bluuebunny/crossref_metadata_2025 huggingface dataset:
Concerning the nature of the cryosectioning process
Authors: Frederik P. M. , Busing W. M. , Persson A.
Published: August 2011

Intentional melting of frozen‐thin‐sections induces ultrastructural alterations. Cross sections through frozen dried and embedded cryosections revealed that even at the surface of such a section no melting artefacts are visible. One surface of the cryosection (obtained at 168 K) has a saw‐teeth appearance whereas the other surface is rather smooth. The rate of freeze‐drying in the cryochamber of the microtome was estimated to be in order 3·3 nm/min at 183 K and 22·5 nm/min at 193 K for sections, about 120 nm thick, of rat kidney frozen in Freon 22 without any chemical fixation or cryoprotection. Although freeze‐drying will normally not interfere with the cryo‐sectioning process itself, it is a process which cannot be neglected during handling of sections. The available evidence indicated that frozen‐thin‐sections represent the frozen tissue with respect to the ultrastructure and probably also with respect to the chemical composition.
Muscle structure, cryo‐methods and image analysis
Authors: Squire John , Edman Anne‐Christine , Freundlich Alan , Harford Jeff , Sjöström Michael
Published: August 2011

Negatively stained cryo‐sections from glutaraldehyde fixed, anti‐freeze treated muscle, quench‐frozen in Freon cooled by liquid nitrogen, show improved preservation of the axial structure of the myofibrils compared with conventional plastic sections. Such sections are being used both to characterize the structural differences in the M‐bands of different vertebrate muscles and fibre types and also to define the axial distribution of myosin crossbridges and non‐myosin proteins in the crossbridge region of the A‐band. Combined with analysis of the transverse A‐band structure from plastic sections, the cryo‐sections are helping to reconstruct a three‐dimensional picture of the molecular architecture of the A‐band. This, in turn, is providing the necessary structural background with which to interpret the wealth of published X‐ray diffraction data on muscle. Such data should reveal the nature of the contractile event itself.

Since good X‐ray diffraction patterns can be obtained from living muscles, these can be compared with optical diffraction patterns from muscle cryo‐sections as a means of testing the degree of preservation in the sections. Muscle is therefore an excellent tissue with which to evaluate new cryo‐techniques.
Quantitative analysis of electrolytes in frozen dried sections
Authors: Rick R. , Dörge A. , Thurau K.
Published: August 2011

During recent years our group has employed the technique of electron microprobe analysis to determine the electrolyte concentrations in various epithelial tissues. The specimen preparation is characterized by shock‐freezing of small tissue pieces in liquid propane/isopentane mixtures at 77 K, cryosectioning of 1 μm thick serial sections at 170 K and subsequent freeze‐drying at 190 K and 10−4 Pa. The analysis of the frozen dried cryosections is performed in a scanning electron microscope which is equipped with an energy dispersive X‐ray detector. The measuring conditions selected are 17–20 kV acceleration voltage and 0·1–0·5 nA probe current. For quantification, the cellular X‐ray spectra are compared with those of an internal albumin standard layer. The evaluation of the characteristic X‐ray intensities is performed using a computer program. Some critical points of this technique will be discussed.
Statistical analysis of support films and the visibility of atoms
Authors: Kübler O. , Downing K. H. , Ruchti P. , Koller T.
Published: August 2011

The statistical properties of an image of an EM support film could be completely specified by evaluating all orders of joint probabilities of the image optical density values. The probability of random fluctuations in the support image interfering with the identification of images of atoms at the vertices of an n‐sided polygon can be determined from the nth order statistics. This probability has been calculated as a function of the OD at specified points, for n= 1, 2 and 3. The first order statistics amount to the probability of finding a particular OD in the image, where the width of the distribution gives the standard deviation of the OD values, or contrast. The second order statistics or covariance (correlation) function provide an intuitive understanding of the granularity of the image. We have measured the first and second order statistics of carbon and aluminum oxide films imaged under a variety of conditions, and have found them useful in quantitative assessment and comparison of the images. The lower contrast of the aluminum oxide films is reflected in both the OD distribution and covariance function. The contrast of both types of films is found to be a non‐monotonic function of film thickness.
Direct electronic visualization of high‐voltage TEM images by sequential pixel acquisition
Authors: Fotino Mircea
Published: August 2011

A hybrid imaging mode is described that allows direct electronic detection and manipulation of TEM images. Such imaging mode should be of particular interest and usefulness in high‐voltage instruments, in which image visibility is notably poor, but it can also be adapted to conventional electron microscopes. Pixels are detected sequentially by electron‐optically sweeping the whole transmitted beam in a two‐dimensional raster past a defining aperture located below the camera. Pixel data are obtained directly in digital form and can thus be conveniently manipulated on‐line or stored for subsequent processing. Preliminary results obtained in one dimension confirm satisfactorily the anticipated characteristics of two‐dimensional images. Possible consequences, applications and extensions of this electronic image acquisition mode are discussed.
Visualization of the nucleoid in living bacteria on poly‐lysine coated surfaces by the immersion technique
Authors: Binnerts J. S. , Woldringh C. L. , Brakenhoff G. J.
Published: August 2011

