Dimensional changes of cultured smooth muscle cells due to preparatory processes for transmission electron microscopy

The change in volume of cultured smooth muscle cells prepared with four different fixation procedures for transmission electron microscopy was studied. Although the cells showed swelling after being embedded in Spurr's embedding medium, the degree of swelling depended on the particular method of fixation procedure used. When the volume of the cells measured using transmission electron microscopy was compared with that of the fresh cell volume, cells prepared by two of the methods showed swelling, and cellular shrinkage was noted in the other two methods. One method which caused the least amount of volume change is recommended for quantitative electron microscopic study of vertebrate smooth muscle cell systems.     

Transmission and scanning electron microscopic examination of intracellular organelles in freeze-substituted Kloeckera and Saccharomyces cerevisiae Yeast Cells

The application of the conventional double-fixation method (glutaraldehyde and osmium tetroxide) to whole yeast cells is difficult because the thick cell wall of the yeast prevents the penetration of osmium tetroxide. However, this problem was solved by using the freeze-substitution fixation method. Therefore, it was possible to examine the intracellular structures of the yeast cells without digestion of the cell wall. In the present method, specimens for transmission electron microscopy and for scanning electron microscopy were prepared simultaneously. By scanning electron microscopic observation, three-dimensional information about internal structures was obtained. In the cytological analysis of the yeasts, intracellular structures were well preserved by using the freeze-substitution fixation method. On the outer leaflet of the nuclear envelope, many ribosomes were attached. The rough endoplasmic reticulum and Golgi apparatus were clearly seen in the yeast cytoplasm. The Golgi stack appeared to consist of smooth membranes, and small vesicles were present beside it. The details of other structures such as the nuclear division apparatus, actinlike filaments, and viruslike particles were also revealed. The present technique can be applied to most species of yeast cells. With this new information, the previous model of a yeast cell was modified.     

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.     

Transmission electron microscopy of yeast

The challenges of sample preparation can limit a researcher's selection of transmission electron microcopy (TEM) for analysis of yeast. However, with the exception of thin sectioning, preparation of well-fixed and infiltrated samples of yeast cells is achievable by any reasonably equipped laboratory. This review presents a general overview of TEM sample preparation methods and detailed protocols for chemical fixation of yeast for ultrastructural analysis and immunolabeling. For ultrastructural analysis, the most commonly used chemical fixation involves treatment with glutaraldehyde followed by either potassium permanganate or osmium. Prior to osmium postfixation, the cell wall must be enzymatically digested to allow optimal fixation and embedding. Freeze substitution methods continue to provide the highest quality of fixation, but equipment needed for these protocols is not generally available to many labs. The low viscosity of Spurr's resin makes it the resin of choice for ultrastructure studies. Immunoelectron microscopy has enjoyed great success in analysis of yeast molecular organization. For immunoelectron microscopy, glutaraldehyde/formaldehyde-fixed cells are embedded in LR White resin. The thin sections are then treated in much the same way as an immunoblot: following blocking, they are incubated in primary antiserum, washed, and then incubated in gold-labeled secondary antiserum.     

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.     

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.     

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.     

Near-infrared optical spectroscopy for pancreas shrinkage estimation with multi synchrosqueezing transform and multivariate regression model

In this article, we proposed a method to estimate pancreas shrinkage with pancreas β cell insulin secretion. The β cells in the pancreas secrete insulin and digestive enzymes after food consumption. Conventionally, the pancreas structure estimation is done with magnetic resonance imaging (MRI) and ultrasound imaging techniques. However, the structure of the pancreas changes due to islet cell death. The presence of islet cells is detected through near infrared (NIR) spectroscopy signal acquired from the epigastric region (pancreas) of the abdomen. Subsequently, the NIR spectroscopy signal from the pancreas is analyzed with multi synchrosqueezing transform (MSST); whereas, the β cell insulin secretion varies for diabetic and nondiabetic persons. The existence of β cell and insulin secretion correlates with Root Mean Square (RMS) and kurtosis via a multivariate regression model to evaluate pancreas shrinkage. In terms of numerical results, NIR spectroscopy signal from the pancreas was obtained for about 20 nondiabetic and 20 diabetic persons. The pancreas shrinkage was estimated with 88% accuracy. The results are validated with MRI pancreas images for earlier detection of the apoptotic pancreas. The pancreas shrinkage causes lower insulin emission and unpredictable blood glucose in diabetic patients. Analysis of NIR spectroscopy signals of the pancreas with MSST was done to obtain higher-order and lower-order frequency components.     

