Scanning electron and light microscope correlation of individual human bone marrow cells before and after culture in nutrient agar.

This study was undertaken with the aim of identifying the different cell types found in human bone marrow by examining their surface morphology. In an attempt to obtain a homogeneous cell population, cells were both fractionated by discontinuous albumin density gradient centrifugation (DADGC) and selectively grown in nutrient agar. Both cell preparations underwent the critical point drying technique before examination under both the scanning electron microscope (SEM) and subsequently the light microscope (LM). When the SEM image of individual cells was compared with the corresponding LM image, it was not easy to identify the different cell types, because of the shrinkage and distortion that occurred during their preparation. The shrinkage observed under the SEM amounted to a 45% reduction in mean cell diameter. This shrinkage was confirmed by comparing the SEM and LM images of the same cell. Although shrinkage occurred throughout the dehydration sequence, critical point drying was responsible for a 25% reduction in mean cell diameter. Furthermore, direct observation under LM of fixed cells drying in air from ethanol, revealed visible contraction of the cell and distortion of the cell membrane. We assume that a similar morphological change occurred during critical point drying. We conclude that the shrinkage and distortion, caused by the dehydration process involved in SEM preparation, severely limit the value of a study of surface morphology by SEM in the identification of the different cell types found in human bone marrow.     

Cell and organelle shrinkage during preparation for scanning electron microscopy: effects of fixation, dehydration and critical point drying.

The critical point drying method of preparing samples for scanning electron microscopy is associated with a variable amount of specimen shrinkage. We studied the causes of this phenomenon is isolated mouse hepatocyte nuclei and in human erythrocytes and found that the critical point drying process itself caused most of the shrinkage that we observed (a 25-30% reduction in diameter in both specimens). Glutaraldehyde fixation and ethanol dehydration caused only minimal size reduction, prior to critical point drying. Substitution of an inert (ethylene glycol-ethylene glycol monethyl ether) dehydration technique did not alter the final result. Previous studies in our laboratory using high resolution SEM and correlative transmission microscopy of isolated nuclei have demonstrated that the shrinkage represents a miniaturization of the organelles in which all structural components retain their usual relationships.     

Volume changes during preparation of mouse embryonic tissue for scanning electron microscopy

This paper describes the results of experiments in which the volume changes in mouse embryo limb samples were followed more or less continuously after fixation through dehydration and critical point drying, with in some instances data relating to post critical point drying shrinkage. 14 and 15 day p. c. mouse embryos were fixed in 3 % glutaraldehyde in cacodylate buffer and stored in this fixative until use. Single specimens were studied using a Quantimet image analysing computer to record the changes in projected area of the unmounted specimens as they were passed through the usual series of reagents according to various commonly used dehydration schedules. The area changes were converted to volume changes for the purposes of presentation in this paper. The Quantimet system could not be used to follow volume changes in the CPD bomb so that most experiments detail the volume in the intermediate fluid before CPD and the size of the specimen immediately after it was removed from the CPD bomb. A few experiments were conducted in which the specimens were measured whilst they were in the CPD bomb. The measurements relating to dehydration and CPD procedures were compared with measurements of air dried and freeze dried specimens. All three drying methods cause considerable shrinkage: freeze drying to 85 % of the glutaraldehyde fixed tissue volume; critical point drying to 41% (after 24 h); and air drying from a volatile solvent to about 18% of the fixed tissue volume. Air drying from water caused a shrinkage to about 12% of the original volume. There was no significant difference between the various commonly used CPD schedules or between GA only and GA + Os O₄ fixed tissue. CPD via cellosolve and CO₂ caused substantially more shrinkage than other methods. Dimensional changes during specimen preparation are probably associated with changes in shape and in relative relationships between organelles, cells and tissues having different compositions. This should be borne in mind by all those interpreting scanning electron micrographs of dried animal soft tissue specimens.     