A method for visualizing the nucleoid in living bacteria is described. The method involves immobilization of the cells by attachment to a poly‐l‐lysine coated slide and subsequent immersion in bovine serum albumin solutions of defined refractive index and osmolality. Dependence of the visibility of the nucleoid on the refractive index of the medium is investigated.
Thermally defined cryomicroscopy and some applications on human leucocytes
Authors: Scheiwe M. W. , Körber Ch.
Published: August 2011

A freezing‐stage has been developed for use on a standard light‐microscope, which can provide reproducible, precisely linear cooling and warming rates in the range from 0·1 to 10,000 K/min. Biological cells in aqueous solutions can be observed during the freeze‐thaw cycle; the volume loss due to osmotic efflux of water and the intracellular crystallization of water are detected by video‐monitoring. The temperature field generated in the observed samples is comparable to extended cylindrical probes and allows the transfer of cryomicroscopic data to technically used vial geometries. Lymphocytes and granulocytes were observed during freezing using the system described. They were separated and washed, and then frozen on the cold stage of the cryomicroscope at cooling rates ranging from 2 to 500 K/min. Shrinkage of the cells was observed up to 100 K/min and intracellular ice formation could be detected starting at 10 K/min. The results show that human leucocytes show excessive shrinkage up to 36% of their initial volume; the probability of intracellular ice formation exhibits a sharp increase from 10 to 100 K/min where nearly all cells contain ice.
Preparation of cryosections with a modified Sorvall MT2B ultramicrotome and cryoattachment
Authors: Biddlecombe W. H. , Jenkinson D. McEwan , McWilliams S. A. , Nicholson W. A. P. , Elder H. Y. , Dempster D. W.
Published: August 2011

The Sorvall MT2B ultramicrotome and cryoattachment were modified to extend the duration of cutting and to overcome problems in tissue preparation. Microtome and cryochamber were encased and surrounded by an atmosphere of dry nitrogen gas at room temperature. A specially designed cold block sited within the box close to the microtome enabled tissues to be trimmed at low temperatures in the dry environment. Tissues could be readily visualized both on the trimming block and in the chamber with the existing microscope modified to improve its working distance and lighting system.

During transfer from storage in liquid nitrogen, through the trimming procedure, to arrival in the cryochamber, the temperature of tissues as determined using thermocouples embedded in PVP on a stub, never exceeded 140 K. Although there was a considerable standing temperature gradient within the cryochamber, the cutting environment, specimen and edge of the knife were thermally stable. Sections could be routinely cut dry and freeze‐dried within the chamber, if desired.
Contribution of cryotechniques to the study of elastin ultrastructure
Authors: Fornieri C. , Ronchetti I. Pasquali , Edman A. C. , Sjöström M.
Published: August 2011

Cryomethods were used in order to investigate the ultrastructure of native elastin fibres from beef ligamentum nuchae. Filaments of diameter 5 nm, running almost in parallel in purified, negatively‐stained elastin preparations, were also seen running along the elastin fibre both in freeze‐fractured and etched elastin, that had been stretched up to 200%, and in cryo‐sectioned elastin that had been stretched and chemically fixed before freezing. Interconnections between elastin filaments were revealed by the freeze‐etching technique. Glycerol treatment, which probably leads to hydration of specimens, resulted, however, in disorganization of filaments and swelling of the elastin fibre. In conclusion, by the use of cryotechniques, it was convincingly demonstrated that elastin molecules are arranged in long interconnecting filaments of about 4–5 nm width.
Location of histological structure of the brain for subsequent monoamine fluorescence with the help of illumination by phase contrast condenser
Authors: Laszlo I.
Published: August 2011

Various histological structures from rat brain prepared by the formaldehyde induced fluorescence method can be visualized with the help of illumination in tungsten light by using phase contrast condenser (without phase contrast objective). The procedure was found useful for the location of areas to be studied by subsequent fluorescence microscopy in order to relate the monoamine fluorescence to the histological structure.
Quantitative analysis of particle sizes: estimation of the most efficient sampling scheme
Authors: Kiss Klara , Pease Robert N.
Published: August 2011

Analysis of variance based on a hierarchical experimental design was used to establish a method finding the most economical replication schemes in quantitative television microscopy of particulates. Two products of greatly different average particle diameters and width of distributions were investigated.

Error contributions were determined for each of three major steps in the procedure and smoothed curves were plotted of error v. particle size. Then cost and overall error were plotted for a variety of possible replication schemes, and a locus of best combinations drawn in.

