Electron microscopy of frozen biological suspensions

The methodology for preparing specimens in the frozen, hydrated state has been assessed using crystals and T4 bacteriophages. The methods have also been demonstrated with lambda bacteriophages, purple membrane of Halobacterium halobium and fibres of DNA. For particles dispersed in an aqueous environment, it is shown that optimum structural preservation is obtained from a thin, quench-frozen film with the bulk aqueous medium in the vitreous state. Crystallization of the bulk water may result in solute segregation and expulsion of the specimen from the film. Contrast measurements can be used to follow directly the state of hydration of a specimen during transition from the fully hydrated to the freeze-dried state and permit direct measurement of the water content of the specimen. By changing the concentration and composition of the aqueous medium the contrast of particles in a vitreous film can be controlled and any state of negative, positive or zero contrast may be obtained. At 100 K, frozen-hydrated, freeze-dried or sugar embedded crystals can withstand a three- to four-fold increase in electron exposure for the same damage when compared with similar sugar-embedded or freeze-dried samples at room temperature.     

Electron diffraction on frozen hydrated polysaccharides

Electron diffraction data were obtained on three different frozen hydrated polysaccharide specimens: pustulan single crystals, algal cell wall fragments consisting of β, 1–3 xylan, and fungal microfibrils of β,1–3 glucan. For pustulan, the diffraction pattern yields two of the crystalline lattice parameters of this polymer: a = 2·44 nm and b= 1·77nm. The results with β,1–3 xylan and β,1–3 glucan are identical to already known X-ray data, but also provide information on the molecular orientation at the micrometre level. In all three cases the hydrated electron diffraction diagrams are compared with the corresponding diagrams of the dried specimen.     

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.     

Immersion fixation of rapidly frozen,untreated tissues and suspensions of cells including spermatozoa

Small pieces of tissue, and cell suspensions in plastic artificial insemination (AI) ‘straws’, were frozen rapidly in Freon-12 at — 155°C, without pre-treatment. Peripheral fragments were thawed directly in 2% glutaraldehyde at 0°C, and processed for transmission electron microscopy (TEM) studies. Preservation of ultrastructure was satisfactory, and freezing artifacts were minimal.     

Low-temperature scanning electron microscopy of frozen hydrated mouse lung

A method is presented by which water is preserved as ice during examination of the lung in the scanning electron microscope (SEM). The lung need only be inflated, frozen, transferred to the microscope and examined with the electron beam. Chemical fixation, solvent dehydration, and drying are not necessary. The low-temperature SEM of Pawley and Norton [11] maintains lung at ?180° C, nearly liquid nitrogen temperature, for extended periods with a Joule-Thomson refrigerator built into the stage. It has an integral high-vacuum preparation chamber attached to the microscope column which allows serial fracture, low-magnification stereo light microscopy, radiant etching, and evaporative coating with gold or carbon. The stage can be tilted from 0° to 45° and rotated a full 360°. It is demonstrated that the air-liquid interface in the lung can be examined and that low-temperature SEM can be used to investigate the shape of alveoli, the patency of the pores of Kohn in the hydrated state, and the shrinkage and distortion of lung with drying.     

Quantitative analysis of intramembranous particles in rapidly frozen 10T1/2 cell monolayers

A simple method for ultrarapid freezing of cell cultures in monolayers was developed. Unfixed and unglycerinated cells were grown on glass substrates. No special treatments of the glass or cells were necessary to facilitate freeze-fracture along the upper plasma membranes. A reliable nonbiased method was developed to detect intramembranous particles (IMP) from the background by totally automatic means using the Cambridge Instruments Quantimet 920 Image Analysis system. Size and density data of IMP from a large number of electron micrographs can be rapidly and objectively quantitated. The automatic determination of locational coordinates for each IMP enables subtle determination of spatial distributional differences by the nearest neighbour function and the differential density distribution function, which are measurements of randomness. Quantitative analysis of the IMP distribution on the fracture face of C3H/10T1/2 mouse embryo fibroblasts upon various drug treatments was demonstrated.     

