bluuebunny/crossref_metadata_2025 huggingface dataset:
Cryofixation methods for ion localization in cells by electron probe microanalysis: a review
Authors: Zierold Karl
Published: August 2011

Electron probe microanalysis data on the intracellular content and distribution of electrolyte ions depends critically on the functional state of the cells at the moment of cryofixation. Whereas tissue specimens often require special in‐situ freezing techniques, isolated and cultured cells can be frozen within their environmental medium under physiologically controlled conditions. Thus, they represent a feasible system to study functional ion‐related intracellular parameters such as the K/Na ratio. Specifically modified freezing devices allow the study of ion shifts related to dynamic processes in cells, for example, locomotion and exocytosis. The time resolution achieved by time‐controlled cryofixation is approximately 1 ms.
Personal‐computer‐based system for electron beam X‐ray microanalysis of biological samples
Authors: Foster Margaret C. , Saubermann Albert J.
Published: August 2011

A system based on a personal computer has been developed which provides a relatively inexpensive way to equip an electron microscopy laboratory for quantitative elemental analyses of cryosectioned biological samples. This system demonstrates the feasibility of making an X‐ray analyser from a personal computer, together with commercially available hardware and software components. Hardware and software have been assembled to drive the beam in a scanning electron microscope, collect and analyse X‐ray spectra, and save, retrieve, and analyse data. Our software provides a menu‐controlled user interface to direct spectra acquisition and analysis. Spot analyses, video images, and quantitative elemental images may be obtained and results transferred in ASCII format to other computers. Wet weight, as well as dry weight, concentrations are calculated, if measurements were made of areas of the hydrated sample before it was freeze‐dried. Grey‐level copies of video and quantitative elemental images may be made on a laser printer.
A simple handling technique for mammalian oocytes and embryos during preparation for transmission electron microscopy
Authors: Britton Ann P. , Moon Young S. , Yuen Basil Ho
Published: August 2011

Due to their small size, mammalian oocytes and embryos pose unique problems during preparation for transmission electron microscopy. This paper outlines a method which combines protein embedding with centrifugation to locate the specimens on the face of a Beem capsule mould. This method facilitates both the processing of oocytes with minimal loss and rapid location of the specimens within the block for simultaneous sectioning, staining and examination.
Contrast in the electron spectroscopic imaging mode of a TEM
Authors: Reimer L. , Rennekamp R. , Fromm I. , Langenfeld M.
Published: August 2011

When the zero‐loss transmission falls below 10−3 for biological sections of mass‐thickness greater than 70 μg/cm2, the energy window in the electron spectroscopic imaging (ESI) mode of an energy‐filtering electron microscope (EFEM) can be shifted to the most‐probable energy loss of the electron energy‐loss spectrum. This enables mass‐thicknesses up to 150 μg/cm2 or thicknesses of 1·5 μm to be examined. Electron energy‐loss spectra of thick carbon films calculated by a Fourier method agree with experimental spectra. Measurements of the electron energy‐loss spectroscopy and ESI image intensities with an additional platinum film confirm a scattering model for the calculation of the image intensity. This model considers the angular broadening at the most‐probable energy loss by introducing an effective illumination aperture of the order of the full‐width at half‐maximum of the angular distribution.
Quantitative analysis of electron energy‐loss spectra from ultrathin‐sectioned biological material
Authors: Sorber C. W. J. , Ketelaars G. A. M. , Gelsema E. S. , Jongkind J. F. , De Bruijn W. C.
Published: August 2011

A computer program for quantitative spectral analysis is proposed for the elemental analysis of biological material by electron energy‐loss spectroscopy in a conventional transmission electron microscope, the Zeiss EM902. Bio‐standards are used to test the performance of this program. The application of a simplex optimization method for curve‐fitting is proposed to separate the ionization edge from the background. Making use of Ce‐, Ca‐ and Fe‐bio‐standards, this method is compared with Egerton's well‐known two‐area method.
The use of low‐cost frame grabber boards on PC‐AT computers for ESI: possibilities and limitations
Authors: Nys B. , Jacob W. , Van Espen P.
Published: August 2011

A Zeiss EM902 microscope was connected to a PC‐AT computer, equipped with a Data Translation DT2861 frame grabber board. Using this system, the three‐window background extrapolation algorithm was implemented to perform elemental mapping in the electron spectroscopic imaging mode of an energy filtering transmission electron microscope.
EELS data acquisition, processing and display for the Zeiss CEM 902 based on lotus 1–2‐3®: application examples from a biological system and inorganic transition metal compounds
Authors: Drechsler M. , Cantow H.‐J.
Published: August 2011

A personal computer combined with lotus 1‐2‐3® software, including the RS232 module of lotus measure® and a 12‐bit ADC, has been used for data acquisition of electron energy‐loss spectroscopy (EELS) spectra with the Zeiss CEM 902. The internal macro language of lotus 1‐2‐3 allows a menu‐driven procedure. Macro‐programs partly combined with external FORTRAN programs can be chosen from the menu for background subtraction, removal of multiple scattering effects by deconvolution, elemental quantification and several utilities. For special applications or conditions the macro programs can easily be modified.

