ranged between 11 and 37 only. The upper bound includes the biological variation of the number of valves among infant lungs.

Some theoretical remarks are also made on the efficiency of the fractionator. It is suggested, for instance, that the initial sampling stages cause more impact on the precision of the final estimator than the subsequent stages, and that an optimal arrangement of fragments submitted to systematic sampling should have the smallest fragments at the ends, with fragment contents increasing smoothly toward the middle of the series.
Curvature measurement with reflected‐light microscopy and its application to fly facet lenses
Authors: Stavenga D. G. , Leertouwer H. L.
Published: August 2011

Two methods for microscopically measuring the curvature of strongly curved surfaces are compared: one using a Michelson interferometer‐type microscope and one using a novel reflectometrical method implemented in an epi‐illumination microscope. The curvature values obtained with the two methods were very similar, but the latter proved to be by far the simplest.

Curvature measurements on the front surface of the facet lenses of various dipteran flies revealed that facet lens diameter and radius of curvature are linearly related over a wide range of facet lens sizes.
Scanning tunnelling microscopy of tobacco mosaic virus on evaporated and sputter‐coated palladium/gold substrates
Authors: Mantovani J. G. , Allison D. P. , Warmack R. J. , Ferrell T. L. , Ford J. R. , Manos R. E. , Thompson J. R. , Reddick B. B. , Jacobson K. Bruce
Published: August 2011

We present scanning tunnelling microscope (STM) images of untreated tobacco mosaic virus (TMV) deposited on thermally evaporated and on sputter‐coated palladium/gold 40:60 (Pd/Au) substrates, and imaged under ordinary atmospheric conditions. The TMV imaged on both evaporated and sputter‐coated substrates was consistently several times wider than the known diameter of the virus. TMV on evaporated Pd/Au became overcoated with Pd/Au material during sample preparation and appeared elevated in STM images, whereas TMV on sputter‐coated Pd/Au appeared as depressions. When naked TMV were intentionally overcoated with Pd/Au, the STM images were found to be similar to those for TMV on evaporated Pd/Au.
Quantitative fluorescence in confocal microscopy
Authors: Van Oostveldt P. , Bauwens S.
Published: August 2011

The relationship between integrated fluorescence intensity and integrated absorbance was measured in Feulgen‐stained pigeon erythrocyte nuclei hydrolysed for different periods of time and stained at different dye concentrations.

In conventional as well as confocal quantitative fluorescence microscopy the relationship between the integrated fluorescence intensity and the integrated absorbance shows a maximum. This is due to inner filtering and re‐absorption of the excitation light and emission light respectively.

In conventional quantitative fluorescence microscopy the relationship is influenced by the numerical aperture of the objective lens.

Under confocal observation, as measured with the BIO‐RAD MRC‐500 Confocal Imaging System, no influence of the numerical aperture of the objective lens on the relationship between the integrated fluorescence intensity and the integrated absorbance could be observed.
The role of detector geometry in confocal imaging
Authors: Wilson T.
Published: August 2011

The key property of confocal microscopes is their ability to produce three‐dimensional images. This arises simply by the use of a point detector. We discuss the use of point detectors, slit detectors, detector arrays and finite‐sized detectors on the strength of the optical sectioning when the systems are operated in both fluorescence and non‐fluorescence modes. We find that the sectioning is sharper in the non‐fluorescence case. We also discuss methods of obtaining differential phase contrast information and show that it is not possible to obtain pure differential phase contrast from a simple confocal microscope. However, a scheme consisting of two, suitably configured, confocal microscopes can give differential phase contrast with optical sectioning.
Optical sectioning with a fluorescence confocal SLM: procedures for determination of the 2‐D digital modulation transfer function and for 3‐D reconstruction by tessellation
Authors: Schormann Thorsten , Jovin Thomas M.
Published: August 2011

We have determined the operational parameters of a confocal scanning laser microscope (CSLM) used for fluorescence imaging. The system performance was characterized by the modulation transfer function (MTF), calculated from an edge response function (ERF) corresponding to a standard test pattern overlaying fluorescence solutions on a microscopic slide. Signal truncation error was avoided by making the ERF continuous at its limits through superposition of a suitable linear curve. We determined an appropriate scanning step density by defining a compromise between the requirements of the sampling theorem with respect to aliasing and the need for minimal suppression of higher spatial frequencies. These procedures for choosing the sampling density of the total digital CSLM permit a systematic optimization of the image acquisition parameters.