The optimal replication curve depends somewhat on the nature of the sample; thus, it is beneficial to carry out a variance analysis whenever microscopic particle size analysis is planned for a large number of samples of a new product.
Cytochemical localization of biliproteins with silicotungstic acid
Authors: Dwarte D. M. , Vesk Maret
Published: August 2011

5% silicotungstic acid (STA) in 6·25% aqueous sodium sulphate has been used as an en bloc cytochemical stain for the localization of biliproteins in the Cryptophyceae. STA also stains a wide range of other proteinaceous structures including the ejectosomes, periplast, pyrenoid, nucleus, cisternae of Golgi and the mitochondrial matrix. The STA technique provides far greater contrast than that of the original phosphotungstic acid (PTA) stain developed by Silverman & Glick (1969a).
X‐ray microtomography
Authors: Elliott J. C. , Dover S. D.
Published: August 2011

A microscope system based on the principles of computerized axial tomography is described for determining the distribution of the X‐ray absorption coefficient in a slice from a solid object without cutting sections. An application is given to determining the distribution at a resolution of about 15 μm through a shell of about 0·5 mm diameter.
Freeze‐fracture electron microscopic and low temperature X‐ray scattering studies of the effect of cryofixation upon serum low density lipoprotein structure
Authors: Aggerbeck Lawrence P. , Gulik‐Krzywicki Tadeusz
Published: August 2011

We report here a correlated X‐ray diffraction and freeze‐fracture electron microscope study of the effects of several cryofixation procedures upon human serum low density lipoprotein (LDL2) structure. Only when the LDL2 solutions contained 75%, by weight, glycerol were the room temperature and post cryofixation low temperature LDL2 X‐ray scattering curves indistinguishable from one another. Other cryofixation procedures, slow or rapid, with or without glycerol, resulted in differences between the room temperature and low temperature LDL2 X‐ray scattering curves, in part due to the effect of quenching upon the solvent. Freezeetching electron microscopy of the slowly cryofixed LDL2 showed marked aggregation of the particles and an unusual morphological appearance. In contrast, after rapid cryofixation or cryofixation in the presence of glycerol, freeze‐etch electron microscopy revealed well‐isolated particles which had a knobby morphology.

The results demonstrate that under certain conditions (in the presence of 75% glycerol) cryofixation results in minimal, if any, structural alteration of, at least, the LDL2 lipid moiety. Further, this study underlines the more general conclusion that any high resolution structural study employing a cryofixation step must be interpreted with caution and the effect of cryofixation upon the sample structure need be evaluated by independent means.
Radiation damage to microdroplets at low temperature
Authors: Roinel Nicole , Malorey Pierre
Published: August 2011

The effect of low temperatures on the stability of characteristic signals during electron probe analysis of microdroplets was investigated. The kinetics of the characteristic Na, K and Cl signals from lyophilized droplets of either KCl or NaCl salts were observed for 1200 s in an electron microprobe whose specimen stage temperature was either 133 K or room temperature. Samples were irradiated with 15 keV electrons, at beam current densities ranging from 7 to 70 A m−2. Initial count rates were lower for the same samples at 133 K than at room temperature. At 133 K all the signals measured increased at the start and then, in some cases, decreased. Increases were more pronounced for measurements made at the lowest beam current density (i.e. the lowest electron dose), and for samples with the highest mass thickness; they were also more marked for KCl than for NaCl samples. At 133 K, measurements after 10 s irradiation showed no proportionality between X‐ray intensity and the concentration of the elements, and proportionality was only restored when droplets had been irradiated for some time and count rates became nearer to the values measured at room temperature. Whether or not these observations reveal the existence of a fundamental physical process or a technical failure is still unknown. In no case was characteristic signal stability improved by the use of a cold stage.
Quantitative analysis of intramembrane particle (IMP) distribution on biomembranes after freeze‐fracture preparation by a computer‐based technique
Authors: Niedermeyer W. , Wilke H.
Published: August 2011

An improved method for the quantitative analysis of intramembrane particles (IMPs) on freeze‐fracture faces of biomembranes is described. The analysis provides, first, the number of IMPs per 1 μm2, second, the coefficient of dispersion (CD) of unweighted and weighted (i.e. the finite size is taken into consideration) IMP distribution, and, third, the diameters of IMPs. Additional data for further statistical evaluation are also derived. These data make it possible to numerically describe and characterize fracture faces of a membrane in a defined physiological state. They allow for comparison of normal membranes with experimentally induced alterations of the same specimen in other experiments, and for observation of differences between normal and malignant cells. Furthermore, such an analysis provides the basis for a dynamic interpretation of biomembrane components seen on the static freeze‐fracture micrographs. With this numerical analysis, descriptive terms for IMP distribution such as ‘random’, ‘aggregated’ or ‘clustered’, which cannot be evaluated or verified, are avoided.

The method, based on the Poisson statistic which provides a means for the description of different particle density distributions, is described. The validity of the method is demonstrated on artificially generated pictures and by an analysis of erythrocyte membranes before and after spectrin depletion.
Quantification for the X‐ray microanalysis of cryosections
Authors: Hall T. A. , Gupta B. L.
Published: August 2011

Some problems of the quantitative analysis of diffusible elements in cryosections are reviewed. The two prevalent methods for obtaining concentrations from X‐ray data, one based on characteristic radiation alone and the other on continuum‐normalization, are recapitulated. Both methods seem suitable at cellular level while the latter seems preferable at finer spatial resolution. Recourse to both methods together is desirable in the analysis of frozen‐hydrated sections especially when there is no peripheral standard.