Spectra from crystals of two inorganic transition metal compounds, ruthenium trichloride and vanadium disulphide, and from a biological sample are presented as examples of the application of this software.
The application of energy‐filtering electron microscopy for the cytochemical localization of Ca2+ ‐ATPase activity in synaptic terminals
Authors: Körtje Karl Heinz , Körtje Doris , Rahmann Hinrich
Published: August 2011

The energy‐filtering electron microscopical modes of electron energy‐loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) have been applied to the cytochemical detection of Ca2+ ‐ATPase activity in synaptic terminals in the brain of a cichlid fish. Using a recently developed modification of an enzyme‐histochemical method, cerium phosphate was precipitated as a marker of high‐affinity Ca2+ ‐ATPase activity. This is considered to be a marker for the plasmalemma‐bound calcium pump, an enzyme which plays a crucial role in the regulation of the cytoplasmic calcium concentrations and therefore of the reactivity of nerve cells. High‐affinity Ca2+ ‐ATPase activity is located preferentially at the inner side of synaptic plasma membranes and enables a discrimination of different types of synapse. It is only by using EELS and ESI that the very small amounts of high‐affinity Ca2+ ‐ATPase reaction product can be analysed reliably and located precisely. These new electron microscopical techniques offer powerful tools for cytochemical studies.
Elemental composition of pyroantimonate precipitates analysed by electron spectroscopic imaging (ESI) and electron energy‐loss spectroscopy (EELS) in vitellogenic ovarian follicles of Drosophila
Authors: Heinrich U.‐R. , Gutzeit H. O. , Kreutz W.
Published: August 2011

Ca2+ was precipitated with potassium antimonate in vitellogenic follicles of the fruit fly Drosophila melanogaster and the distribution of the precipitates formed was studied by electron microscopy. The microvilli of the oolemma in mid‐ and late vitellogenic follicles were lined with precipitates. The chemical composition of the precipitates was analysed by electron spectroscopic imaging (ESI). The images produced by inelastically scattered electrons at specific ionization edges were compared, and the nonspecific background signals were subtracted by an image processing system. The presence of Ca2+, antimony and oxygen in the precipitates formed could be demonstrated. The elemental composition of the precipitates and of yolk spheres was also analysed by electron energy‐loss spectroscopy (EELS). With respect to the precipitates, signals at the calcium L2,3‐edge, the oxygen K‐edge and the antimony M4,5‐edge were recorded without deconvolution and background subtraction. The yolk spheres, which were free of precipitates, gave the characteristic signal of the nitrogen K‐edge. The applied techniques combine good ultrastructural resolution with the possibility of analysing the elemental composition of histochemical reaction products and cellular structures.
Progress in electron microscopic diagnostics: semi‐quantitative determination of precipitable calcium in different cell types of the organ of Corti in the guinea‐pig
Authors: Heinrich U.‐R. , Maurer J. , Mann W. , Kreutz W.
Published: August 2011

Potassium antimonate was used to precipitate calcium in the cochlea of the guinea‐pig. The distribution of the calcium antimonate precipitates was analysed by electron microscopy. The precipitate density was determined in different cell types in the organ of Corti by counting the number of calcium binding sites in a 10‐μm2 area. The size of the precipitates varied considerably, and thus the relative amount of the precipitable calcium was estimated only semi‐quantitatively. As the prominent carbon signal is superimposed over the nearby small Ca2+ ‐edge signals, the combined signal of the antimony M4,5‐edge and the oxygen K‐edge of the calcium antimonate salt formed was chosen for the semi‐quantitative estimation. Images of the inelastically scattered electrons of the precipitates at ΔE = 570 eV were recorded by electron spectroscopic imaging. The area covered by the calcium precipitates within a given cell type was determined in different ultrathin sections of the same organ of Corti by an image processing system.
Ultrastructural and electron spectroscopic analyses of cyanobacteria and bacteria
Authors: Golecki Jochen R. , Heinrich Ulf‐Rüdiger
Published: August 2011

The extracellular sheath material and some intracellular cell components of cyanobacteria and phosphate‐accumulating sewage bacteria were analysed by electron spectroscopic imaging (ESI) and electron energy‐loss spectroscopy (EELS). The specimens were embedded in water‐soluble Nanoplast resin without any previous fixation and ultrathin sections were examined in a Zeiss CEM 902 microscope. A high sulphur content was detected in the inner sheath of the cyanobacterium Gloeothece. The elemental composition of some cell components and inclusion bodies, such as carboxysomes and cyanophycin, was determined by ESI and EELS. In addition, the phosphate content in specific granules of phosphate‐accumulating sewage bacteria was estimated by EELS and nuclear magnetic resonance spectroscopy.
A method for the characterization of surface‐modified asbestos fibres by electron energy‐loss spectroscopy and electron spectroscopic imaging
Authors: Xhoffer Chris , Berghmans Patrick , Muir Ian , Jacob Wim , Van Grieken René , Adams Freddy
Published: August 2011

