Electron microscopy of frozen water and aqueous solutions

Thin layers of pure water or aqueous solutions are frozen in the vitreous state or with the water phase in the form of hexagonal or cubic crystals, either by using a spray-freezing method or by spreading the liquid on alkylamine treated films. The specimens are observed in a conventional and in a scanning transmission electron microscope at temperatures down to 25 K. In general, the formation of crystals and segregation of solutes during freezing, devitrification and evaporation upon warming, take place as foreseen by previous X-ray, thermal, optical and electron microscopical studies. Electron beam damage appears in three forms. The devitrification of vitreous ice. The slow loss of material for the specimen at a rate of about one molecule of pure water for every sixty electrons. The bubbling in solutions of organic material for doses in the range of thousands of e nm^?2. We propose a possible model for the mechanism of beam damage in aqueous solutions. The structural and thermal properties of pure frozen water important for electron microscopy are summarized in an appendix.     

Antiseizure properties of aqueous solutions of compounds forming liquid crystalline structures

Amphiphilic compounds composed of a hydrophilic part and a hydrophobic alkyl chain were investigated. The compounds—sodium lauryl sulfate (SLS) and ethoxylated sodium lauryl sulfate (ESLS)—exhibit strong affinity for solid surfaces and form liquid crystalline structures in water. It is expected that they may become effective additives that could significantly modify antiseizure properties of water. They may extend the possibilities for applying water as an ecological base for lubricating substances. Rheological and structural X-ray studies as well as measurements of conductance and wetting angle were carried out. Their aim was to verify and validate the structures being formed in aqueous solutions of the compounds studied. Tribological studies were conducted to measure friction torque as a function of linearly increasing load by means of a four-ball apparatus. The tests were followed by measurement of the wear scar diameter on the balls. Based on the results obtained, antiwear properties were characterized by seizure and scuffing loads as well as by limiting pressure of seizure. The test results are highly surprising. The systems in which simple one-component aqueous solutions were the lubricating substance did not undergo seizure up to the maximum load of 8 kN designed for the tribological tester used. A number of typical antiwear additives in suitable oil bases do not exhibit such good characteristics. Also, the quantities characterizing seizure show relatively high values. Local maxima for the two compounds tested are formed in the area of low concentrations in the dependences of antiseizure properties as a function of concentration. It can be assumed that this is due to ordered structures being formed in the surface phase. The hexagonal phase of high viscosity of the order of several thousand Pa·s which forms in the bulk phase for ESLS and SLS solutions has no visible effect on antiseizure properties, whereas the existence of the lamellar phase for a 70% solution of ESLS affects an increase in the values being measured.     

Growth of ice crystals in frozen specimens

The formation of ice crystals, which might be the possible artefact in cryo-techniques for electron microscopy, was examined during the rewarming process of rapidly frozen erythrocytes. Intracellular ice formation, which is usually found in cells suspended in saline by rapid freezing, was inhibited by the addition of 30% glycerol. When such glycerinated cells, having no ice crystals at liquid nitrogen temperature, were rewarmed to higher temperatures above ? 80° C, recrystallization of ice occurred. Ice particles became visible within the cells even at ? 80°C and grew larger with a temperature rise. From the results obtained in the morphological and physiological investigations, it also became evident that the recrystallization of ice appeared prior to the increase in haemolysis during the rewarming process.     

The measurement of water distribution in frozen specimens

There are three techniques to measure local water fractions in the cryomicroscope. First, water content may be measured by a direct analysis of oxygen in bulk samples using a windowless detector. Secondly, mass thickness may be estimated in frozen-hydrated, then frozen-dried sections. This technique offers unrivalled spatial resolution, especially if the radiation dose in the frozen-hydrated state is kept low by the use of electron scattering techniques instead of an X-ray microanalytical background determination. External water content standards can be used instead of frozen-hydrated sections and the whole analysis can even be performed exclusively on frozen-dried sections at room temperature. Thirdly, local water fractions can be evaluated from X-ray microanalytical measurements of element concentrations per mass in the frozen-hydrated and frozen-dried state. Corrections necessary for the other techniques cancel out. However, the high radiation dose required for a fully quantitative analysis excludes the use of these methods in thin or ultrathin sections.     

