bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chemical cryoprotection for structural studies
Authors: Skaer Helen 
Published: August 2011

SUMMARYWith the ascendency of techniques for ultrarapid cooling and the successful control of ice crystal damage by purely physical means, it has become necessary to make a case for the continued use of chemical antifreeze agents in any circumstances. These circumstances include the need to explore tissues deeper than those superficial layers, whose morphology can be preserved by ultrarapid cooling, the avoidance of superficial areas of damage inflicted by dissection and tissue slicing, and situations where the growth of ice crystals must be controlled throughout the specimen, as for example for the cutting of frozen sections.The control of ice crystal damage would ideally be by the avoidance of any freezing at all but in practice can be seen in terms of the control of ice crystal size, which in turn depends on the density of nuclei that develop in the specimen and the rate of accretion of water molecules to the crystallites that form on these nuclei. Chemical antifreeze agents act in a variety of ways to increase the density of nuclei and/or to reduce the rate of growth of ice crystals: by promoting nucleation, by enhancing subcooling and by increasing the viscosity of the extracellular medium. In this way, large numbers of small ice crystals are produced. The different types of cryofixative agent each have their own advocates, advantages, special applications and drawbacks and these must be taken into account when considering the range of methods available for the analysis of cryofixed material.
Cryoultramicrotomy: evidence against melting and the use of a low temperature cement for specimen orientation
Authors: Karp R. D. , Silcox J. C. , Somlyo A. V. 
Published: August 2011

SUMMARYA technique is presented showing the use of toluene as a low temperature cement to attach portions of frozen tissues in the desired orientation on to the cryoultramicrotome chuck. Examples of transverse sections of skeletal and vascular smooth muscle are illustrated. Electron probe analysis of these sections indicates that the distribution of elements is maintained.Evidence is presented against through section melting based on sectioning toluene at a temperature just below its melting point (178 K). Toluene was sectioned (about 100 nm thickness) successfully at three block temperatures (177, 163 and 113 K) when the knife temperature was by 1 degree or more colder than the m.p. of toluene. In all cases the toluene sections melted when the knife temperature reached 178 K. We conclude that sufficient heat is not generated and/or transferred to the toluene sections during cryosectioning under our conditions to raise the temperature of a 100 nm section by 1 K or more.
Concerning the nature of the cryosectioning process
Authors: Frederik P. M. , Busing W. M. , Persson A. 
Published: August 2011

SUMMARYIntentional 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

SUMMARYNegatively 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

SUMMARYDuring 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

SUMMARYThe 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

SUMMARYA 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

SUMMARYA 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

SUMMARYA 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

SUMMARYThe 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

SUMMARYCryomethods 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

SUMMARYVarious 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

SUMMARYAnalysis 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

SUMMARY5% 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

SUMMARYA 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

SUMMARYWe 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

SUMMARYThe 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

SUMMARYAn 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

SUMMARYSome 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

SUMMARYThe 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.
Software for scanning Auger microscopy
Authors: Prutton M. , Peacock D. C. 
Published: August 2011

SUMMARYThe 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.