A method for the characterization of surface‐treated asbestos fibres with electron microscopy is presented. Electron spectroscopic imaging (ESI) of organosilane‐treated chrysotile asbestos fibres has been carried out. Initially, the region below the carbon edge was inspected in ESI mode for its effectiveness as a background correction. Elemental mapping was performed on standard untreated fibres to take into account non‐characteristic signals from extrapolation errors and camera artefacts. The highest resulting pixel value that results from non‐characteristic signals was used as a threshold for further background correction in the net images. Samples for electron energy‐loss spectroscopy were prepared in two different ways, either by gluing on grids, or by using perforated carbon foils. The results show that the use of a conducting carbon film is necessary for the analysis of such electrically insulating asbestos fibres. Focusing of the electron beam on the individual fibres results in a thermal effect promoting the evaporation of the organosilane reaction products.
Image analysis of macroporous polystyrene sorbents using electron spectroscopic imaging to enhance mass contrast. The architectural relationship between polymer porosity and absorption capacity
Authors: Huxham I. M. , Rowatt B. , Tetley L. , Sherrington D. C.
Published: August 2011

We describe the microscopical analysis of two polystyrene divinylbenzene copolymers (PS‐DVB) made using 20 and 55% (v/v) DVB cross‐linker respectively, in which the hydrated structure of each polymeric material solvated in water is preserved by employing very rapid freezing and low‐temperature freeze‐drying techniques. Using the zero‐loss mode of electron spectroscopic imaging to enhance the contrast of unstained polymer sections, we introduce some novel methods for the application of computer‐assisted image analysis for routine examination of polymer pore structure. We also relate these parameters to the ability of the two polymers to absorb nanometre‐sized particles—neutral colloidal gold particles and cationized ferritin. Both methods will be invaluable in the structural analysis of porous polymers, but particularly for comparative analyses of macroporous sorbents.
Stereological estimation of the mean and variance of nuclear volume from vertical sections
Authors: Sørensen Flemming Brandt
Published: August 2011

The application of assumption‐free, unbiased stereological techniques for estimation of the volume‐weighted mean nuclear volume, nuclear v̄V, from vertical sections of benign and malignant nuclear aggregates in melanocytic skin tumours is described. Combining sampling of nuclei with uniform probability in a physical disector and Cavalieri's direct estimator of volume, the unbiased, number‐weighted mean nuclear volume, nuclear v̄N, of the same benign and malignant nuclear populations is also estimated. Having obtained estimates of nuclear volume in both the volume‐and number distribution of volume, a detailed investigation of nuclear size variability is possible. Benign and malignant nuclear populations show approximately the same relative variability with regard to nuclear volume, and the presented data are compatible with a simple size transformation from the smaller benign nuclei to the larger malignant nuclei. Finally, the variance in the volume distribution of nuclear volume is estimated by shape‐independent estimates of the volume‐weighted second moment of the nuclear volume, ***v̄2v, using both a manual and a computer‐assisted approach. The working procedure for the description of 3‐D size variability within benign and malignant nuclear populations can for all practical purposes be reduced to 2‐D measurement of nuclear profile areas. These new powerful stereological estimators of nuclear volume and nuclear size variability provide an attractive approach to quantitative and reproducible malignancy grading of cancers.
Review and outline of environmental SEM at present
Authors: Danilatos G. D.
Published: August 2011

The environmental scanning electron microscope (ESEM) allows the examination of specimens in a gaseous environment. It is based on an integration of efficient differential pumping with a new design of electron optics and detection systems. Backscattered, cathodoluminescence and X‐ray detectors can be designed to fit and to perform optimally in the ESEM. The secondary electron signal can be detected with the gaseous detector device, which is a new multipurpose detector. Insulating, uncoated, wet and generally both treated or untreated specimens can be studied.
Cryoultramicrotomy of muscle: improved preservation and resolution of muscle ultrastructure using negatively stained ultrathin cryosections
Authors: Sjöström Michael , Squire John M. , Luther Pradeep , Morris Ed , Edman Anne‐Christine
Published: August 2011