Electron microscope autoradiography of a diffusible intracellular constituent, using freeze-dried frozen sections of mammalian intestinal epithelium.

Autoradiograms were prepared from freeze-dried frozen sections of rat intestinal epithelium, previously incubated in vitro with 3H-galactose. Although the sections showed evidence of disruption by ice-crystal growth during freezing, they retained sufficient recognizable ultrastructure to facilitate the identification of several subcellular compartments. Statistical analysis of the grain distribution indicated that a significant non-uniform distribution of the labelled galactose occurs in the absorptive cells during transport, and is maintained in the sections throughout the various stages of autoradiography.     

Electron microscopy of frozen biological objects: a study using cryosectioning and cryosubstitution

Freezing of bulk biological objects was investigated by X-ray cryodiffraction. Freezing at atmospheric pressure of most microscopic biological samples gives rise to large hexagonal crystals and leads to poor structural preservation of these specimens. High-pressure freezing induces the formation of different ices (hexagonal, cubic and a high-pressure form) consisting of crystals having sizes smaller than those formed at atmospheric pressure. With both freezing methods, a cryoprotectant has to be added to the biological object to avoid the formation of ice crystals. However, special cases can be encountered: some biological objects contain large amounts of natural cryoprotectant or have a low water content. In these cases, vitrification can be achieved, especially using high-pressure freezing. Cryo-sectioning can be performed on vitrified samples, and the sections studied by electron cryomicroscopy. Images and electron diffraction patterns having a resolution better than 2 and 0.2 nm, respectively, can be obtained with such sections. Because samples containing crystalline ices cannot be cryosectioned, their structure has to be studied using cryosubstitution and resin embedding. We show that bacteria, yeast, and ciliate and marine worm elytrum have cellular compartments with an organization that has not been described by classical techniques relying on chemical fixation of the tissues. A high-pressure artefact affecting the Paramecium trichocysts is described. Such artefacts are not general; for example, we show that 70% of high-pressure frozen yeast cells survive successive high-pressure freezing and thawing steps.     

Electron spectroscopic study (ESI,EELS) of Nanoplast-embedded mammalian lung

The potential of Nanoplast melamine resin embedding for the study of mammalian lung parenchyma was examined by means of electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). Samples were either fixed with glutaralde-hyde-paraformaldehyde or glutaraldehyde-tannic acid, or were directly transferred to the embedding medium without prior fixation. Organic dehydrants, as well as fixatives containing heavy metals and stains, were omitted. A very high level of ultrastructural detail of chromatin, ribosomes, mitochondria and plasma membranes was achieved by ESI from the Nanoplast-embedded samples. The most prominent gain in ultrastructural detail was achieved when moving from an energy loss just below the L_2,3 edge of phosphorus at 132 eV to an energy loss just beyond this edge. This reflects the prominent P L_2,3 edge observed by EELS of Nanoplast-embedded samples in comparison with conventionally processed samples. Thus, taking into account possible sectioning artefacts, excellent heterochromatin images which rely on the phosphorus distribution can be obtained from Nanoplast-embedded samples by computer-assisted analysis of electron spectroscopic images. In this respect glutaraldehyde-paraformaldehyde fixation is preferable to glutaraldehyde-tannic acid fixation because the presence of silicon, revealed by EELS, in tannic-acid-fixed samples may introduce artefacts in phosphorus distribution images obtained by the three-window method because of the close proximity of the L_2,3 edges of silicon and phosphorus.     

Ultrastructure of red cells frozen with hydroxyethyl starch.

