Effect of fixative on chromatin structure and DNA detection

In this study, we analyzed the chromatin ultrastructure in interphase cells after different chemical fixations. In light of the fact that there is little information regarding the fixation of biological samples in combination with molecular biology methods (such as DNA extraction and in situ hybridization methods) we analyzed the ultrastructure of chromatin in interphase cells fixed with different fixatives and tested under the same conditions for both DNA extraction and in situ hybridization. The results showed that, among the different combinations and concentrations we analyzed, the solution of 4% paraformaldehyde/0.1% glutaraldehyde was the best compromise in order to achieve a well-preserved morphology, successful DNA extraction, and specific signaling of in situ hybridization, suggesting a low interference of this fixative with the chromatin organization.     

Effects of glutaraldehyde fixative osmolarities on smooth muscle cell volume,and osmotic reactivity of the cells after fixation

The contribution of glutaraldehyde (GA) to the effective osmolarity of GA fixatives, the osmotic reactivity of the cells after fixation in GA, and also the duration of fixation in GA on cell volume, were investigated using cultured smooth muscle cells (SMC) and spiral aortic strips. Four fixation procedures were studied. We found that GA contributes to the total effective osmolarity of the fixatives, and that the type of buffers used for the fixatives can also affect the cell volume differently during GA fixation. After GA fixation, the cells were still osmotically reactive, regardless of the buffer types for making up the GA fixatives, so that the osmolarity of the wash buffer after GA fixation is important. However, OsO₄ eliminates osmotic responses, thus the osmolarity of OsO₄ fixative and wash buffer have negligible influence on the cell volume. Longer fixation time up to 4 h had no effect on the cell volume.     

Monitoring of the course of fixation of plant cells

The fixation of cells of petiolar hairs of tomato was monitored by phase contrast and Nomarski interference contrast microscopy. Because the cuticle covering these hairs is relatively impermeable some fixatives enter only the base of the hair so that a ‘fixation front’ can be followed through a single cell. The basal end of such a cell may be immobilized by the fixative while the apical end is still streaming. A variety of fixatives were tested at room temperature and at 273–278 K. All procedures tested failed to stabilize a pleomorphic canalicular system that is a dominant feature of these cells so that no clue to the presence of this system can be seen in sectioned material. It is therefore very desirable that images of fixed cells be compared to the structure of similar cells which are still alive.     

Cytochrome oxidase activity and confocal laser scanning microscopic analysis of the hamster submandibular gland using microwave irradiated fixation

Submandibular glands of the hamster were irradiated in 2% paraformaldehyde (pFA)-0.5% pure glutaraldehyde (PGA) with a microwave (MW) processor at temperatures of 10 degrees and 37 degrees C. Electron microscopy showed that cytochrome oxidase activity was taking place in the mitochondrial intermembrane-intracristal space of the granular duct cell when the temperature of the MW-irradiated fixatives was at 10 degrees C. However, a decrease of this activity was observed when we took care to keep the temperature of the MW-irradiated fixatives at 37 degrees C. The distinct reduction of cytochrome oxidase activity allowed by MW irradiation seems to be due the thermal affects of fixatives. Of course, the possibility cannot be excluded that MW irradiation caused other undetectable membrane damage. Then, we used confocal laser scanning microscopy for the preservation check of the mitochondrial membrane for cytochemistry with MW-irradiated fixation. The fluorescence of rhodamine 123 was observed in the inner spaces of the mitochondria at temperatures of 10 degrees and 37 degrees C. When the same tissues were fixed with 2% pFA using an MW processor as the sole fixative at 10 degrees C, no mitochondrial fluorescence was observed. Cytochrome oxidase activity, by contrast, could be seen in the mitochondrial intermembrane-intracristal spaces in the same condition. Formaldehyde is not the best aldehyde for the purpose of ultrastructural preservation. On the other hand, light and electron microscopy showed that the endogenous peroxidase activity was localized in the nuclear envelope, endoplasmic reticulum, secretory granules, and Golgi apparatus of the hamster submandibular gland using 2% pFA-0.5% PGA fixative with and without MW irradiations at temperatures of 10 degrees and 37 degrees C. Some of the same cells were fixed with only 2% pFA under MW irradiation at 10 degrees C; however, marked diffuseness of the peroxidase activity was observed. Therefore, these results indicated that cytochrome oxidase activity was sensitive to heat with MW-irradiated fixation. Peroxidase activity was very resistant to heat with MW-irradiated fixation but not with pFA solo fixation, therefore, PGA had to be used.     

Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

Trace metals play important roles in biological function, and x‐ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side‐by‐side comparison shows that plunge‐freezing‐based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. When chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x‐ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.     

An integrated approach to the study of living cells by atomic force microscopy

We describe a technique for studying living cells with the atomic force microscope (AFM) in tapping mode using a thermostated, controlled-environment culture system. We also describe the integration of the AFM with bright field, epifluorescence and surface interference microscopy, achieving the highest level of integration for the AFM thus far described. We succeeded in the continuous, long-term imaging of relatively flat but very fragile cytoplasmic regions of COS cells at a lateral resolution of about 70 nm and a vertical resolution of about 3 nm. In addition, we demonstrate the applicability of our technology for continuous force volume imaging of cultured vertebrate cells.
The hybrid instrument we describe can be used to collect simultaneously a diverse variety of physical, chemical and morphological data on living vertebrate cells. The integration of light microscopy with AFM and steady-state culture methods for vertebrate cells represents a new approach for studies in cell biology and physiology.     

High-resolution three-dimensional imaging of the rich membrane structures of bone marrow-derived mast cells

Atomic force microscopy (AFM) enables high-resolution three-dimensional (3D) imaging of cultured bone marrow-derived mast cells. Cells were immobilized by a quick centrifugation and fixation to preserve their transient cellular morphologies followed by AFM characterization in buffer. This "fix-and-look" approach preserves the structural integrity of individual cells. Well-known membrane morphologies, such as ridges and microvilli, are visualized, consistent with prior electron microscopy observations. Additional information including the 3D measurements of these characteristic features are attained from AFM topographs. Filopodia and lamellopodia, associated with cell spreading, were captured and visualized in three dimensions. New morphologies are also revealed, such as high-density ridges and micro-craters. This investigation demonstrates that the "fix-and-look" approach followed by AFM imaging provides an effective means to characterize the membrane structure of hydrated cells with high resolution. The quantitative imaging and measurements pave the way for systematic correlation of membrane structural features with the biological status of individual cells.     

Intercellular interaction observed by atomic force microscopy

The cultured myoblasts, focusing on the microprocesses related to the intercellular interaction, were observed by taking topological images. For atomic force microscopy (AFM), cells were fixed and either dried as in usual scanning electron microscopy or kept in the buffer. The dried cells were used for observing intercellular interactions related to the fusion. The prefusing myoblasts aligned in a chain were mostly spindle in shape and were characterized by the presence of many microprocesses along the facing edges of adjacent aligned myoblasts. The space between fusing myoblasts and between myotubes and myoblasts were often traversed by filopodia and cellular bridges formed by the connection of microvilli. These results suggest that microprocesses may be involved in the fusion of myoblasts. The best images of the fixed cell in liquid were obtained using the contact mode of AFM. AFM observation is an efficient tool in the study on the interaction between cells, and the fixation, imaging in liquid is a good approach to understand the cellular dynamics.     

Using the atomic force microscope to observe and study the ultrastructure of the living BIU-87 cells of the human bladder cancer

In this study, the ultrastructure of living BIU-87 cells of human bladder cancer was mapped using atomic force microscopy to reveal the dynamic change of single cancerous cell division. Simultaneously, the feasibility and functional reliability of the atomic force microscope (AFM) were established and a laboratory model using AFM to study living cancerous cells was created. In this experiment, BIU-87 cells of human bladder cancer were cultured by conventional methods and grown in gelatin-treated dishes. A thermostat was used for preserving the cell's living temperature. Scanning of these cells using AFM was carried out in physiologic condition. The AFM images of the ultrastructure of living BIU-87 cells as well as those of the cell's membrane and cytoskeleton were very clear. The dynamic phenomenon of single cell division was observed. It was concluded that the AFM was able to observe and depict the ultrastructure of living cells of human bladder cancer directly and in real time. This experimental model is expected to play an important role in elucidating the cancerous mechanism of bladder normal cells at the atomic or nanometer level.     

