Quantitation of shrinkage during preparation for scanning electron microscopy: Human lung

Human lung tissue is found to shrink considerably with preparation for SEM. Fifty-one blocks of glutar-aldehyde-fixed and inflated lung, approximately 2.5 cm × 2.5 cm × 1 cm, shrank a mean of 19% (± 4.0% SD) linear dimension through post fixation, dehydration and critical point drying. Shrinkage with fixation was not measured. Blocks of lung were observed to shrink equally in length (L) and width (W), L = 19.4% ± 2.7 SD, W = 19.0% ± 4.0 SD. Final shrinkage was the same whether samples were dehydrated in acetone or ethanol, although with acetone more of the shrinkage occurred during the dehydration process and less occurred during critical point drying.     

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.     

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.     

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.     

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.     

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.     

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.     

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).     

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).     

干燥带对绝缘子温度场影响的研究

对干燥带对污秽绝缘子温度场分布进行了研究,介绍了绝缘子干燥带形成原因,利用绘图软件,绘制了绝缘子分析模型; 然后分析了干燥带的产生过程; 最后运用ANSYS软件电热耦合的方法,从干燥带产生位置、干燥带产生条数、干燥带产生长度、干燥带产生宽度4个方面,分析污秽绝缘子径向温度分布情况。研究结果表明,干燥带产生位置、干燥带产生长度、干燥带产生宽度、干燥带产生条数不同,绝缘子径向温度分布特征差异较大。     

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.     

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.     

A synchrotron X-ray study of the changes occurring in the corneal stroma during processing for electron microscopy

Using a high-intensity synchrotron X-ray source, the structural changes occurring in the corneal stroma were monitored during each stage of several different processing runs for the transmission electron microscope (TEM) and scanning electron microscope (SEM). The parameters studied were interfibrillar spacing, intermolecular spacing, D-periodicity and fibril diameter. The processing schedule that produced the least changes in spacings for TEM specimens involved extended fixation in glutaraldehyde followed by low-temperature embedding in Lowicryl K4M resin. However, interfibrillar material was better preserved after embedding in LR White resin or Nanoplast. Almost every processing stage for electron microscopy produced significant changes in one or more structural parameters in the cornea. Glutaraldehyde fixation significantly increased the intermolecular spacings, while resin infiltration and resin polymerization each resulted in shrinkage of all the spacings monitored. Critical-point drying for SEM specimens resulted in considerable shrinkage in all three spacings, but was still preferable to air drying, which caused reduction in the order of the fibril packing, resulting in loss of the interfibrillar X-ray pattern. Perhaps the most drastic effect was caused by post-fixation in osmium tetroxide, which resulted in loss of the intermolecular pattern, and also increased the amount of shrinkage in the interfibrillar spacings and the D-periodicity which occurred during later stages of processing.     

Pressure waves in the aorta during isolated abdominal belt loading: the magnitude, phasing, and attenuation

While rupture of the aorta is a leading cause of sudden death following motor vehicle crashes, the specific mechanism that causes this injury is not currently well understood. Aortic ruptures occurring in the field are likely due to a complex combination of contributing factors such as acceleration, compression of the chest, and increased pressure within the aorta. The objective of the current study was to investigate one of these factors in more detail than has been done previously; specifically, to investigate the in situ intra-aortic pressure generated during isolated belt loading to the abdomen. Ten juvenile swine were subjected to dynamic belt loads applied to the abdomen. Intraaortic pressure was measured at multiple locations to assess the magnitude and propagation of the resulting blood pressure wave. The greatest average peak pressure (113.6 +/- 43.5 kPa) was measured in the abdominal aorta. Pressures measured in the thoracic aorta and aortic arch were 70 per cent and 50 per cent, respectively, that measured in the abdominal aorta. No macroscopic aortic trauma was observed. To the authors' knowledge the present study is the first one to document the presence, propagation, and attenuation of a transient pressure wave in the aorta generated by abdominal belt loading. The superiorly moving wave is sufficient to generate hydrostatic and intimal shear stress in the aorta, possibly contributing to the hypothesized mechanisms of traumatic aortic rupture.     