Selective local contamination is a particular hazard in the analysis of chlorine. In the case of sodium, physical parameters set restrictive limits to the minimum concentration measurable by ‘energy‐dispersive’ X‐ray spectrometry (about 20 mm kg−1) and to the spatial resolution attainable by diffractive X‐ray spectrometry (∼0·2 μm).

One obvious danger to meaningful quantitative analysis is inadvertent redistribution of diffusible elements during the moments preceding the freeze‐quenching of a tiny piece of tissue. Data are presented to show that concentration changes due to simple evaporation are a real hazard prior to the quenching of sub‐millimetre size samples.
The detection quantum efficiency of electronic image recording systems
Authors: Herrmann K.‐H. , Krahl D.
Published: August 2011

The principles in design and operation of TV electron‐image recording systems are elucidated. Using the ‘detection quantum efficiency’ (DQE) as a figure of merit, the influence of various parameters is discussed, such as single electron response, read‐out noise and saturation of the camera tubes as well as the properties of digital frame stores. The analogue signal processing mode gives a DQE of about 0·8 and can be adapted to a wide range of operating conditions. Counting of normalized electron signals in digital storages allows an almost ideal image detection, which can, however, at best only be applied with a current density below 10−13 A cm−2. Compared to photographic recording, electronic systems offer real‐time response, compatibility with computers and great flexibility of stored information. Their DQE is similar in the analogue mode but superior in the counting mode. The storage capacity, on the other hand—particularly the number of pixels—is presently still inferior to the photoplate.
Application of an array processor to image processing in electron microscopy
Authors: Pitt T. J.
Published: August 2011

It is now possible to obtain a number of so‐called array processors to attach to existing computer systems, to increase the computing power and speed available. In this paper, the application of one such array processor to image processing in electron microscopy is considered, and some of the practical experience thus gained is reported.
Software for scanning Auger microscopy
Authors: Prutton M. , Peacock D. C.
Published: August 2011

The procedures embodied in a program suite designed for use with a computer controlled digital scanning Auger electron microscope are elucidated. These include techniques for the collection and subsequent processing of spectra, linescans and images. Schemes found to be the most efficient for the normalization of data to correct for both incident beam current fluctuations and sample surface topography are explained. Averaging and economical smoothing algorithms are considered as a means of improving the signal‐to‐noise ratio when working with high spatial resolution (probe diameter less than 150 nm), which necessarily limits the available beam current to less than about 3 nA. Emphasis is laid upon the desirability of obtaining quantitative information and to this end software routines for use in ascertaining surface chemical composition from spectra are described. These include numerical differentiation of spectra and modelling and subtraction of the secondary electron background from beneath an Auger feature. The transfer of data to a faster computer and more lengthy processing operations, for improving the quality of Auger maps, are also discussed.
The correlation averaging of a regularly arranged bacterial cell envelope protein
Authors: Saxton W. O. , Baumeister W.
Published: August 2011

An adaptation of the ‘correlation averaging’ method is described which allows reliable and almost fully automatic image averaging in the case of near‐periodic structures notwithstanding the presence of substantial crystal imperfections; methods for assessing resolution and symmetry without reliance on crystallinity are also discussed. Electron micrographs of negatively stained and rotary shadowed preparations of the HPI‐layer protein from the cell envelope of Micrococcus radiodurans have been averaged using the method, and the projected structure is described to a resolution of about 1·9 nm.
An automatic focusing and astigmatism correction system for the SEM and CTEM
Authors: Erasmus S. J. , Smith K. C. A.
Published: August 2011

Focusing and correcting the astigmatism of an electron microscope can be time consuming and difficult to achieve accurately. An automatic method of performing these tasks using an on‐line computer system and a digital framestore has been developed. The method is applicable to noisy images in both scanning and conventional transmission electron microscopes. It is capable of setting focus and correcting astigmatism at least as accurately as can be achieved by an experienced operator.
An improved specimen table for the Balzers freeze etching system BAF 400
Authors: Wisse D. M. , Spies F.
Published: August 2011

A time saving, simple and inexpensive adaptation of a Balzers specimen table is described. This specimen table is used in combination with the small specimen holders originally developed for the Denton Freeze‐etching module. They are in particular suited for cell suspensions, and are easy to handle for freezing procedures. Ten replicas are produced routinely per freeze fracture run, with the aid of this combined system.
Observations on the real space computation of dynamical electron diffraction intensities
Authors: Self P. G.
Published: August 2011

The computational procedures and theoretical approximations used in the real space approach to dynamical electron diffraction calculations are discussed. Although the method is theoretically sound it is found to be unsuitable for rapid and accurate computation of electron wavefunctions.
A new technique for perfusion fixation and contrast enhancement of foetal lamb myocardium for electron microscopy
Authors: Dae Michael W. , Heymann Michael A. , Jones Albert L.
Published: August 2011