Ultrathin sections of rapidly frozen, briefly pre‐treated muscle tissue are cut and thereafter are thawed and contrasted using a negative staining technique. The method has provided micrographs in which the in‐vivo order in the muscle fibres has been preserved well enough to enable both a more complete interpretation of X‐ray diffraction evidence from muscle, and also a gain of new ultrastructural information on aspects of myofibril and myofilament architecture in different types of fibre. Examples here are taken from chicken, rabbit and fish muscles and show both the M‐band and the bridge region of the A‐band in great detail. To enhance the detail in the original images, one‐dimensional (1‐D) and 2‐D averaging techniques (lateral smearing and step averaging, respectively) are used. Although there is major shrinkage in section thickness to about one‐third of its original value, demonstrated here for the first time is the fact that the characteristic A‐band lattice planes are preserved in these sections in 3‐D. This confirms the usefulness of cryosections not just for 1‐D and 2‐D image processing, but also for 3‐D reconstruction. Thus, in combination with techniques of image processing, cryoultramicrotomy can give the muscle morphologist the detailed data that are needed to match the molecular biologists, biochemists and immunologists in the interpretation of their data about physiological and pathophysiological events in muscle fibres at the macromolecular level.
Platinum/iridium/carbon: a high‐resolution shadowing material for TEM, STM and SEM of biological macromolecular structures
Authors: Wepf Roger , Amrein Matthias , Bürkli Urs , Gross Heinz
Published: August 2011

Thin Pt/Ir/C coating films (1.5 nm) show a fine granularity and provide a high structural resolution in the transmission electron microscope (TEM) when applied to freeze‐dried biological macromolecules. They keep their structure when exposed to atmospheric conditions, without the need of an additional stabilizing carbon layer, in contrast to conventional high‐resolution shadowing materials such as Ta/W and Pt/C. However, the correct ratio of the components has turned out to be crucial. When evaporating Pt/Ir/C from the source electrode in an electron‐beam‐heated evaporator, the ratio of the three elements changes progressively, and, consequently, the properties of such films depend strongly on the mass that has been pre‐evaporated.

In this paper we present a quantitative analysis of the composition of Pt/Ir/C films by wavelength‐dispersive X‐ray analysis (WDX) undertaken in association with TEM experiments. We applied Pt/Ir/C shadowing to two regular biological test specimens, the phage T4 type III polyhead and the HPI‐layer of Deinococcus radiodurans. It turns out that Pt/Ir/C films containing at least 25% C are three‐dimensionally stable on the freeze‐dried macromolecular samples.

By the dramatically improved resolution power of the latest scanning electron microscopes (SEM) and the invention of the scanning tunnelling microscope (STM), two new surface‐sensitive tools for the investigation of biological macromolecular structures became available. The Pt/Ir/C coating has proved to be well suited for STM and SEM imaging of freeze‐dried biological structures because of its good electrical conductivity and its direct three‐dimensional stability. We compare STM, SEM and TEM images of freeze‐dried and Pt/Ir/C‐coated polyheads.
Gap‐junction quantification in biological tissues: freeze‐fracture replicas versus thin sections
Authors: Ryerse J. S. , Nagel B. A.
Published: August 2011

The relative efficiency of freeze‐fracture replicas versus thin sections for the visualization and quantification of gap junctions in biological tissues has been evaluated. Both methods may underestimate gap‐junction number—thin sections for reasons of tissue resolution and freeze‐fracture replicas due to the mechanics of the fracturing process. Freeze‐fracture misses gap junctions in regions of plasma membrane which are highly contoured, such as the overlapping basal cell processes of Drosophila imaginal wing discs and the interdigitating lateral membrane plications of intercalated discs in cardiac tissue. If the missed gap junctions are relatively large, as they are in both of these examples, freeze‐fracture significantly underestimates the total gap‐junctional area. Thin sections may miss small gap junctions, but in tissues which contain a range of gap‐junction sizes the lost junctions constitute a relatively small fraction of the total junctional area. In neoplastic imaginal wing discs, thin sections were as efficient as freeze‐fracture replicas in identifying even the smallest gap junctions. Although freeze‐fracture may be the better technique for the qualitative and quantitative documentation of small gap junctions in tissues with relatively flat to gently contoured plasma membranes, and thin sections may be the superior method for gap‐junction quantification in tissues containing a range of gap‐junctional sizes and highly contoured cellular processes, the data suggest that a combination of the two approaches should be utilized whenever possible.
Cell surfaces and fractal dimensions
Authors: Keough K. M. W. , Hyam P. , Pink D. A. , Quinn B.
Published: August 2011

The perimeters of the surface membranes of some different cell types have been digitized from electron micrographs and the data analysed in order to discover whether the perimeter can be described by a fractal dimension, df. Micrographs obtained at various magnifications and subsequently enlarged by different amounts have been used. Values of df ranging from 1.02 to 1.34 were manifested over a scale length of about one order of magnitude. Values of df were independent of the magnification, and were the same for cells of the same type. Possible implications of these results are discussed.
Optical sectioning strength of the direct‐view microscope employing finite‐sized pin‐hole arrays
Authors: Wilson T. , Hewlett S. J.
Published: August 2011

We discuss how the strength of the optical sectioning property of the direct‐view reflection microscope varies with the size and distribution of the pin‐holes in the source and detector arrays. Finite‐sized circular pin‐holes are considered in both bright‐field and fluorescence imaging and the results are compared with those of the corresponding scanning optical microscope. We discuss the effect of using both incoherent and coherent illumination.
A confocal beam scanning white‐light microscope
Authors: Hell S. , Witting S. , v. Schickfus M. , van Resandt R. W. Wijnaendts , Hunklinger S. , Smolka E. , Neiger M.
Published: August 2011