人脐静脉内皮细胞形貌与表面粘附力的原子力显微镜表征

目的:探讨原子力显微镜(AFM)在研究人脐静脉内皮细胞(ECV304)表面形貌、超微结构及纳米机械性质等方面的应用,讨论ECV304超微结构和机械性质与其功能的关系。方法:利用AFM对ECV304细胞的表面形貌及生物机械性质进行表征与测量。结果:在AFM下观察到用普通光学显微镜难以观察到的ECV304细胞的独特的形态结构,如细胞骨架、伪足及细胞边缘微丝等。ECV304细胞呈现长梭形、多角形、圆形等多种形态,细胞表面平均粗糙度为320.52±75.98 nm,表面均匀分布微绒毛,细胞周围有铺展的圆盘状物质。力曲线定量分析得出针尖与细胞表面的非特异性粘附力为75±14 pN。结论:通过AFM成像和力曲线测量表明,ECV304细胞呈圆形,多角形,梭形等多种形态,针尖与细胞膜表面问的粘附力比较小,约75±14pN。     

Quantitative assessment of chemical fixation,including the use of malachite green,for electron microscopy of vacuolate mesophyll cells from leaves of a grass

Various chemical fixation regimes were studied using leaves of the grass Lolium multiflorum Lam. as a test system. To evaluate the quality of fixation for electron microscopy two parameters were chosen; the extent of cell damage to mesophyll cells and damage and change in size of chloroplasts. Reducing the concentration of glutaraldehyde from 3% to 1% resulted in extensive cell damage. The addition of paraformaldehyde to 1% glutaraldehyde did not totally eliminate cell damage and resulted in dilation of thylakoids within the chloroplasts. In tissue fixed and washed in solutions matched to the total osmolality of the primary fixative solution there was little cell damage but the chloroplasts were larger, diameters 4.5times2.9 μm, than when only the buffer vehicle osmolality was maintained during subsequent washings, diameters 3.5times1.6 μm. Malachite green added to the primary fixative solution abolished cell damage regardless of buffer concentration and resulted in the appearance of osmiophilic droplets on the surface of and within the chloroplasts which were not present in the absence of malachite green. It is suggested that malachite green stabilizes cell membranes during primary fixation.     

Vacuolar structures can be identified by AFM elasticity mapping

Fluid-filled organelles like vesicles, endosomes and pinosomes are inevitable parts of cellular signalling and transport. Endothelial cells, building a barrier between blood and tissue, can form vacuolar organelles. These structures are implicated in upregulated fluid transport across the endothelium under inflammatory conditions. Vacuolar organelles have been described by transmission electron microscopy so far. Here, we present a method that images and mechanically characterizes intracellular structures in whole cells by atomic force microscopy (AFM). After crosslinking the cellular proteins with the fixative glutaraldehyde, plasma membrane depressions become observable, which are scattered around the cell nucleus. Nanomechanical analysis identifies them as spots of reduced stiffness. Scanning electron microscopy confirms their pit-like appearance. In addition, fluorescence microscopy detects an analogous pattern of protein-poor spots, thereby confirming mechanical rigidity to arise from crosslinked proteins. This AFM application opens up a mechanical dimension for the investigation of intracellular organelles.     

Atomic force microscopy applied to study macromolecular content of embedded biological material

We demonstrate that atomic force microscopy represents a powerful tool for the estimation of structural preservation of biological samples embedded in epoxy resin, in terms of their macromolecular distribution and architecture. The comparison of atomic force microscopy (AFM) and transmission electron microscopy (TEM) images of a biosample (Caenorhabditis elegans) prepared following to different types of freeze-substitution protocols (conventional OsO4 fixation, epoxy fixation) led to the conclusion that high TEM stainability of the sample results from a low macromolecular density of the cellular matrix. We propose a novel procedure aimed to obtain AFM and TEM images of the same particular organelle, which strongly facilitates AFM image interpretation and reveals new ultrastructural aspects (mainly protein arrangement) of a biosample in addition to TEM data.     