A technique of perfusion fixation following elective diastolic cardiac arrest was developed to obtain excellent preservation of foetal lamb hearts for morphological studies with electron microscopy. A technique was developed to markedly enhance the contrast of embedded material by treating thin sections with tannic acid prior to lead staining. This technique has been particularly useful for quantitative morphometric analysis.
On‐line image processing in high resolution electron microscopy
Authors: Boyes E. D. , Muggridge B. J. , Goringe M. J.
Published: August 2011

On‐line processing and analysis of high resolution electron microscope (HREM) images has been implemented using a software controlled image processor based on a commercial digital video framestore. It directly digitizes the output from the low light TV camera on the HREM and applies any processing necessary. The conditions under which images from amorphous specimens are being obtained can be established on‐line. The fast fourier transforms (FFTs) of 256 times 256 pixels produced in 15 s with the system off‐line are comparable to those from diffraction analysis of the same data on a laser optical bench. The precision of analysis of defocus, aberrations and astigmatism on‐line is discussed, together with possible approaches to routine analysis of crystalline specimens and for interactive control.
Resolution and contrast in the electron microscope: an historical review
Authors: Cosslett V. E.
Published: August 2011

The development of quantitative interpretation of electron micrographs, in respect of contrast as well as resolution, has followed similar lines to those in optical microscopy, though on a faster time scale. A thorough understanding of phase contrast came relatively late in both disciplines. The definition of resolution is more complicated in the case of the electron image on account of the severe effect of lens aberrations, especially spherical aberration. Experimental measurement of the performance of an electron microscope requires an operational definition of resolving power, which must include contrast considerations as well as the limitations imposed by lack of spatial and temporal coherence of the electron source. Agreement on suitable test procedures is now being reached, at least for a very thin specimen.
The contribution of electron microscopic cytochemistry to an understanding of the biogenesis of the endoplasmic reticulum of rat hepatocytes
Authors: Higgins Joan A.
Published: August 2011

The endoplasmic reticulum has the enzymic machinery for the synthesis of both protein and phospholipid and hence plays a central role in its own biogenesis and that of other cellular membranes. The evidence available concerning the biogenesis of the phospholipid bilayer of the endoplasmic reticulum, particularly from the application of fine structural cytochemical methods for the localization of acyltransferases, is reviewed. The observations are consistent with a model in which phospholipid is synthesized in situ at the site of membrane growth. Synthesis is asymmetric, with most enzymes located at the cytoplasmic side of the membrane, and controlled transmembrane movement of phospholipid results in an asymmetric bilayer.
Quantitative light and electron microscopical studies of the epithelial‐connective tissue junction in intraoral mucosae
Authors: Bale Elizabeth , White F. H.
Published: August 2011

The present study was designed to clarify the morphological nature of epithelial‐connective tissue attachment by quantitative comparison of palatal and buccal mucosae. Tissue samples were obtained from ferrets and, following a strict sampling regime, sections were obtained for quantitative light and electron microscopy. Electron micrographs from the epithelial‐connective tissue junction were subjected to analysis by stereological intersection counting. Quantitative results show that palatal epithelium has a comparatively convoluted interface, and is twice as thick as buccal epithelium. Stereological data are presented for relative surface area of basal plasma membrane occupied by hemidesmosomes, mean hemidesmosomal diameter and numerical density of hemidesmosomes per unit area of basal plasma membrane. Larger hemidesmosomes are found in the palatal epithelium. Results suggest that a prominent rete‐ridge pattern, as seen in the palatal epithelium, along with a greater specialized surface for adhesion, may be responsible for providing epithelial‐connective tissue stability in regions subjected to high mechanical stress.
Specificity of stain distribution in electron micrographs of protein molecules contrasted with uranyl acetate
Authors: Steven Alasdair C. , Navia Manuel A.
Published: August 2011

The mechanism of contrast enhancement of protein molecules by negative staining with uranyl acetate has been investigated by analysing electron micrographs of microcrystals of the human immunoglobulin Dob. Digitally filtered micrographs were compared systematically with idealized reference images which were constructed computationally, starting from knowledge of the primary sequence and three‐dimensional crystal structure of this IgG molecule. By separately modelling negative staining as bulk exclusion of heavy metals, and positive staining as the specific decoration of charged amino acid residues, and then combining these simulated images, we were able to assess quantitatively the amount of positive staining present in micrographs of ostensibly ‘negatively stained’ proteins. At a resolution of 2 nm, we find that the experimental images do indeed exhibit predominantly negative staining, the best matches being obtained by simulations which also include a minor contribution (10–40%) of positive staining. We have also compared two independent measures for the significant resolution present in images of periodic biological specimens: (i) the outermost visible orders of optical diffraction patterns, and (ii) the band‐limited resolutions of the idealized simulations when they most closely match the experimental images. These criteria observe close correspondence, thus vindicating the traditional practice of inferring resolution from the optical diffraction spectra of indirectly represented (stained) objects.
Electron scattering in ice and organic materials
Authors: Eusemann R. , Rose H. , Dubochet J.
Published: August 2011