A quantitative study of the preparation of rabbit aortic endothelial cells for scanning electron microscopy

Quantitative studies were done with the scanning electron microscope (SEM) on aortic endothelial cells from ten rabbits. Of these, five were plastic casts and five were dehydrated with three different, but standard, techniques. The results indicated that all forms of dehydration caused significant shrinkage artefacts and that these were different in different directions in both the thoracic and abdominal aorta. The greatest shrinkage was found with the critical point drying technique, 45% in the abdominal aorta and 31% in the thoracic aorta. In the abdominal aorta this shrinkage was mainly due to a shrinkage in length (36%) rather than a shrinkage in width (15%). In comparison, in the thoracic aorta critical point drying resulted in a 15% shrinkage in length and a 19% shrinkage in width. Air drying and alcohol dehydration caused considerable shrinkage (29% and 18% respectively in the thoracic aorta, 29% and 36% respectively in the abdominal aorta). Directional differences were also found with these techniques, for instance alcohol dehydration in the thoracic aorta resulted in 0% shrinkage in length and 18% shrinkage in width.     

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.     

Measurements of critical point shrinkage of glutaraldehyde fixed mouse liver

The dimensional changes of small cubes of glutaraldehyde fixed mouse liver tissue were measured using a light microscope image projected into the Quantimet 720 Image Analysing computer system. The dimensional changes occurring in the critical drying bomb could be followed at all stages when violent turbulence was not occurring. The results show that liver tissue blocks shrink in four stages whilst in the critical point drying bomb: (1) during substitution of the intermediate solvent with the transitional fluid; (2) when the transitional fluid is warmed above the critical temperature; (3) when the transitional fluid, now a gas, is allowed to escape from the CPD bomb – the rate of shrinkage increasing as atmospheric pressure is approached; (4) at atmospheric pressure when all the gas has been allowed to escape from the bomb. Taken together with the authors' previous findings, it would seem that substantial shrinkage of animal soft tissue specimens must occur whilst they are undergoing “critical point drying”. This fact should be taken into account when interpreting SEM images of CPD tissues.     

Description of a coverslip holder for,and observations on,the critical point drying of cell cultures

The elimination of artefact during the preparation of cell cultures for scanning electron microscopy is difficult. Collapse of cellular projections, cytoplasmic cracks, perforations and fracturing of cell-cell processes and cell-substrate attachments occur during fixation, dehydration and critical point drying. Coating and storage may cause further artefact. A specimen holder which serves to minimize turbulence in the critical point dryer and which allows for the simultaneous processing of up to five coverslips, as well as a reproducible technique for the preparation of cell cultures are described.     

A comparative survey of techniques for preparing plant surfaces for the scanning electron microscope

A comparative investigation of techniques for the preparation of soft botanical tissue for the scanning electron microscope has been carried out using the leaves and petals of Pelargonium zonale as test specimens. Twelve different preparative procedures involving combinations of fixation, dehydration, air drying, freeze drying, critical point drying, coating methods, replicas and a temperature controlled specimen stage were tested.     

Iron-polyethylene glycol impregnation: An attempt to reduce shrinkage of specimens in SEM preparation

Glutaraldehyde-fixed HeLa cells were soaked in a mixture of fine cationic iron colloid and polyethylene glycol, immersed in tannic acid solution containing guanidine hydrochloride, and stained with osmic acid. The treated cells showed little shrinkage in the scanning electron microscope even after ethanol dehydration and CO₂ critical point drying. On the assumption that every HeLa cell maintained contact with each other, preservation rate was computed as 0.975 × 0.0033 in linear dimension. Microvilli on the cell surface were well preserved, and few undersirable deposits were noted on the specimen surface. This treatment was also applicable to bulk staining of tissue blocks, such as rat kidneys. The podocyte foot processes and endothelial micropores of the glomerulus were well preserved; the epithelial cells of the Bowman's urinary capsule were not collapsed; the microvilli of the brush border of the proximal convoluted urinary tubule kept their ordinary length (2 μm).     

A cleaning method ensuring recovery of delicate freeze-fracture replicas of cereal leaf cells

Normal human leucocytes, successively treated with glutaraldehyde-tannic acid-osmium tetroxide-thiocarbohydrazide-osmium tetroxide-thiocarbohydrazide-osmium tetroxide, were prepared for scanning electron microscopy observation. These cells produced well-contrasted, non-charging scanning images compatible with metal-evaporated material. Further, the mononuclear and polymorphonuclear cells resisted shrinkage during dehydration and critical point drying, thus allowing much improved images at high magnification than those covered with evaporated metal. In all cases at least a second thiocarbohydrazide-osmium tetroxide treatment could not be avoided.     