Automatic analysis of aqueous specimens for phytoplankton structure recognition and population estimation

An automatic microscope image acquisition, evaluation, and recognition system was developed for the analysis of Uterm?hl plankton chambers in terms of taxonomic algae recognition. The system called PLASA (Plankton Structure Analysis) comprises (1) fully automatic archiving (optical fixation) of aqueous specimens as digital bright field and fluorescence images, (2) phytoplankton analysis and recognition, and (3) training facilities for new taxa. It enables characterization of aqueous specimens by their populations. The system is described in detail with emphasis on image analytical aspects. Plankton chambers are scanned by sizable grids, divers objective(s), and up to four fluorescence spectral bands. Acquisition positions are focused and digitized by a TV camera and archived on disk. The image data sets are evaluated by a large set of quantitative features. Automatic classifications for a number of organisms are developed and embedded in the program. Interactive programs for the design of training sets were additionally implemented. A long-term sampling period of 23 weeks from two ponds at two different locations each was performed to generate a reliable data set for training and testing purposes. These data were used to present this system's results for phytoplankton structure characterization. PLASA represents an automatic system, comprising all steps from specimen processing to algae identification up to species level and quantification.     

Microwave thawing of frozen parenteral solutions

A commercially available microwave oven modified for use at medication stations throughout hospitals allows timely thawing of frozen parenteral solutions. The inherent problems of safety and uniform heating have been overcome, thus making possible the preparation, storage, and distribution of admixtures on a regional basis and ensuring the integrity of the product. Most parenteral medications are not degraded by microwave energy, and thawing by microwave energy permits timely administration and allows coordination of medication for a series of patients.     

Transfer,observation and analysis of frozen hydrated specimens

Hitherto, the observation of frozen hydrated specimens in transmission electron microscopes has been inhibited due to the technical difficulties experienced in transferring the specimen to the microscope and maintaining it at a low temperature during observation. This has resulted in loss of the primary advantage of freezing since the frozen water had to be removed from the specimen before it could be introduced into the electron microscope. The cryo-transfer system overcomes these objections and provides a means to transfer frozen hydrated specimens from any preparation equipment into the microscope without ice condensation on the specimen. The cryo-transfer system consists of a cryo-transfer unit, a cryo-specimen holder and a temperature control unit.     

Digital image processing of crystalline specimens examined by electron microscopy

Crystalline specimens imaged in the electron microscope are analysed using digital processing. Some principles of structural analysis using the method of Fourier decomposition are discussed. Complementary techniques, such as enhancement by gradient and Laplacian operators, have been found useful in analysing electron micrographs. The application of these techniques to some problems in Materials Science and Biology are reviewed. By selecting and phase-correcting spots in the computed diffraction pattern, it was possible to localize atoms, molecules, and their defective arrangement in evaporated gold, sputter-deposited tungsten films, and single crystals of cadmium selenide. Digital processing based on the theory of helical diffraction was used to explore the three-dimensional arrangement of molecules in cellular components of alveolar soft part sarcoma, Hirano bodies, and neurofibrillar tangles in the human brain.     