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.     

Functional morphology of the secretory pathway organelles in yeast

The glycoprotein secretory pathway of yeast serves mainly for cell surface growth and cell division. It involves a centrifugal transport of transit macromolecules among organelles, whose membranes contain resident proteins needed for driving the transport. These resident membrane proteins return by retrograde vesicular transport. Apart from this, the pathway involves endocytosis. The model yeast Saccharomyces cerevisiae and vertebrate cells were found to contain very similar gene products regulating the molecular mechanism of glycoprotein transport, and the cellular mechanism of their secretion pathways was therefore also presumed to be identical. Biochemists have postulated that, in S. cerevisiae, the translocation of peptides through the endoplasmic reticulum membranes into the lumen of ER cisternae and the core glycosylation is followed by a vector-mediated transport into the functional cascade of the Golgi system cisternae and between them. This is the site of maturation and sorting of glycoproteins, before the ultimate transport by other vectors involving either secretion from the cells (exocytosis across the plasmalemma into the cell wall) or transport into the lysosome-like vacuole via a prevacuolar compartment, which serves at the same time as a primary endosome. The established cellular model of secretion deals with budding yeast; interphase yeast cells, in which the secretion is limited and which predominate in exponential cultures, have not been taken into consideration. The quality of organelle imaging in S. cerevisiae ultra-thin sections depends on the fixation technique used and on specimen contrasting by metals. The results achieved by combinations of different techniques differ mostly in the imaging of bilayers of membrane interfaces and the transparence of the matrix phase. Fixation procedures are decisive for the results of topochemical localisations of cellular antigenic components or enzyme activities, which form the basis of the following survey of functional morphology of organelles involved in the yeast secretory pathway. The existing results of these studies do not confirm all aspects of the vertebrate model of the Golgi apparatus proposed by molecular geneticists to hold for S. cerevisiae, and alternative models of the cellular mechanism of secretion in this yeast are, therefore, also discussed.     

Atomic force microscopy of BHK-21 cells: an investigation of cell fixation techniques

The atomic force microscope (AFM) has been used to image a wide variety of biological samples, including cultured cells, in air. Whilst cultured cells have been prepared for AFM analysis using a variety of matrices and fixatives, a definitive study of sample preparation and its effects on cell morphology has not, as far as the authors are aware, previously been reported. Although a considerable number of cell fixatives exist, no single fixative is ideal for all investigations. Prior to the performance of specialised techniques, such as atomic force microscopy of cultured cells in air, the cell fixation method must be investigated and optimised. The fixative abilities of 2% paraformaldehyde-lysine-periodate, 0.25% glutaraldehyde, paraformaldehyde-glutaraldehyde, 4% phosphate-buffered formal saline, 1% formaldehyde, methanol:acetone, formal saline, 4% paraformaldehyde and ethanol:acetic acid were assessed in this study. A qualitative assessment system was used to evaluate the efficacy of the above fixatives using conventional fixation criteria (i.e. the presence of fibroblastic morphology consistent with optical microscopy and the absence of fixation artifacts). The optimal fixative was identified as 4% paraformaldehyde, which was capable of providing optically consistent images of BHK-21 (fibroblastic) cells, whose heights remained within the measurement capability of the AFM instrument used in this study.     