Scattering cross‐sections for the interpretation of the scattering‐absorption contrast of microscopical images of amorphous biological specimens are calculated. The constituent atoms are treated as independent particles and their atomic potential is approximated by a modified Lenz‐Wentzel potential. Both elastic and inelastic scattering processes are considered. Formulas of the effective elastic and inelastic atom scattering cross‐sections are derived for both a CTEM with hollow‐cone illumination and a STEM using an annular detector. The scattering‐absorption contrast is plotted as a function of the atomic number for a CTEM applying parallel illumination and a STEM using a dark‐field detector with maximum collection efficiency. The presented results are valid for acceleration voltages U in the range 20 kV<U<150 kV. Employing these data the ‘angular mass thicknesses’ of representative constituents of biological materials are determined. The application of the listed data is demonstrated by means of a special example.
The evaluation of autofluorescence emission spectra derived from neuronal lipopigment
Authors: Dowson J. H.
Published: August 2011

A method for measuring the emission spectra from regions of neuronal lipopigment in tissue sections is described and illustrated. Each emission spectrum was derived from the means of six sets of readings, from either six regions of lipopigment from six neurones which were presumed to be from a homogeneous cell population, or from one region of one neurone. Characteristics of the emission spectra from lipopigment in various forms of neuronal ceroid‐lipofusinoses (NCLs) and in brains without evidence of NCL are presented and discussed. The results indicate that the classification of lipopigments should not be restricted to the two categories of ‘lipofuscin’ and ‘ceroid’.

This method may aid the identification of various pathogenic mechanisms in neurones, and provide another means of investigating the effects of certain drugs on cerebral function.
Low temperature techniques applied for CTEM and STEM analysis of cellular components at a molecular level
Authors: Sjöstrand Fritiof S.
Published: August 2011

One of the most important problems in tissue preparation for electron microscopic analysis at a molecular level involves the preservation of the tissue without introducing extensive denaturation of the proteins. Low temperature is a most efficient condition for the inhibition of protein denaturation and freeze‐drying offers favourable conditions for transferring proteins to a dry state with minimal denaturation of the proteins. However, the embedding of the dried tissue in a plastic leads to extensive denaturation of the proteins when performed in the conventional way. This eliminates very efficiently the advantages of the method. The situation becomes even worse when subjecting the tissue to freeze‐substitution. To eliminate as far as possible the denaturing effect of plastic embedding, freeze‐drying can be combined with low temperature embedding in a plastic.

Freeze‐fracturing allows a most efficient use of low temperature to reduce conformation changes in proteins. The value of the freeze‐fracturing technique depends entirely on a precise knowledge of the location of the fracture planes. Since this location is not known, it must be determined on the basis of a deduction. If this deduction is wrong, the method becomes misleading. Two methods which allow a certain testing of the correctness of the deduced location of the fracture planes are mentioned.
Imaging of outer segment periodicities in unstained cryoultramicrotomy sections of the frog retina
Authors: Kretzer Frank , Cohen Harold
Published: August 2011

Frog retinas were examined following cryoultramicrotomy. The extent of glutaraldehyde cross‐linking ranged from maximally 2% for 2 h to no cross‐linking. When the tissue was cryo‐protected, the effects of hypotonic, isotonic and hypertonic media on outer segment banding periodicities were studied. The specimen was cryosectioned at 193 K to 113 K with sections collected on a dry knife (213 K to 123 K). The sections were freeze‐dried and only rehydrated with phosphotungstic acid section stain when transmission electron microscopy was used to establish the extent of gross retinal morphology for each fixation protocol employed. As the extreme of no cross‐linking and no cryoprotection was approached and reached, conventional sections which displayed extensive retinal morphology were not obtained. Instead, fragments of retinal tissue resulted which showed periodicities of alternating light and dark bands. These periodicities are interpreted as photoreceptor outer segment discs with contrast formation depending upon an interplay between three factors: mass density differences between disc and interdisc space, section cutting angle, and section thickness.
A polychrome stain for conventionally fixed paraffin‐embedded woody plant tissues
Authors: Sam Sterling J.
Published: August 2011

A method for the differential staining of conventionally fixed paraffin‐embedded woody plant tissue is described. A five‐component stain combination is used with Bismarck Brown, included to avoid the stain variability, precipitation, and overstaining problems often encountered when using safranin.
Holographic methods in microscopy
Authors: Smith R. W.
Published: August 2011

Holography can be used to record, on a flat photographic plate, information about a three‐dimensional object. In conventional microscopy a thin slice of an object is observed in focus and recorded. By combining microscopy and holography it is possible to encode, on the same flat record, all the depth information in a three‐dimensional microscopic object, not just a single infocus section. Any section of the three‐dimensional object may be subsequently reconstructed and brought into focus by using a suitable viewing system to decode the hologram. Arrangements for doing this are described. It is shown that in order to achieve the highest resolution imagery of a three‐dimensional object, reversed wave reconstruction is necessary.