Vibratory cavitation erosion in aqueous solutions

Vibratory cavitation erosion tests on AISI-SAE 1018 carbon steel in tap water and in mild (0.1 M) aqueous solutions of CaCO₃, CaO, NaHCO₃ and NaOH were conducted at a temperature of 80 °F (27 °C), a double amplitude of 1.38 × 10^?3 in (35.1 μm) and a pressure of 1 atm. For the maximum (150 min) test duration the weight loss in tap water (no additive) is the smallest. However, this is not the case for shorter test times. The biggest difference between weight losses among the various solutions is about 10% – 30%, which is somewhat beyond natural data scatter for such vibratory tests. Released gases and also particles may play an important role in the results.There are three easily distinguishable damage regions for all cavitated surfaces, i.e. generally undamaged rim, central heavily damaged region and transition region, as for most vibratory tests. The relative areas of the three regions are about 53.5%, 0.13% and 46.4% respectively for the present tests.The erosion rate and extent of the damaged regions do not depend substantially on the solute tested. The very small area of the heavily damaged central region is presumably due to the relatively low horn amplitude used in these tests. The increase in damage rate with respect to tap water is about 50% for the maximum test duration.Surface photographs and scanning electron microscopy photomicrographs (for a test duration of 150 min) are presented. Cracks, intercrystalline fractures and single-blow craters are most concentrated in the central region, as would be expected.     

Analysis of photographic emulsions for electron microscopy of two-dimensional crystalline specimens

Images recorded on photographic media are affected by several parameters which are characteristic of the particular emulsion used. The influence of the emulsion on the quality of the image of a two-dimensional crystalline specimen can, in particular, be predicted with a knowledge of the speed, the modulation transfer function, and the exposure dependence of the noise power spectrum of the emulsion. We have measured these parameters for several photographic emulsions used in electron microscopy, and use the results to predict signal-to-noise ratios in the Fourier transforms of images. No single emulsion provides the best results under all conditions. We present our data in a form which accounts for the interplay between the emulsion and the recording conditions, allowing optimization of the recording process.     

Freezing of aqueous specimens: an X-ray diffraction study

The effects on water of two cooling methods, immersion in a liquid cryogen and high-pressure freezing, were studied by X-ray cryodiffraction on different sucrose solutions. The nature of the ice formed by each method depends on both the sucrose concentration and the specimen thickness. In order to compare the two methods, we mainly studied specimens having a thickness of 0.2 mm. Under these conditions, freezing by immersion gives rise to hexagonal (IH), cubic (IC) and amorphous (IV) ices when the sucrose concentration (weight/weight) has a value within the range 0–30%, 30–60%, 60% and higher, respectively. The temperature of the phase transitions IV–IC, IC–IH depends on the sucrose concentration. High-pressure freezing gives rise to two specific forms of ice: an amorphous and a crystalline ice (ice III). Ice III is observed when pure water samples are high-pressure frozen provided that the sample temperature does not rise above −150 °C. Above this temperature, ice III transforms into hexagonal ice. Amorphous ice is formed when the sucrose concentration is higher than 20%. The amorphous ice formed under high pressure has a similar, but not identical, X-ray diffraction pattern to that of amorphous ice formed at atmospheric pressure. While the X-ray diffraction pattern of amorphous ice formed at atmospheric pressure (IV) shows a broad ring at a position corresponding to 0.37 nm, that of high-pressure amorphous ice (IVHP) shows a broader ring, located at 0.35 nm. IVHP presents a phase transition (IVHP–IV) at temperatures that depend on the sucrose concentration. We also observed that some precautions have to be taken in order to minimize the alcohol contamination of high-pressure frozen samples. The ice-phase diagram presented in this paper should be taken into account in all methods dedicated to the structural study of frozen biological specimens.     

Rubber friction in aqueous solutions containing ions

Lubricants ordinarily operate by keeping two sliding surfaces separated. The efficiency of a lubricant may be expressed by equations of hydrodynamics where the mechanical properties of the films of lubricants are adequately defined by the properties of bulk lubricant.In the case of water lubrication of black rubber at slow speeds and low pressures the lubricating ability of water is shown to be enhanced by addition of electrolytes to the water. It is thought that negative ions from the electrolytic solution collect on each sliding surface, repel each other and prevent the close approach of two sliding surfaces. Thus a thicker water film exists between the sliding surfaces than if the ion layers did not exist. The thicker film results in a reduced viscous drag force, lower than can be accounted for by conventional hydrodynamics.     

