bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative light microscopy of combined perfusion and freezing processes
Authors: Walcerz Douglas B. , Diller Kenneth R. 
Published: August 2011

SUMMARYThe rational design of cryopreservation protocols for living tissues demands an understanding of the mechanisms of mass transport between cells and their environment throughout the entire process. We have developed a new microscope stage to enable a specimen to be viewed continuously during a preservation protocol, including the addition and removal of cryoprotective additives and freezing and thawing. The specimen is contained in a sealed chamber having inlet and outlet ports for admitting and collecting perfusate solution, the entire volume of which may be exchanged with a time constant of 1–5 s, depending on the solution viscosity. The temperature of the active area of the stage is regulated by the standard techniques of convection cryo‐microscopy over a range in excess of 50 to — 100°C.A series of experiments has been performed on this system to measure the osmotic behaviour of rat pancreas islets during the addition and removal of dimethyl sulphoxide at temperatures between 25 and — 10°C. The technique involves mounting a single islet onto the low‐temperature stage so that it is constrained from lateral movement by a specially sized mesh. Both the system temperature and chemical composition are monitored and controlled simultaneously and independently; as a consequence, virtually any defined cryopreservation protocol may be imposed on the specimen. For making permeability measurements, the bathing medium of the specimen may be changed very rapidly to produce a defined osmotic stress. Alternatively, the specimen may be subcooled to a specific and fixed subzero temperature, at which point ice is nucleated in the extracellular medium, creating a near instantaneous change in composition. The temporal alteration in specimen size is monitored by video microscopy and quantified by computer vision analysis methods. One of several mass transfer models is fitted to the data to estimate the membrane permeability based on the assumption of either transport dominated by the movement of water or simultaneous coupled flows of water and cryoprotective agent.
Intracellular ice formation: cryomicroscopical observation and calorimetric measurement
Authors: Körber Christoph , Englich Sabine , Rau Günter 
Published: August 2011

SUMMARYThe formation of ice crystals within biological cells is generally deleterious and results in a severe loss of cellular viability and function. With the aim of circumventing this lethal event, the mechanisms of nucleation and their dependence on governing parameters such as temperature, cooling rate and solute and/or additive concentration, and the correlation with the osmotically induced water transport across the cell membrane were investigated. Quantitative low‐temperature light microscopy was used for this purpose as it offers the major advantage of studying the dynamics of the involved processes. To substantiate further the visual observations of the morphological changes associated with intracellular ice formation, supplementary studies by differential scanning calorimetry (DSC) were performed under comparable conditions to measure the quantity of water actually transformed into the crystalline state due to the evolution of latent heat.Human lymphocytes were used as a biological model cell. In particular it could be shown that the twitching type of intracellular ice formation which is evident but difficult to observe under the cryomicroscope can be attributed to a liquid‐solid phase change within the cells as determined by DSC. Good agreement was obtained between the results measured by both techniques with respect to the following dependencies of governing parameters: the fraction of cells exhibiting intracellular ice determined as a function of the cooling rate shows a sharp demarcation zone with an increase from 0 to 100% at about the same threshold cooling rate. On the other hand, the temperatures at which intracellular ice forms were found to be only weakly dependent on the cooling rate. With respect to the effect of cryo‐additive concentration at a fixed value of the cooling rate, the crystallization temperatures were seen to decrease with concentration. The DSC results may hence be regarded as a validation of the microscopic observations.
Early results using high‐resolution, low‐voltage, low‐temperature SEM
Authors: Pawley James B. , Walther Paul , Shih Shian‐Jiun , Malecki Marek 
Published: August 2011

SUMMARYRecent advances in the design of the scanning electron microscope (SEM) column, such as the coupling of a field‐emission gun to a low‐aberration immersion lens and the availability of a high‐stability cryo‐transfer stage, make low‐temperature, low‐voltage SEM (LTLVSEM) possible at very high resolution. We have used this combination to obtain results with uncoated biological specimens.The trichocyst from a Paramecium was used as a test specimen to observe the shrinkage of this structure as the temperature is raised from 170 K to room temperature following freeze‐drying. High‐magnification stereo images were obtained of trichocysts that had been prepared by freezing, freeze‐substitution and critical‐point drying and which were subsequently viewed by LTLVSEM to reduce beam damage and contamination.
Cryofixation methods for ion localization in cells by electron probe microanalysis: a review
Authors: Zierold Karl 
Published: August 2011

SUMMARYElectron 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

SUMMARYA 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

SUMMARYDue 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

SUMMARYWhen 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

SUMMARYA 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

SUMMARYA 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

SUMMARYA 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

SUMMARYThe 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

SUMMARYCa2+ 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

SUMMARYPotassium 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

SUMMARYThe 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

SUMMARYA 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

SUMMARYWe 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

SUMMARYThe 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

SUMMARYThe 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

SUMMARYUltrathin 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.