Air-drying of human leucocytes for scanning electron microscopy using the GTGO procedure

The utilization of tannic acid and guanidine hydrochloride as mordants for better osmium binding has been shown to serve as an excellent alternative to metal coating of organ tissue specimens for scanning electron microscopy (SEM). The present report describes the GTGO procedure, a modification of the TAO technique introduced by Murakami et al. (1977, 1978), which we have found successful for the preparation of air dried peripheral blood leucocytes for SEM studies. Air dried, GTGO-treated leucocytes show excellent preservation of surface features with minimal cell shrinkage. When critical point dried, GTGO-treated cells are examined, they also show less shrinkage than cells prepared with standard glutaraldehyde fixation and critical point drying. The potential application of this air drying procedure (GTGO-AD) to other soft biological specimens is currently under investigation. This technique is recommended as a new and effective air drying procedure for the successful preparation of cells for SEM.     

Water loss at normal enamel histological points during air drying at room temperature

This in vitro study aimed to quantify water loss at histological points in ground sections of normal enamel during air drying at room temperature (25°C) and relative humidity of 50%. From each of 10 ground sections of erupted permanent human normal enamel, three histological points (n = 30) located at 100, 300 and 500 μm from enamel surface and along a transversal following prisms paths were characterized regarding the mineral, organic and water volumes. Water loss during air drying was from 0 to 48 h. Drying occurred with both falling and constant‐drying rates, and drying stabilization times (T_eq) ranged from 0.5 to 11 h with a mean 0.26 (±0.12)% weight loss. In some samples (n = 5; 15 points), T_eq increased as a function of the distance from the enamel surface, and drying occurred at an apparent diffusion rate of 3.47 × 10^?8 cm² s^?1. Our data provide evidence of air drying resulting in air replacing enamel's loosely bound water in prisms sheaths following a unidirectional water diffusion rate of 3.47 × 10^?8 cm² s^?1 (from the original enamel surface inward), not necessarily resulting in water evaporating directly into air, with important implications for transport processes and optical and mechanical properties.     

Improved ultrastructural preservation of yeast cells for scanning electron microscopy

The processing of yeast cells for scanning electron microscopy by conventional sequential fixation with glutaralde-hyde and osmium tetroxide and subsequent dehydration and critical point-drying caused pronounced deformation and visible shrinkage in all basidiomycetous and ascomy-cetous yeast strains studied. The mean cell diameter decreased to nearly 60 and 70%, respectively. After an additional sequential fixation with 1% tannic acid and 0–5% uranyl acetate the cell shrinkage was significantly reduced, but the most important result was a considerable reduction of wrinkling and deformation of the yeast cells.     

Improvement to critical point drying technique for SEM

We have developed an improved method for dehydration and critical point drying which leads to a marked reduction in morphological artefact in at least two classes of problematical specimen: Foetal enamel and avian embryonic heads. Water is replaced by ethanol and ethanol by C₂C1₃F₃ by refluxing in a Soxhlet apparatus. Containers are designed to prevent air drying on transfer to the CPD bomb. We concluded that the thorough removal of both water and ethanol prior to CPD can reduce the types of artefact associated with post-CPD shrinkage (superdrying).     

The effects of glycol methacrylate as a dehydrating agent on the dimensional changes of liver tissue

The dimensional changes of liver sections during the course of processing with glycol methacrylate (GMA) or with ethanol are described. Tissue processing with ethanol served as a control. During prolonged processing steps (24 h each), linear shrinkage of tissue specimens dehydrated with GMA at room temperature was 13.2%. Subsequent infiltration with GMA resulted in trivial swelling, and polymerization in slight shrinkage (2.3%). In comparison, processing with cold GMA resulted in shrinkage during dehydration (about 10.8%), a slight swelling in pure GMA, followed by shrinkage during polymerization (2.2%). Short routine processing schedules resulted in similar shrinkage/swelling patterns, although precise values differed slightly. In all experiments, ethanolic dehydration resulted in smaller dimensional tissue changes than did GMA dehydration. The dimensional changes of tissue sections during stretching on water, mounting and drying compensated for the major part of the shrinkage manifested during processing.     

