Patrick Echlin

Advanced Scanning Electron Microscopy and X-Ray Microanalysis

1. Modeling Electron Beam-Specimen Interactions.- 2. SEM Microcharacterization of Semiconductors.- 3. Electron Channeling Contrast in the SEM.- 4. Magnetic Contrast in the SEM.- 5. Computer-Aided Imaging and Interpretation.- 6. Alternative Microanalytical Techniques.- 7. Specimen Coating.- 8. Advances in Specimen Preparation for Biological SEM.- 9. Cryomicroscopy.- References.

Polymer cryoprotectants in the preservation of biological ultrastructure. I. Low temperature states of aqueous solutions of hydrophilic polymers.

The solid states formed by vitrified and frozen aqueous solutions of some hydrophilic polymers, able to act as biological cryoproteetants, have been studied by differential scanning calorimetry and freeze fracture electron microscopy. Glass transitions, devitrification, recrystallization and melting behaviour of aqueous solutions of polyvinylpyrrolidone, hydroxyethyl starch and dextran have been established. The vitrified polymer solutions exhibit a characteristic microspherical morphology which is not induced by the quench cooling process but is an inherent feature of the solutions themselves.The solid states formed by vitrified and frozen aqueous solutions of some hydrophilic polymers, able to act as biological cryoprotectants, have been studied by differential scanning calorimetry and freeze fracture electron microscopy. Glass transitions, devitrification, recrystallization and melting behaviour of aqueous solutions of polyvinylpyrrolidone, hydroxyethyl starch and dextran have been established. The vitrified polymer solutions exhibit a characteristic microspheral morphology which is not induced by the quench cooling process but is an inherent feature of the solutions themselves.

Freeze Substitution and Low-Temperature Embedding

Although freeze substitution and low-temperature embedding are two distinct processes, they are considered together because they complement each other and are frequently used sequentially during sample preparation. Freeze substitution is a chemical dehydration process in which ice in frozen-hydrated specimens is removed and replaced by an organic solvent. This procedure is in sharp contrast to the process of freeze-drying discussed in the previous chapter, where the water is removed by a purely physical process and is not replaced. Low-temperature embedding seeks to replace either the spaces once occupied by water in a freeze-dried sample or the organic fluid that has been used during freeze substitution, by infiltrating the sample at low temperatures with resins, which are, in turn, polymerized at low temperatures. The temperatures at which these processes are carried out are critical. If it is too high, the rate of ice recrystallization will damage the sample. If it is too low, the organic solutions become so viscous that they will not adequately penetrate the specimen. The compromise is to use lower temperature (193–173 K) for substitution and somewhat higher temperatures (253–238 K) for embedding.*

Polymeric cryoproteetants in the preservation of biological ultrastructure

A study has been made of the physiological effects of three non‐penetrating polymeric cryoprotective agents on sixteen different plant and animal cells and tissues. The cryoproteetants, when used at concentrations at which they are effective in preventing ice‐crystal formation, generally have a lower toxicity to cells and tissue than similar concentrations of glycerol. The relatively low toxicity of these substances suggests that they would be more suitable as cryoproteetants for morphological and analytical studies than the commonly used low molecular weight compounds.

SEM Microcharacterization of Semiconductors

The electronics industry is now one of the principal users of the scanning electron microscope, with as many as half of all new SEMs being bought, directly or indirectly, for semiconductor applications. There are several reasons for this popularity. First, the SEM provides a variety of contrast modes which are of great value in qualitatively and quantitatively assessing the properties of semiconductor materials. Second, the SEM offers modes which allow the operation of devices such as switches, transistors, and even complete integrated circuits, to be observed under conditions which approximate those of normal use. As the size of devices has been reduced to the micrometer scale, and as devices themselves become more complex, the fact that the SEM can combine imaging and chemical microanalysis with such facilities as the ability to identify electrically active defects, or measure voltages, makes it in many cases the most versatile tool for characterization, diagnosis, and failure analysis. The major techniques in current use for semiconductor studies are discused below, but other modes of operation including electron channeling and x-ray microanalysis are also of value and the chapters dealing with these topics should be consulted as well.