A reproducible digital image was obtained, an important prerequisite for subsequent 3‐D image reconstruction. The latter was accomplished by first developing and applying a maximally automated algorithm for finding closed and distinct contours (corresponding to objects in the 2‐D optical sections). The missing information between contour loops was then interpolated by tessellation (triangulation) using a minimal polygon edge length criterion capable of describing closed surfaces even for adjacent contours with highly dissimilar geometries. In this procedure, we define an average shift length which compensates for the inherent disadvantage of run length encoding algorithms applied to different starting points in successive planes. The surface segments were used to calculate 3‐D representations by applying a shading model, in the present applications specifically to chromosome I and the nucleolus of polytenized Chironomus thummi salivary gland nuclei.
Three‐dimensional optical microscopy using tilted views
Authors: Shaw P. J.
Published: August 2011

The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the x–y plane. This is true of conventional or confocal microscopes. To alleviate this problem we used multiple tilted views to supply the ‘missing data’ and thus increase the resolution in z. A special tilting stage was constructed which allowed specimens to be rotated through large angles. The relative, translation, rotation and z‐spacing between data sets were determined by a novel Wiener/phase cross‐correlation function. Once brought to a common coordinate system the data sets can be combined by Fourier space techniques similar to those used in X‐ray crystallography. We applied this technique to metaphase chromosomes from intact embryos of Drosophila melanogaster. As determined from significant intensity in the Fourier transform, the resolution of the final reconstruction was about 0ċ25 μm in x and y, and 0ċ4 μm in z.
Measurement of osteoclastic resorption pits with a tandem scanning microscope
Authors: Boyde A. , Dillon C. E. , Jones S. J.
Published: August 2011

The functional capacity of isolated osteoclasts is measured by determining the volume of bone or dentine which they can resorb in vitro when cultured on flat tissue slices. Previous SEM stereophotogrammetric evaluations showed that the measurement of total resorptive area per sample, number of pits per sample, area per pit, etc., may be insufficient and that the volume to area ratio may be of utmost importance. A new procedure for the 3‐D description and measurement of resorption pits using confocal microscopy has been established. Osteoclasts were cultured on slices of dentine which were prepared as for SEM. They were imaged with a Tracor Northern TSM equipped with piezo‐electric translators for the objective lens focusing motion, interfaced to a TN8500 computer, using a TV camera for image transfer. The procedure consisted of generating a z map image of the focus depth at which maximum brightness was found at each pixel. Constructing binary masks corresponding to (i) the edge of the resorption pit to define the area beneath which the volume is to be calculated, and (ii) a uniform width at a small distance outside the edge of the pit, enabled the average height of the original surface to be measured and the slice above the pit to be subtracted. After subtraction of the volume element which lay above the top of the original specimen surface, the z map is a measure of both the area and volume of the pit.
Study of Golgi‐impregnated material using the confocal tandem scanning reflected light microscope
Authors: Freire M. , Boyde A.
Published: August 2011

The tandem scanning reflected‐light microscope (TSM) is a real‐time, direct‐view confocal microscope. Only those points in the specimen situated in the focal plane contribute information to the image. A Tracor Northern TMS with piezo‐electric control of the objective lens was used to generate 3‐D images from Golgi‐impregnated hamster cerebral cortex. Stereoscopic pairs of images were recorded as 35‐mm colour film transparencies by photographing while automatically through‐focusing along inclined axes. Transferring the image via a TV camera to the computer, stereo‐pairs were obtained by oblique through‐focusing and summing, displaying maximum intensity data in each line of sight. Pseudocolour topographic displays were generated by assigning the pixel value in a z map image as the focal depth at which the back‐scattered light signal was maximal. The TSM was also modified so that a conventional transmitted‐light image with a large depth of field could be obtained simultaneously as the very shallow depth of field confocal back‐scattered‐light image seen at any focus level. The conventional image is a silhouette of the impregnated neurons: the top surface of the cell is not visible and the relationships of processes that cross over cell bodies cannot be discerned. TSM gives a high‐contrast image. The Golgi precipitate over the neuronal surface is resolved as globular or ovoid, coloured particles. The smaller particles also cover the dendritic spines. All the confocal range (extended focus) image display methods satisfactorily demonstrated the 3‐D arrangement of cell bodies and processes in the chosen volume.