An alkali digestion method to expose connective tissue fibers: A scanning electron microscopy study of rat lung

Alkali digestion has been used to remove cellular elements of tissues thus exposing the underlying connective tissue framework. We studied the action of this severe alkali treatment on the delicate tissues of rat lung. The lungs of male Sprague-Dawley rats were perfused with saline to remove blood and then inflated by fixation through the airways at 20 cm pressure. Sections of lung 2 × 5 × 5 mm were immersed in 2.5 M NaOH at 25°C for 6 h, 16 h, 24 h, 48 h, and 72 h. The alkali was changed daily. Tissues were washed to neutral with water (24 h), treated with tannic acid (1%, 3h), post-fixed with osmic acid (1%, 3 h) and processed for SEM. At 6 h, epithelial cells started to peel off the alveolar surface. At 16 h the digestion process was well advanced. At 48 h the cells were completely removed revealing the lattice network of connective tissue fibers within the alveolar surface. The method allows the complete removal of cellular elements of the lung while retaining the very fine 3D structure of the connective tissue matrix.     

Experimental study on the thermal performance of a small-scale loop heat pipe with polypropylene wick

A small-scale loop heat pipe (LHP) with polypropylene wick was fabricated and tested for investigation of its thermal performance. The container and tubing of the system were made of stainless steel and several working fluids were tested including methanol, ethanol, and acetone. The heating area was 35 mm×35 mm and nine axial grooves were provided in the evaporator to provide vapor passages. The pore size of the polypropylene wick inside the evaporator was varied from 0.5 μm to 25 μm. The inner diameter of liquid and vapor transport lines were 2.0 mm and 4.0 mm, respectively and the length of which were 0.5 m. The size of condenser was 40 mm (W) ×50 mm (L) in which ten coolant paths were provided. Start-up characteristics as well as steady-state performance was analyzed and discussed. The minimum thermal load of 10W (0.8W/cm²) and maximum thermal load of 80 W (6.5W/cm²) were achieved using methanol as working fluid with the condenser temperature of 20°C with horizontal position.     

Dimensional stability in tissues dehydrated with 2,2-dimethoxypropane for electron microscopy

Dimensions of tissues fixed in glutaraldehyde-osmium tetroxide mixture, osmium tetroxide and uranyl acetate and then dehydrated in 2,2-dimethoxypropane (DMP) were measured using transmission and scanning electron microscopy. Rat cardiac muscle, kidney and other tissues were examined in this study. The mean dimensions of characteristic ultrastructural features of material prepared by this method are similar or larger than those reported in the literature for conventionally processed samples. Critical point drying of specimens dehydrated with DMP does not produce abnormal shrinkage. Simultaneous primary fixation of lipids and proteins in a glutaraldehyde osmium tetroxide mixture and omission of the water wash after uranyl acetate appear to be important in stabilizing the tissue for rapid dehydration. The rapid reaction of DMP and water yielding the products acetone and methanol does not appear to denature tissue components to a greater extent than conventional solvent exchange dehydration.     

The fixation,dehydration, drying and coating of cultured cells for SEM

Cultivated cells form a valuable model system for studies on the effects of various preparative protocols for scanning electron microscopy (SEM). The various effects of each preparative step can be followed in detail in the light microscope and no diffusion gradients complicate the fixation and other procedures as in the case of solid tissues. Studies on cultivated cells indicate that the glutaraldehyde component of a glutaraldehyde-based fixative does not contribute to the effective osmotic pressure of the fixative and thus the osmolarity of the buffer, and other components, must be equalized to that of the medium in which the cells grow. Even small deviations from this ideal effective osmotic pressure will result in osmotically induced artefacts. Disturbances of pH and temperature of the cultures prior to and during fixation will result in changes in the appearance of many cellular structures such as microspikes and ruffles. We find that osmium fixation is advisable in most instances for best possible membrane preservation and that even long periods of glutaraldehyde fixation do not compensate for osmium fixation. Dehydration always results in shrinkage. Freeze drying (FD) and critical point drying (CPD) also give rise to shrinkage, the former to a lesser degree than the latter. A gold-palladium alloy gives a less granular coating that does gold alone. When cultured cells are studied, a metal thickness of between 5 and 15 nm is usually sufficient to give rise to an adequate secondary electron production and to avoid charging even at accelerating voltages of 30–40 kV. Without treatment with OsO₄ a thicker metal coating is required.     

用顶空气相色谱内标法测定子仁霜中溶剂残留量

本文报道了用丙酮作为内标物的顶空气相色谱法测定子仁霜中溶剂残留量。此法对样品处理条件要求不严,对生产上批量测定具有操作方便,结果准确等特点。其相关系数r=0.9998,相对标准偏差RSD=2.3％。可作为新药生产的质量标准测定。