We report on a confocal beam scanning microscope utilizing a continuous Xe short‐arc lamp operating in the visible spectrum with unprecedented radiance. Measurements of lateral and vertical resolution will be presented and compared with those of an equivalent scanning laser microscope. Resolution of the white‐light microscope is equivalent to that of the scanning laser microscope. White‐light microscope images positively stand out from those of the scanning laser microscope by their lack of artefacts caused by interference.
Topological description of the densification of a granular medium
Authors: Prasad P. Bhanu , Jernot J. P.
Published: August 2011

The densification of a granular medium is described in terms of topological parameters. Two successive cases are quantitatively analysed starting from regular three‐dimensional (3‐D) packings (in continuous and digitized space) and simulated random 3‐D packings of monosized spheres.
Electron microscopic study of the atomic structure of OMVPE‐grown GaInAs/InP quantum wells
Authors: Hsu Tung , Wang T. Y. , Stringfellow G. B.
Published: August 2011

The as‐grown (001) surfaces of InP single crystals and GaInAs/InP multilayers grown under different conditions by organometallic vapour phase epitaxy (OMVPE) have been studied using reflection electron microscopy (REM). It was found that atomically smooth surfaces can be grown by OMVPE using the proper growth conditions. A small density of elliptical defects also found on these surfaces may be attributed to abnormal growth due to defects formed during the growth process.
In defence of aldehyde—osmium fixation and critical‐point drying for characterization of seed‐storage fungi by scanning electron microscopy
Authors: Mycock D. J. , Berjak Patricia
Published: August 2011

Low‐temperature scanning electron microscopy cannot be used as a general approach when resolution of definitive surface features is required for fungal characterization, because of the production of a pervading exudate by some of the species, strains or varieties. As the resolution of the light microscope is obviously limiting, scanning electron microscopy remains the best microscopical mode. Results are presented for four species within the Aspergillus flavus group which indicate that minimal (if any) distortion or collapse can be achieved with concomitant preservation of surface morphology, when particular precautions are taken during fixation and critical‐point drying. Details of the steps pertaining to preparation for scanning electron microscopy following critical‐point drying that are considered collectively to contribute to the success of the approach are discussed.
A strategy for the reconstruction of structures possessing axial symmetry: sectioned axonemes in sperm flagella
Authors: Lanzavecchia Salvatore , Bellon Pier Luigi , Afzelius Björn A.
Published: August 2011

The images of complex biological structures seen in the electron microscope, possessing an n‐fold rotational symmetry, can be enhanced by averaging the axially repeating motif in order to improve their signal‐to‐noise ratio; this requires that the slices with n‐fold symmetry do not exhibit distortions relative to one another.

A strategy is proposed to detect the relative distortions and to remove them in order to obtain reliable results from the averaging process. Extensive use is made of cross‐correlation analysis and of interpolation; because the procedure involves iterated resampling of the image, it is essential to adopt interpolation algorithms which preserve the spectral power.

The procedure is illustrated by the analysis of transverse sections of the sperm flagellum axoneme of a stick insect; it might be used for the reconstruction of microtubules, nuclear pore complexes, virus capsids, and other supramolecular aggregates. The reconstructed images of axoneme sections reveal new information about the interactions between adjacent doublets; in particular, a double conjunction connecting the inner dynein arm with the nearest B‐tubule has been consistently observed.
A note on stereological estimation of the volume‐weighted second moment of particle volume
Authors: Jensen E. B. , Sørensen F. B.
Published: August 2011

It is shown that for a variety of biological particle shapes, the volume‐weighted second moment of particle volume can be estimated stereologically using only the areas of particle transects, which can be estimated manually by point‐counting.
A method for rapid fixation and dehydration of nematode tissue for transmission electron microscopy
Authors: Jones John T. , Gwynn Iolo Ap
Published: August 2011

A method is described using a microwave oven and acidified 2,2‐dimethoxypropane to fix and dehydrate nematode tissue rapidly in preparation for transmission electron microscopy. Nematode tissue prepared using this method is well preserved compared with tissue fixed by conventional methods. The technique also enabled fixation of unhatched nematodes in the egg, a process that is almost impossible to achieve using conventional methods of fixation. Using the method described, differences were found between the cuticles of different nematode stages.
Suggestions for the quantitative X‐ray microanalysis of thin sections of frozen‐dried and embedded biological tissues
Authors: Hall T. A.
Published: August 2011

When a microregion in a thin section of frozen‐dried and embedded tissue is analysed by the conventional electron‐probe X‐ray continuum‐normalization method, the measured quantity is in mmol of element per kg of embedded specimen. As each microregion contains an unknown amount of embedding medium, this quantity generally lies indeterminately somewhere within the wide range between mmol of element per kg of hydrated tissue and mmol of element per kg of dehydrated tissue. However, if a ‘tag’ element is incorporated in the embedding medium, the contribution of the medium to the local continuum count in each probed field should be measurable, and the X‐ray data may then unambiguously yield mmol of element per kg of dehydrated tissue. This result should not be affected by shrinkage on freeze‐drying or by incomplete replacement of water by embedding medium.