The use of a simple method to avoid cell shrinkage during SEM preparation

In the course of preparing specimens for scanning electron microscopy, both glutaraldehyde and OsO₄-fixed cells exhibit a considerable shrinkage with a reduction of the mean cellular diameter of about 45% after critical point drying. However, if cells are successively treated with glutaraldehyde, OsO₄, tannic acid and uranyl acetate solutions, cellular shrinkage of only 5% is observed.     

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.     

Research on micro‐damage behavior of wool fabrics drying in domestic dryer

To evaluate the performance degradation of fabric drying in domestic air‐vented dryer, the appearance, mechanical, and microscopic properties of the wool fabric after different drying cycles were studied. Pilling and dimensional stability tests were performed according to established international standards. Microscopic studies were carried out by Scanning Electron Microscopy (SEM) and X‐ray (XRD). The results show that pilling is the dominant damage mechanism and gradually increased during lower number of drying cycles (0–10 cycles). The results of dimensional stability showed that the highest dimensional shrinkage of knit wool fabric was first five drying cycles, and then increased at a relatively slow rate with the increase in drying cycles. And fabric shrinkage takes place mainly in the warp direction. The SEM images of wool fiber showed that the scales on the fiber surface were intact in the first 15 drying cycles but start to flake after 20 drying cycles. In the drying process, fabrics can suffer several fractures such as cut fracture, split fracture, partial fracture and distortion and even scale flaking and interior structural distortion of fiber. The damages occur quite randomly, but complexity and severity of damages increased with increase in drying cycles. XRD analysis showed that drying had little effect on fiber crystallinity, indicating that drying only leads to changes in the physical properties of wool fabrics instead of changes in the chemical composition of the fiber. And found SEM analysis is not only an effective method to characterize the change in the surface morphology of the fibers resulting from the drying treatments, but also explain the cause of fabric performance degradation such as pilling and strength reduction during drying.     

Sublimation rates of ice in a cryo-ultramicrotome

Sublimation rates of ice in the nitrogen-filled cryochamber of an ultramicrotome have been measured. The values are four to five orders of magnitude smaller than vacuum sublimation rates at the same temperatures. This is a consequence of the much shorter mean free path length of water molecules in nitrogen at atmospheric pressure, as compared to freeze drying under vacuum. The partial pressure at the phase boundary and gas diffusion and convection determine the rate of freeze drying. This has implications for section handling and the design of transfer equipment. Generally, it means that the danger of even partial dehydration of semi-thick (0·5–1 μm) sections used mainly for X-ray microanalysis is less serious than commonly assumed.     

New application of air-drying techniques for studying Ephemeroptera and Plecoptera eggs by scanning electron microscopy

Hexamethyldisilizane (HMDS) and tetramethylsilane are organic compounds that are volatile at ambient temperature and which can therefore be used for air-drying biological samples for SEM studies. The techniques using these compounds provide results that are comparable with those obtained by critical point drying, but which involve a very simple process that saves time and money. Both techniques were applied to SEM studies of Ephemeroptera and Plecoptera eggs in order to assess their suitability as alternative methods to critical point drying for these kinds of biological material. The results show no morphological differences between eggs HMDS air-dried and critical point-dried.     

Positive displacement holder for critical point drying of small particle materials*

A specimen holder has been designed specifically to contain small particulate matter, such as microorganisms, fibres or powders, during the fluid exchange cycle and final evaporation step of the critical point drying process. The mode of fluid exchange is positive displacement rather than diffusion. The holder can accommodate up to ten different samples without loss of material or cross-contamination during the dehydration process. The samples are separated by silver membrane filters, and fluid exchange is accomplished in minimal time by only slight positive pressure. Experiments with soluble dyes have shown that any fluid can be displaced completely with another in less than 2 min, whereas diffusion requires more than 30 min. The holder and individual capsules have been tested with different materials and a standard water/ethyl alcohol/amyl acetate/carbon dioxide fluid sequence. This paper describes the design, production, and testing of the holder and capsules, and presents selected examples of results.