As holograms are made and reconstructed using a coherent laser light source, holographic microscopes are easily adapted for interferometry and an example of this is described. The differences between coherent and conventional imagery are briefly considered. The coherence of the illumination gives rise to the problems of coherent noise and speckle. Coherent noise is due to stray reflections in the optical system and can be reduced by using as few surfaces as possible or by using holographic lenses. A speckle reduction technique employing a new type of holographic optical element is described and its application to the stereomicroscopy of fossil ostracods considered.
Some improvements in the phase contrast microscope
Authors: Yamamoto K. , Taira A.
Published: August 2011

Some considerations on the improvement of the image quality of the phase contrast microscope are presented. The significance of glare and image fidelity discussed theoretically and the dependence of image fidelity and contrast on the phase plate characteristics are investigated on the basis of images calculated for different parameters of the annular phase plate. This paper also describes an anti‐reflection technique for the phase plate and demonstrates its effectiveness in the elimination of glare.
An evaluation of methods for preparing easily damaged cuticular surfaces of plants for scanning electron microscopy
Authors: Eveling D. W. , McCall R. D.
Published: August 2011

Techniques were tested to ascertain the best method for preparing the surfaces of delicate plant cuticles for microscopic examination at magnifications not exceeding 10,000. Using scanning electron microscopy, comparative examinations were made of cuticles of fresh material, ambient dried, freeze dried, critical point dried, and frozen material kept at low temperature. Micrographs were compared with material examined using light microscopy which acted as a control at low magnification. Cuticles of the leaves of runner bean, Coleus and the petals of Nicotiana, showed best surface preservation and least wrinkling when frozen, held at low temperature, and examined on a cryostage.
Vascular reactions to perfusion fixation
Authors: Thorball N. , Tranum‐Jensen J.
Published: August 2011

Vascular reactions to fixation with a 2·5% w/v glutaraldehyde fixative were studied in a constant flow perfusion system on isolated rabbit intestines and hind limb preparations. Reactions induced by the fixative were compared to reactions induced by noradrenaline in each single preparation. Changes in vascular resistance were assessed from continuous recordings of arterial and venous pressures and perfusion flow. Changes in vascular filtration and macro‐molecular permeability during fixation were approximated from recordings of flow and measurements of concentrations of dextran in the perfusate at the arterial inlet and at the venous outlet.

Our results indicate that the vascular bed of the preparations studied can be reliably fixed in a high resistance state of smooth muscle activity induced by noradrenaline as well as in a low resistance state in the absence of noradrenaline, provided that hydrodynamic parameters are strictly controlled. The vascular filtration decreases during fixation. In optimally perfused preparations 5–10 min are required to fully saturate the binding capacity of the tissue for glutaraldehyde.

Data on viscosity and colloid osmotic pressure of Tyrode solution containing dextran T‐70 in various concentrations, and of various aldehyde‐containing fixatives in 0·1 M sodium phosphate buffer with and without dextran added, are given.
Fractal analysis and stereological evaluation of microstructures
Authors: Wright K. , Karlsson B.
Published: August 2011

Fractal properties and the concept of fractal dimension has been studied. Emphasis is given to the applicability in structure analysis. Comparison between different measurement procedures, analyses of mathematically defined lines and surfaces as well as measurements on real surfaces have been performed. The stereological consequences have been considered.

A restrictive use of the fractal analysis results as an indicator of size, shape and self‐similarity is recommended. If results obtained by quantitative microscopy at different magnification‐resolution levels are to be compared, fractal analysis may be of advantage. The actual choice of resolution should yet be determined from the physical relevance of the geometrical details.
Extraction of membrane lipids during fixation, dehydration and embedding of Acholeplasma laidlawii‐cells for electron microscopy
Authors: Weibull C. , Christiansson A. , Carlemalm E.
Published: August 2011

The aim of the present investigation was to study the extent to which lipids are extracted from biological membranes during dehydration and embedding procedures carried out at high or low temperatures. Cells of Acholeplasma laidlawii were used as experimental material, since the lipids of this bacterium easily can be radioactively labelled without labelling the rest of the cell, and the lipids are almost entirely located in the cytoplasmic membrane. The cells were fixed at 277 K with glutaraldehyde, sequentially with this reagent and osmium tetroxide, or with glutaraldehyde, osmium tetroxide and uranyl acetate in that order. Loss of lipid during these procedures was negligible.

When cells fixed with glutaraldehyde and osmium tetroxide were dehydrated with ethanol at room temperature and embedded in Epon at 333 K, i.e. subjected to a conventional treatment, about 90% of the lipid content of the cells was extracted. The loss was reduced to c. 20% when treatment with uranyl acetate was included in the procedure and the non‐polar methacrylate resin Lowicryl HM20 was substituted for Epon. When cells fixed with glutaraldehyde and osmium tetroxide were dehydrated with ethanol at 238 K and embedded in Lowicryl HM20 at room temperature, practically no lipid was extracted. Substitution of the polar methacrylate‐acrylate resin Lowicryl K4M for Lowicryl HM20 resulted in the loss of about half of the lipid content of the cells. The use of ethanediol as dehydrating agent instead of ethanol did not diminish the extraction. Cells fixed solely with glutaraldehyde lost about half of their lipid content, even when both dehydration and embedding was performed at 238 K.