The same X‐ray data can additionally provide estimates of mmol of element per unit volume, mmol of element per kg of hydrated tissue and local dry‐mass fraction. However, these estimates are subject to errors due to tissue shrinkage, incomplete replacement of water and beam damage.
A comparison of some methods for measuring the size distribution of two‐dimensionally clustered latex particles in TEM images using mathematical morphology
Authors: Froehling P. E.
Published: August 2011

The particle size distribution of polymer latexes of, for example, polybutadiene can be measured on transmission electron micrographs using image analysis methods. By taking suitable measures in preparing the sample it can be ensured that the image only consists of projections of two‐dimensional clusters of adhering latex spheres. For the deagglomeration of these clusters a number of methods based on mathematical morphology are discussed. Speed and accuracy of the methods were tested on a personal‐computer‐based image analysis system and compared to manual measurements.

Repeated openings or erosions applied to a binary image are relatively fast global methods with a low accuracy due to the fact that particle diameters are measured in whole numbers of pixels; only the horizontal diameters of rectangular pixels are measured.

With an iterative method the individual components of the clusters are found by repeated conditional thickening of the erosion kernels. The method is accurate but very slow due to the large number of processing steps.

The watershed method as applied to the original grey‐scale transmission electron micrograph makes use of the sharp local maxima which arise at the interface of the projection of two adhering particles. This method is as accurate as the iterative method, but about eight times as fast.
On the contraction and dilation of the plastic support membranes under electron‐beam irradiation in the transmission electron microscope
Authors: Dimitriu Corneliu
Published: August 2011

Time relation curves are given for Formvar membrane contraction or collodion membrane dilation under electron‐beam irradiation. Also, numerical values are given for the contraction or dilation measured in different zones of the Formvar or collodion support membranes.
A method using an on‐line digital computer for the improvement of resolution of backscattered electron images
Authors: Oho Eisaku , Ogihara Akinori , Kanaya Koichi
Published: August 2011

In principle, the resolution of backscattered electron (BSE) images can be little improved, even though an infinitely small beam size is achieved by various improvements in the intrinsic instrument. In order to circumvent this problem, a method is proposed which utilizes an on‐line digital computer for the image recording and processing. The major image‐processing tools are reduction, expansion, super‐imposition with matching of the images, and high‐emphasis filtering in the Fourier domain. By using various combinations of these techniques, the resolution of BSE images has been significantly improved. The validity of these improved images has been confirmed. In the case of a BSE image with too wide a dynamic range, both the present method and digital homomorphic filtering provide successful results.
Anisotropy analysis for particle systems
Authors: Stoyan Dietrich , Beneš Victor
Published: August 2011

This paper discusses some methods for the quantitative description of anisotropy of planar structures. These methods can be applied to systems of particles randomly distributed. It is also possible to detect anisotropics in particle arrangements. Finally, a simple Monte Carlo test of an isotropy hypothesis is suggested. The methods are applied to a planar section of a Cd‐Zn eutecticum with clear anisotropy both in particle orientation and arrangement.
Increased depth of field and stereo pairs of fluorescence micrographs via inverse filtering and maximum‐likelihood estimation
Authors: Holmes Timothy J. , Liu Yi‐Hwa , Khosla Deepak , Agard David A.
Published: August 2011

Image processing methods are presented for effectively increasing the depth of field and for generating stereo pairs of fluorescence micrographs from a conventional optical microscope. In developing these methods the slice theorem of computed tomography is used. In this way the image reconstruction problem is reduced to one of processing only two‐dimensional arrays rather than three‐dimensional arrays and the classical difficult problem of restoring missing Fourier components within the missing cone region is circumvented. Two different approaches to such processing are presented. One approach is based on inverse filtering. Another approach is based on previous development of iterative image restoration algorithms for quantum‐limited incoherent imagery, founded on maximum‐likelihood estimation. Limited experimentation with real micrographs shows that both approaches work well. Some preliminary comparisons are made between the different variations of the methods tested, which point out the advantages and present limitations. Both methods can be implemented on IBM‐AT‐compatible computers with relatively fast execution times. Advantages that these methods have over confocal microscopy are (i) the optical and computing equipment required is less expensive, and (ii) a conventional microscope when set up properly can have much better light sensitivity than a confocal microscope.
Effect of refraction on optical microscopic measurement of internal blood‐vessel diameter and its correction
Authors: Chen Eric Q. , Lam Chan F. , Periasamy Ammafi
Published: August 2011