The freeze fracture appearance of red cells frozen in the presence of varying concentrations of hydroxyethyl starch (HES) is described. A technique is used which allows examination of a small portion of cells from a larger unit. The frozen cells appear distorted probably as a result of osmotic dehydration but indicate no evidence of intracellular ice. The frozen mixture with HES has three phases--a particulate phase consisting of the concentrated HES (and other salts), a sculptured ice phase and the red cells. When the concentration of HES is increased, the particulate phase becomes more prominent and at 14% HES appears to surround nearly all cells. In cells frozen in saline alone and 4% HES, the cytoplasm in a majority of cells has numerous cavities and depressions. Since such units haemolyse badly when thawed, it is possible that these regions indicate structural damage. In contrast, those units frozen with 14% HES (in which nearly 85% of the cells survive freeze-thaw) possess cells which only infrequently have such regions in the cytoplasm.     

Digital photogrammetric quantification of surface area and volume on scanning electron micrographs of frozen hydrated lung tissue

A digital video plotter (DVP, Leica), the personal computer equivalent of an analytical plotter, was used to measure the coordinates of points chosen from stereo pair images of the surface of frozen hydrated lung imaged at magnifications of 2000 and 5000x with a low-temperature scanning electron microscope (SEM). Rat lung tissue was frozen in vivo with a liquid nitrogen cryoprobe under carefully controlled physiologic conditions. At slow freezing rates, water in the aqueous layer at the surface of the lung segregates into ice crystals (dendrites) which branch in the plane of the surface. Coordinates of points on dendrite surfaces were measured by the DVP and passed to TERRAMODEL (Plus III Software, a land modeling program) where they were used to generate a three-dimensional model, from which surface area and planimetric area of the lung surface were calculated. Additional measurements were made at the top and bottom of the ice structures and a Basic language program was written to calculate the volume of ice on the lung surface. Digital photogrammetry coupled with low-temperature SEM of frozen samples allows measurement of water and water-containing microstructures ubiquitous in biology.     

快速烧结NdFeB的研究

利用放电等离子烧结系统（SPS）对烧结NdFeB进行了研究。试验表明，比冷等静压（CIP-S）保温时间仅为其1／60烧结温度低180℃时烧结的NdFeB密度接近了理论密度7.6g／cm^3，高于CIP-S达到的密度（7.5glcm^3），从断口形貌上看，900℃下已经达到致密；NdFeB在800℃下经SPS烧结后的相组成与原粉末相近，而在900℃烧结后两元素相组成明显减少，三元素相组成显著增多，如Nd2Fe14B，Fe20Nd3，Nd4，Fe4B4，其中Nd2Fe14B成为主要相。     

Electron probe x-ray microanalysis of frozen-hydrated biological specimens.

A technique is described for preparing frozen-hydrated bulk samples of biological specimens for electron probe X-ray microanalysis. The method allows reproducible quantitative analyses to be made. Specimens are rapidly frozen, transferred to a vacuum evaporator, fractured under high vacuum at - 180 degrees C and coated with 20 nm of chromium. Transferal to the cryostage of a scanning electron microscope is accomplished without exposure to the atmosphere and without the specimen temperature rising above -120 degrees C. Analyses are made at a temperature of -145 degrees C. Contamination by frost does not occur. Etching and charging of the specimen are eliminated. Specimen charging is shown to be related to temperature. It can be eliminated at low temperature by coating with carbon, aluminium or chromium but consistent elimination could only be achieved with chromium. The chromium coat does not appear to have an absorption effect on quantitative analysis.     

Structure and function of frozen cells: freezing patterns and post-thaw survival.

Freezing patterns and post-thaw survival of cells varies with different cooling rates. The optimal cooling rates, indicating the highest percentage survival, were different in yeast and red blood cells. A difference of freezing patterns was also noticed in preparations frozen above and below the optimal cooling rate for each cell, namely, cell shrinkage at lower rates and intracellular ice formation at higher rates which showed similar trends in both the cells, even though there was some shifting of the optimum. Ultra-rapid freezing and addition of cryoprotectants are useful ways to minimize ice crystal formation and to cause such ice formations to approach the vitreous state. Ice crystals are hardly detectable in yeast cells as well as in erythrocytes, when these cells are frozen ultra-rapidly in the presence of cryoprotective agents in moderate concentration.     

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.