The preparation, examination and analysis of frozen hydrated tissue sections by scanning transmission electron microscopy and x-ray microanalysis.

A method is reported for preparing, examining and analysing frozen hydrated tissue sections using transmission electron microscopy and X‐ray microanalysis. Use of this method permits localization and measurement of water soluble or diffusible elements within the hydrated cell matrix. Since any change in total fresh weight of the specimen will affect the concentration of all components, great care has been taken to demonstrate that the mass neither increases nor decreases and to ensure that the tissue remains frozen‐hydrated. Criteria for assessing whether or not the tissue remains frozen‐hydrated are reported.

Low temperature scanning electron microscopy: a review.

Low temperature scanning electron microscopy is useful for morphological and analytical studies both in situations where low temperature techniques are used during specimen preparation and where low temperature stages are used for specimen examination and analysis. Examples are given of different low temperature specimen preparation techniques and how they may be applied to different types of specimen. There are still a number of problems associated with morphological identification in fully frozen‐hydrated samples and it is important to carry out parallel studies using more conventional transmission electron microscopy and light microscopy preparation techniques. A number of criteria are presented, some or all of which may be used to establish the existence of the frozen‐hydrated state.

The preparation and X‐ray microanalysis of bulk frozen hydrated vacuolate plant tissue

A series of modifications have been devised which allow the peak to background ratio X‐ray analytical method to be used more effectively to measure elemental concentrations in large vacuolate plant cells. Planar, frozen‐hydrated fracture faces of bulk plant tissue are coated with a thin film of evaporated chromium, which prevents surface charging. Provided the film is sufficiently thin, c. 5–10 nm, there is no attenuation of the electron beam and only a small absorption of soft X‐rays. The chromium makes a small but measurable contribution to the spectral background and suitable corrections may be made to the quantitative results. An improved back‐scattered imaging system is described, which helps to overcome the problem of spurious X‐ray signals from rough surfaces. The microscope column has been modified to permit a continuous readout of beam current, sensu stricta, during X‐ray microanalysis and to allow rapid exchange of the electron gun assembly during low temperature operation. Calculations are given relating the size of the X‐ray interactive volume to electron penetration and X‐ray emission in both frozen hydrated and frozen dried cells. The problems of X‐ray microanalysis are discussed in relation to the highly vacuolate cells found in most mature plant tissues and an example given of the distribution of four major cations in tobacco leaves.

Low-temperature X-ray microanalysis of the differentiating vascular tissue in root tips of Lemna minor L

The fracture faces of bulk‐frozen tissue offer a number of advantages for the analysis of diffusible elements. They are easy to prepare, remain uncontaminated, and, unlike most frozen‐hydrated sections, can be shown to exist in a fully hydrated state throughout examination and analysis. Root tips of Lemna minor briefly treated with a polymeric cryoprotectant are quench frozen in melting nitrogen. Fractures are prepared using the AMRAY Biochamber, lightly etched if necessary to reveal surface detail and carbon coated while maintaining the specimen at 110 K. The frozen‐hydrated fracture faces are analysed at 110 K using the P/B ratio method which is less sensitive to changes in surface geometry and variations in beam current. The method has been used to investigate the distribution of seven elements (Na+, Mg++, P, S, Cl−, K+ and Ca++) in the developing vascular tissue of the root tip. The microprobe can measure relative elemental ratios at the cellular level and the results from this present study reveal important variations in different parts of the root. The younger, more actively dividing cells, appear to have a slightly higher concentration of diffusible ions in comparison to the somewhat older tissues which have begun to differentiate into what are presumed to be functional vascular elements.

The biology of Glaucocystis nostochinearum

A study has been made of the morphology and fine structure of young and old cells of the apoplastidic alga Glaucocystis nostochinearum Itzigsohn which contains endophytic blue-green algae. Experiments have been described which indicate that the blue-green algae form a symbiotic association with the host algae, acting as ‘chloroplasts’. The findings have been discussed in the light of recent work on this alga, particularly in relation to the vexing question of the taxonomic position of the organism.