In optical microscopic measurement of internal blood‐vessel diameters, the effect of refraction must be taken into account to ensure accuracy of the result. This effect is discussed and an analytical correction formula derived. Phantom blood vessels with known internal and external diameters were used to test the validity of the correction formula. The errors obtained prior to correction were reduced significantly after correction.
Characterization of biological macromolecules by combined mass mapping and electron energy‐loss spectroscopy
Authors: Leapman R. D. , Andrews S. B.
Published: August 2011

The combination of scanning transmission electron microscopy (STEM) and parallel‐detection energy‐loss spectroscopy (EELS) was used to detect specific bound elements within macromolecules and macromolecular assemblies prepared by direct freezing. After cryotransferring and freeze‐drying in situ, samples were re‐cooled to liquid nitrogen temperature and low‐dose (about 103 e/nm2) digital dark‐field images were obtained with single‐electron sensitivity using a beam energy of approximately 100 keV and a probe current of approximately 5 pA. These maps provided a means of characterizing the molecular weights of the structures at low dose. The probe current was subsequently increased to about 5 nA in order to perform elemental analysis. The 320 copper atoms in a keyhole limpet haemocyanin molecule (mol.wt = 8 MDa) were detected with a sensitivity of ± 30 atoms in an acquisition time of 200 s. Phosphorus was detected in an approximately 10‐nm length of single‐stranded RNA contained in a tobacco mosaic virus particle (mol.wt = 130 kDa/nm) with a sensitivity of ± 25 atoms. Near single‐atom sensitivity was achieved for the detection of iron in one haemoglobin molecule (mol.wt = 65 kDa, containing four Fe atoms). Such detection limits are only feasible if special processing methods are employed, as is demonstrated by the use of the second‐difference acquisition technique and multiple least‐squares fitting of reference spectra. Moreover, an extremely high electron dose (about 1010 e/nm2) is required resulting in mass loss that may be attributable to ‘knock‐on’ radiation damage.
Distribution of projection angles for single‐axis‐tilt electron microscope tomography of extended thin planar specimens
Authors: Levy Henri A. , Olins Ada L. , Olins Donald E.
Published: August 2011

An optimized distribution of tilt angles for tomography of specimens of non‐circular cross‐section is derived and tested with reconstructions of a phantom model.
The use of polarization analysis in the quantification of fluorescent emission: general principles
Authors: Entwistle A. , Noble M.
Published: August 2011

Fluorescent emission and fluorophore concentration are only linearly related below particular concentrations of fluorophore. Theoretically, analysis of fluorescent polarization might allow identification of situations in which local concentrations of fluorophore are above the range of linear response. Using a confocal scanning laser microscope, we demonstrate that progressive depolarization of fluorescent emission from fluorescein and fluorescein analogues occurs over the concentration range where linearity is lost. Critically, depolarization of emission is first seen at concentrations of fluorophore slightly below those at which linearity is lost. Thus, polarization analysis can be used to determine whether the local concentration of fluorophore is such that quantitative analysis can be carried out.
The isector: a simple and direct method for generating isotropic, uniform random sections from small specimens
Authors: Nyengaard Jens R. , Gundersen Hans Jørgen G.
Published: August 2011

The very simple and strong principle of vertical sections devised by Baddeley et al. has been a major advance in stereology when any kind of anisotropy is present in the specimen under study. On the other hand, some important stereological estimators still require isotropic, uniform random sections. This paper deals with a simple technique for embedding specimens in rubber moulds with spherical cavities. After the embedding, any handling of the resulting sphere independent of the specimen will induce isotropy of the final histological sections.
Molecular histochemistry and plant biology: a review
Authors: Harris N.
Published: August 2011

This review considers some of the recent advances in the applications of molecular histochemical techniques to the analysis of the temporal and spatial patterns of gene expression associated with differentiation and development in plant tissues. A range of methods is outlined, and those for tissue preparation and the isotopic and non‐isotopic labelling of probes for in situ hybridization studies are discussed, taking account of the various approaches which can be used for the localization of specifically bound probes.

An overview of some successful applications of molecular histochemistry includes the elucidation of the roles of homeotic genes in floral biology, the ontogeny and differentiation of the photosynthetic apparatus, and aspects of seed development, including storage protein gene regulation during embryogenesis.
Vacuolar sequestration of fluorescent probes in plant cells: a review
Authors: Oparka Karl J. , Hawes Chris
Published: August 2011

The use of fluorescent probes as indicator and tracer molecules is becoming an important aspect of plant cell biology. In many cases the dye, whether introduced directly into the cytosol or sequestered by the cell from its external environment, is preferentially transferred to the vacuole. In the light of increasing evidence for endocytosis in plant cells, the sequestration of high‐molecular‐weight fluorescent dextrans and the membrane‐impermeant dye Lucifer Yellow‐CH into the vacuole has been cited as evidence supporting the presence of a fluid‐phase endocytic pathway. In this review we consider these recent reports of vacuolar sequestration in the light of new evidence arising on the mechanisms underlying dye uptake.
Wavelength considerations in confocal microscopy of botanical specimens
Authors: Fricker M. D. , White N. S.
Published: August 2011