The lipid material extracted from glutaraldehyde‐fixed cells contained slightly more saturated fatty acids than that remaining in the cells. The reverse was true for osmium tetroxide‐fixed cells. With respect to lipid species, the extractions were generally rather unspecific.
High resolution and conventional electron microscopy studies of repeated wedge microtwins in monoclinic rare earth sesquioxides
Authors: Boulesteix C. , Yangui B. , Nihoul G. , Bourret A.
Published: August 2011

High resolution and conventional studies have been made on the inclined twin boundaries occurring near the tip of wedge microtwins in the case of repeated wedge microtwins occurring at twin intersections. These inclined boundaries appear to be continuous and all the atomic planes are also continuous so that we suggest the use of the term ‘inclined coherent boundaries’ instead of ‘incoherent’ which one generally employs, in analogy to the case of precipitates in a matrix. This is different from what occurs in the case of isolated wedge microtwins for the same material and for the same kind of twins where the inclined twin boundary is really not coherent, being made of non‐inclined coherent regions separated by steps containing a dislocation.

The coherent inclined boundaries of repeated wedge microtwins appear to be rotated about two perpendicular axes of the habit plane (tilt and twist) giving rise to a distribution of microdislocations whose role has been studied. The values of the tilt and twist angles are related to a minimization of the total energy by a mechanism that is likely to appear only in the case of thin foils. A comparative study of the different roles related to the different structures of inclined twin boundaries for isolated and repeated wedge microtwins has been made, and also a comparative study of the mechanisms of stress relaxation in each case. The studies have been made on (313) twins of a monoclinic samarium sesquioxide.
Coherency strain fields: magnitude and symmetry
Authors: Porter A. J. , Ecob R. C. , Ricks R. A.
Published: August 2011

This paper considers the various methods available for the measurement of small values of in‐situ lattice misfit between a coherent particle and the matrix. Examination of such misfits by Asby‐Brown contrast imaging, moiré fringe measurements, lattice fringe imaging and convergent beam electron diffraction (C.B.E.D.) is considered, and the results obtained by these techniques are compared. In addition the nature of the strain fields in the matrix adjacent to a coherent misfitting particle is examined using C.B.E.D.
Topographic quantification of non‐planar localized surfaces
Authors: Wright K. , Karlsson B.
Published: August 2011

The topography of non‐planar localized surfaces can be quantified by roughness indices as well as by height and angular distributions of the surface elements. For the roughness indices general relationships between the profile and spatial quantities including overlapping have been established. Some measurement methods are briefly considered. The profile analysis has proved to be a reliable, easy and fast way to characterize the topography of surfaces.
A routine flat embedding method for electron microscopy of microorganisms allowing selection and precisely orientated sectioning of single cells by light microscopy
Authors: Reymond Olivier L. , Pickett‐Heaps Jeremy D.
Published: August 2011

A simple method is described to embed material in resin, in the form of microscope slides, to observe it with high resolution light microscopy, to select, orient and section it for TEM. This method can be applied to many kinds of material but is particularly useful for the study of rare or tiny plant or animal microorganisms from field or culture. A diamond scriber, translucent hydrosoluble resin release agent, translucent and smooth resin stubs and a longitudinally perforated block‐holder for ultramicrotome are the specific tools of this method.
Calibration of transmission electron microscopic mass determination using objects with known mass distribution
Authors: Linders P. W. J. , Stols A. L. H. , Stadhouders A. M.
Published: August 2011

An improved calibration procedure is described for dry mass determination methods, that are based on microdensitometry of transmission electron micrographs. Polystyrene latex spheres, that are supposed to have a homogeneous mass distribution, are used for the calibration procedure. By computer fitting of the theoretically expected mass distribution to the distribution measured in a line through the centre of a sphere, a mass calibration factor is obtained. The number of measurements required for a predetermined calibration accuracy can be decreased considerably as compared to other calibration methods. Accurate calibration and reproducible adjustment of the electron optical magnification are required.
Electron trajectory‐linked phenomena in thin foil X‐ray microanalysis
Authors: Jones I. P. , Nicholls A. W.
Published: August 2011

The methods of calculating electron trajectories are reviewed. Approximate methods may be necessary because of computing time problems on small computers. The various situations in microanalysis where electron trajectories are important are discussed in turn. It appears that the single scattering theory of Goldstein et al. (1977) is useful for situations dominated by high scattering angle events whereas the Gaussian theory of Doig et al. (1981a) may be appropriate to multiple low angle scattering. This type of calculation might turn out to be unnecessary if coincident counting techniques are exploited.