Computation of contrasts in atomic resolution electron spectroscopic images of planar defects in crystalline specimens

The image obtained in a conventional transmission electron microscope contains contributions from elastically and from inelastically scattered electrons. The electron spectroscopic imaging mode of an energy-filtering transmission electron microscope allows us to separate these two different contributions by inserting an energy-selecting slit in the energy-dispersive plane of an imaging energy filter. Selecting a specific energy loss corresponding to the ionization of the inner shell of a particular element one can obtain information on the distribution of the element within the specimen. The contrast is then caused by inelastically scattered electrons. For crystalline specimens, however, the contrast will be influenced additionally by the elastic contrast. This elastic contrast arises from electron diffraction and increases with increasing crystal thickness. Therefore the intensity distribution in the image cannot directly be interpreted as an elemental map. For a reliable interpretation of contrast formation in elemental maps it is therefore necessary to compute theoretical energy-loss images for various crystal thicknesses and compare these images with the experimental images. As an example we discuss the influence of electron diffraction effects on energy-loss images of two crystals with planar defects. Linescans are computed for various thicknesses of these crystals. Our calculations are performed using first-order perturbation theory to describe the transitions between the Bloch-wave states of the incident electron. The computed linescans for various crystal thicknesses show clearly that the influence of the elastic contrast on an image increases when we investigate thicker specimens. Furthermore, the comparison between elastic and energy-loss images demonstrates the partial preservation of the elastic contrast as a function of thickness. We find that for specimens thicker than about one third of the extinction length (here approximately 80-100 A) it is impossible to interpret an energy-loss image directly as elemental map.     

A simple technique for examining frozen hydrated specimens in the scanning electron microscope.

The use of a wide angle backscattered electron detector in a scanning electron microscope, which has the capability of the specimen chamber pressure being controlled independently of the column pressure, provides a simple technique for examining frozen hydrated specimens. Large specimens have been examined within 1 min of being placed on the stub and have been examined for many hours without charging artefacts or distortion due to dehydration.     

Instrumentation and specimen preparation for electron beam X-ray microanalysis of frozen hydrated bulk specimens

A cooling chain for the handling of frozen hydrated bulk specimens is described. Use of this method permits the specimen to be kept fully hydrated. After quench freezing, the specimen is transferred to a freeze etch apparatus, freeze fractured, carbon coated and transferred onto the precooled cold stage of the SEM by means of an airlock. The specimen is examined in the secondary electron mode and analysed using an energy dispersive X-ray analyser. The midgut of Chironomus thummi larvae and frog skin epithelium were used to test the performance.     

High-resolution low-temperature scanning electron microscopy for observing intracellular structures of quick frozen biological specimens

Intracellular structures of rapidly frozen biological tissues were observed in 3-D under a low-temperature scanning electron microscope using a newly developed side-entry type cryo-holder. The present low-temperature SEM is simple, easy to operate and effective for observing biological materials at high magnification. Biological tissues (the pancreas, small intestine, brown adipose tissue and Harderian gland) freshly removed from the mouse were immediately frozen in liquid propane cooled with liquid nitrogen, and their surfaces were manually fractured using a precooled razor blade in liquid nitrogen before introducing the cryo-holder into the SEM. When intracellular structures were revealed after appropriate sublimation, the specimens were coated with gold using a metal evaporator fitted to the side of the microscope column at one of the specimen chamber ports. The cryo-holder was connected to a copper braid coming from a liquid nitrogen reservoir to maintain a low temperature. Using this method, intracellular structures such as the mitochondria and endoplasmic reticulum were demonstrated at high magnifications. Ribosomal granules were discerned on the rough endoplasmic reticulum of the pancreatic acinar cells. Granular substances, presumably elementary particles, were also recognized on the mitochondrial cristae of the brown adipose tissue. The method was particularly effective for studying the 3-D configuration of lipid droplets which had been difficult to preserve by chemical fixation.