We have used a multiple‐laser confocal microscope with lines at 325, 442, 488, 514 and 633 nm to investigate optical sectioning of botanical specimens over a wide range of wavelengths. The 442‐nm line allowed efficient excitation of Chromomycin A3, with minimal background autofluorescence, to visualize GC‐rich heterochromatin as an aid to chromosome identification. Sequential excitation with 442‐ and 488‐nm light enabled ratio imaging of cytosolic pH using BCECF. The red HeNe laser penetrated deep into intact plant tissues, being less prone to scattering than shorter blue lines, and was also used to image fluorescent samples in reflection, prior to fluorescence measurements, to reduce photobleaching. Chromatic corrections are more important in confocal microscope optics than in conventional microscopy. Measured focus differences between blue, green and red wavelengths, for commonly used objectives, were up to half the optical section thickness for both our multi‐laser system and a multi‐line single‐laser instrument. This limited high‐resolution sectioning at visible wavelengths caused a loss in signal. For ultraviolet excitation the focus shift was much larger and had to be corrected by pre‐focusing the illumination. With this system we have imaged DAPI‐stained nuclei, callose in pollen tubes using Aniline Blue and the calcium probe Indo‐1.
Confocal microscopy and image processing in the study of plant nuclear structure
Authors: Shaw Peter , Highett Martin , Rawlins David
Published: August 2011

We describe measurements of the point spread function (PSF) for a confocal microscope and compare them with the PSF for a conventional (wide‐field) fluorescence microscope. In situ hybridization with probes to telomere and ribosomal rDNA sequences, combined with three‐dimensional (3‐D) microscopy, has been used to study interphase nuclei in root tissue of Pisum sativum and Vicia faba. Nearly all the telomeres in both species are located at the nuclear envelope, and are highly clustered in the Vicia tissues, suggesting specific binding interactions. rDNA labelling in P. sativum shows four brightly staining knobs, corresponding to condensed regions of the rDNA genes from the two pairs of nucleolar organizer genes in this species, arranged approximately tetrahedrally around each nucleolus. Deconvolution using the measured PSFs can be used to improve these images, revealing a fibrous substructure in the perinucleolar knobs, and a large amount of interconnecting internal structure, which we suggest represents rDNA both in the fibrillar centres and also more diffuse, widely dispersed rDNA. Finally we show that accurate conventional data coupled with deconvolution can produce 3‐D reconstructions comparable to those obtainable with confocal microscopy, but that the clearest images are obtained by applying deconvolution to the confocal data.
Co‐localization of the cellulose framework and cell‐wall matrix in helicoidal constructions
Authors: Vian B. , Temsah M. , Reis D. , Roland J. C.
Published: August 2011

Affinity methods (enzymes, lectins and antibodies used as specific probes) were applied in order to target cellulose and the major matrix components in cell walls of dicotyledons built up as helicoids. The probes were the enzyme cellobiohydrolase, CBH1, for cellulose, polyclonal antibodies and the lectin RCA (Ricinus communis agglutinin) for xyloglucans, and monoclonal antibodies JIM5 or JIM7 for homogalac‐turonan sequences (according to their degree of esterification). Observations were performed: (i) in muro and/or on heteromolecular fractions following controlled cell‐wall dissociation experiments; and (ii) at the light microscope level and/or at the electron miscroscope level by means of various visualization markers. Affinity labelling was complemented by subtractive cytochemistry and by the labelling of available anionic groups by means of cationic gold particles. Data confirm the importance of using a variety of probes, the combination of which allows the acquisition of convergent and complementary results. Concerning the particular case of helicoidal walls of elongating cells, it was shown that cellulose was always co‐localized with xyloglucans and homogalacturonan polymers in zones where the cholesteric order was well defined. Cellulose was always associated with compatible hemicellulose polymers capable of binding tightly. Moreover, residual charges were always present along the microfibrils, forming an anionic coat able to repel the adjacent cellulose microfibrils. A possible role of the heteromolecular association of xyloglucan and pectate as a surfactant allowing the cholesteric assembly is hypothesized.
Contrast enhancement and depth perception in three‐dimensional representations of differential interference contrast and confocal scanning laser microscope images
Authors: Schormann Thorsten , Jovin Thomas M.
Published: August 2011

A commonly used three‐dimensional reconstruction method in confocal scanning laser microscopy (CSLM) involves the generation of stereo views by stacking optical sections. Under certain conditions the resulting stereo pairs exhibit inferior quality with respect to contrast and depth perception. A method based on digital image processing is described in which the individual images are enhanced prior to reconstruction, thereby increasing the number of usable optical sections by a factor of up to five. Furthermore, we introduce a new contrast enhancement transformation based upon local statistics and a grey‐level probability density function that provides improved depth perception. Digital image processing methods map a discrete grey scale onto a continuous, unbounded interval. Inasmuch as a closed, discrete grey scale is required for computer display purposes, we present an appropriate mapping function derived from an entropy criterion.