Analysis of shrinkage and warpage in an injection-molded part with a thin shell feature using the response surface methodology

This paper presents a systematic methodology to analyze the shrinkage and warpage in an injection-molded part with a thin shell feature during the injection molding process. The systematic experimental design based on the response surface methodology (RSM) is applied to identify the effects of machining parameters on the performance of shrinkage and warpage. The experiment plan adopts the centered central composite design (CCD). The quadratic model of RSM associated sequential approximation optimization (SAO) method is used to find the optimum value of machining parameters. One real case study in the injection molding process of polycarbonate/acrylonitrile butadiene styrene (PC/ABS) cell phone shell has been performed to verify the proposed optimum procedure. The mold temperature (M _T), packing time (P _t), packing pressure (P _P) and cooling time (C _t) in the packing stage are considered as machining parameters. The results of analysis of variance (ANOVA) and conducting confirmation experiments demonstrate that the quadratic models of the shrinkage and warpage are fairly well fitted with the experimental values. The individual influences of all machining parameters on the shrinkage and warpage have been analyzed and predicted by the obtained mathematical models. For the manufacture of PC/ABS cell phone shell, the values of shrinkage and warpage present the reduction of 37.8 and 53.9%, respectively, using this optimal procedure.     

Investigation of the morphology, viability and mechanical properties of yeast cells in environmental SEM

The mechanical properties of biological cells at nanoscale may be characterized using an environmental scanning electron microscopy (ESEM) combined with a force measurement device. However, the electron beam radiation in an ESEM may damage a specimen. So far, little is known about the radiation damage to biological cells. In this work, single yeast cells were imaged using an ESEM under both high and low vacuum modes. The changes in their morphology and viability were monitored as a function of radiation time for a given beam current of 538 pA corresponding to 10 kV accelerating voltage and spot size 4. Under the two modes, the radiation damage to the morphology of yeast cells became evident after an exposure time of 3 min, but under the low vacuum mode, the damage to their morphology was more severe. However, all cells lost their viability after 5 min under the high vacuum mode with the electron beam off from an initial viability of 95+/-1%. In contrast, the viability of cells under the low vacuum mode was found to be approximately 20% after 20 min. In addition, a newly developed ESEM-based nanomanipulation technique was applied to measure the force imposed on single yeast cells and their deformation, including contact diameter and central lateral diameter for the compression of single yeast cells to a given displacement within a time frame of 1 min, and the data obtained may be used to validate mathematical modeling of the stress-strain relationship for the compression of cells in order to determine their intrinsic mechanical property parameters.     

Freeze-drying process of biological specimens observed with a scanning electron microscope

A pit membrane was observed with a cryo-SEM during the course of dehydration at low temperatures. The freeze-drying process of sea-urchin eggs and parenchyma cells of higher plants was also examined with this microscope. Conditions for observation of frozen specimens in the native state were discussed on the basis of morphological studies of alterations such as shrinkage or deformation which appeared during the freeze-drying process.     

Effect of ambient humidity on the strength of the adhesion force of single yeast cell inside environmental-SEM

A novel method for measuring an adhesion force of single yeast cell is proposed based on a nanorobotic manipulation system inside an environmental scanning electron microscope (ESEM). The effect of ambient humidity on a single yeast cell adhesion force was studied. Ambient humidity was controlled by adjusting the chamber pressure and temperature inside the ESEM. It has been demonstrated that a thicker water film was formed at a higher humidity condition. The adhesion force between an atomic force microscopy (AFM) cantilever and a tungsten probe which later on known as a substrate was evaluated at various humidity conditions. A micro-puller was fabricated from an AFM cantilever by use of focused ion beam (FIB) etching. The adhesion force of a single yeast cell (W303) to the substrate was measured using the micro-puller at the three humidity conditions: 100%, 70%, and 40%. The results showed that the adhesion force between the single yeast cell and the substrate is much smaller at higher humidity condition. The yeast cells were still alive after being observed and manipulated inside ESEM based on the result obtained from the re-culturing of the single yeast cell. The results from this work would help us to understand the ESEM system better and its potential benefit to the single cell analysis research.     

"Large" and "small" nuclear pore complexes; the influence of glutaraldehyde.

Aliquots of lymphocyte cell suspensions were pretreated according to the following three schedules before freeze fracturing: (a) prefixed with 2% glutaraldehyde before infiltration with 25% glycerol in medium RPMI-1640; (b) frozen in medium RPMI-1640 without additional pretreatment; and (c) frozen after pretreatment with 25% glycerol in medium RPMI-1640. The diameters of the fractured nuclear pore complexes of cells prefixed with glutaraldehyde were normally distributed within the range 70-120 nm (median 90 nm). The nuclear envelopes of 66-75% of cells processed through schedules b and c, which omitted glutaraldehyde fixation, had 70-120 nm diameter pores, while the remainder had pores with diameters in the range 120-175 nm. The large pores were structurally similar to the smaller pores except for their dimensions. These results indicate that glutaraldehyde gives rise to shrinkage of the larger pores to the minimum, smaller, diameter. Apparent orifices of at least 30 nm diameter were sometimes observed at the centres of these large pore complexes. We propose that the variation in pore diameters may indicate opening and closure of this orifice, and that the widely reported "central granule" of the nuclear pore complex corresponds with the orifice in a closed configuration.     

Localization of acid and alkaline phosphatase

Ultrathin frozen sections of glutaraldehyde fixed yeast cells have been successfully used for the demonstration of acid and alkaline phosphatase. Acid phosphatase was localized over the cell wall of both the mother cell and the bud as well as over the newly forming cross wall (septum). Cytoplasmic vesicles (vacuoles, lysosomes?) located close to the cell wall showed a positive reaction for acid phosphatase as well. After 3 h glutaraldehyde fixation an activation of the nuclear acid phosphatase was observed. Lead precipitates were predominantly found over the nucleolar material of ‘resting’ and budding cells. Alkaline phosphatase could be demonstrated in the ‘yeast-mesosome’ and within the plasmalemma invaginations. After separation of the bud, small vesicles, probably derived from the endoplasmic reticulum showed a strong positive reaction for alkaline phosphatase. In frozen sections incubated for alkaline phosphatase, non-specific lead precipitates were found in the nucleus and along the plasmalemma invaginations.     

Effect of glutaraldehyde fixation on bacterial cells observed by atomic force microscopy

Atomic force microscopy (AFM) is a promising microscopy technique that can provide high-resolution images of bacterial cells without fixation. Three species of bacteria, Xanthomonas campestris, Pseudomonas syringae, and Bacillus subtilis, were used in this study. AFM images were obtained from unfixed and glutaraldehyde-fixed cells, and cell height was measured. The mean height of bacterial cells prepared by fixation was higher than that of those prepared by nonfixation. However, the height changes were different between Gram-negative and Gram-positive bacteria: the mean height of two fixed Gram-negative bacteria, X. campestris and P. syringae, increased by 112.31 and 84.08%, respectively, whereas Gram-positive bacterium, B. subtilis, increased only by 38.79%. The results above indicated that glutaraldehyde fixation could affect the measured height of cells imaged by AFM; further more, the effect of glutaraldehyde fixation on the measured height of Gram-negative bacterial cells imaged by AFM seemed much more than on that of Gram-positive bacterial cells.     

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.     

磁悬浮飞轮锁紧装置及其优化设计

针对磁悬浮飞轮用一次性锁紧装置不可重复锁紧/释放的缺点,提出了一种基于电机、碳纤维弹片和钢丝绳的可重复锁紧装置,并给出了锁紧装置的结构和工作原理。根据执行锁紧过程碳纤维变形状况,将其分为弯曲变形和受压变形两个阶段,并在此基础上对锁紧装置进行了受力分析。利用多学科优化软件iSIGHT集成有限元分析软件对碳纤维弹片进行了优化设计,以材料强度、结构反力和共振频率多学科要求同时作为约束条件,采用针对离散变量的全域遍数法嵌套连续变量的序列二次规划法对碳纤维弹片质量进行优化。优化结果显示,碳纤维弹片个数为12时,碳纤维质量达到最小为46g,比初始质量112g减少了59%。结果表明,该优化方法提高了锁紧装置设计的可靠性和效率,对飞轮系统整体优化设计有重要意义。