Atomic force microscopy imaging of living cells: a preliminary study of the disruptive effect of the cantilever tip on cell morphology

Recent studies have demonstrated that atomic force microscopy (AFM) is a potential tool for studying important dynamic cellular processes in real time. However, the interactions between the cantilever tip and the cell surface are not well understood, and the disruptive effect of the cantilever tip on cell morphology has not been well characterized. In this study, the disruptive effect of the scanning cantilever tip on cell morphology, in the AFM contact mode, has been investigated. The aims of this study are to identify what kinds of cell morphological changes generally occurred under normal AFM imaging conditions and to find out how long cells remain viable during scanning. Two cell lines, SK-N-SH (human neuroblastoma cells) and AV12 (Syrian hamster cells) were studied in the experiment because these are widely used in biomedical research as an expression system for studying cellular functions of neuronal receptors. The experimental results suggest that the sensitivity of cells to the cantilever disruptive effect is dependent on cell type and that there are patterns observed in the changes of cell morphology induced by the cantilever force in these two cell lines.     

SNOM and AFM microscopy techniques to study the effect of non-ionizing radiation on the morphological and biochemical properties of human keratinocytes cell line (HaCaT)

In this study we have employed atomic force microscopy (AFM) and scanning near‐field optical microscopy (SNOM) techniques to study the effect of the interaction between human keratinocytes (HaCaT) and electromagnetic fields at low frequency. HaCaT cells were exposed to a sinusoidal magnetic field at a density of 50 Hz, 1 mT. AFM analysis revealed modification in shape and morphology in exposed cells with an increase in the areas of adhesion between cells. This latter finding was confirmed by SNOM indirect immunofluorescence analysis performed with a fluorescent antibody against the adhesion marker β4 integrin, which revealed an increase of β4 integrin segregation in the cell membrane of 50‐Hz exposed cells, suggesting that a higher percentage of these cells shows a modified pattern of this adhesion marker.     

Direct measurement of the osmotic effects of buffers and fixatives in Nitella flexilis

The pressure probe has been used to investigate the amount and time course of changes in turgor induced by agents used in fixation of biological material for electron microscopy. All buffers tested decreased the turgor pressure of internodal cells of the green alga Nitella flexilis in proportion to their osmotic concentration. With the exception of s-collidine, the buffers were impermeant, as shown by the stability of the reduced turgor. The fixatives formaldehyde and glutaraldehyde caused a rapid reduction in turgor followed by recovery of turgor as the aldehyde penetrated the cell. Plasmolysis of cells can be avoided by a procedure involving stepwise increase of fixative concentration.     

利用原子力显微技术对神经胶质细胞超微结构及其相互间的纳米连接结构的观察

本研究利用原子力显微技术（AFM）观察原代培养的神经胶质细胞及其相互间的纳米连接结构。选择生长良好的神经胶质细胞用戊二醛固定30分钟,固定于AFM基底上进行扫描成像,用AFM脱机软件（SPM OFFLINE 2．20）进行检测。观察到胶质细胞平铺于培养皿的底部,胞体形状不规则,表面较扁平。突起丰富,但没有极性,无轴突树突之分,还观察到两胶质细胞间存在长程纤维管状连接结构。     

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.     

Alteration of cartilage matrix morphology with histological processing

An interlacunar network in the extracellular matrix of femoral head articular cartilage of neontal rats was seen by light microscopy to: (1) consist of elements, 0·5 μm thick, which occurred as individual elements, as bundles of elements, and as fused elements, (2) stain intensely with toluidine blue, methylene blue, and safranin O, and (3) connect chondrocytes by inserting on the chondrocyte capsules which were composed of morphologically and cytochemically similar material. By electron microscopy, the single elements were seen to be composed of thicker, denser staining areas of the honeycomb appearing matrix and the fused elements appeared as non-membrane bound channels containing granular material. Articular cartilage was processed using combinations of fixatives, dehydrating agents, and embedding media. Regardless of fixation, demineralization, or embedding, the network was not seen after dehydration of the cartilage with methanol, ethanol, acetone or tert-butanol but was seen after dehydration with aqueous solutions of glycol methacrylate, propylene oxide, 2-propanol or 2,2-dimethoxypropane. Network visualization following a variety of methods demonstrated that no single fixative, dehydrating agent, or embedding medium caused its formation. The presence of the network in different cartilage zones, its consistent morphology by light and electron microscopy, the uniformity of the elements in their connection with the chondrocytes, and presence in fresh-frozen sections suggest the network may be real, but rigorous evidence for its existence in vivo is still required. Since cartilage morphology was altered by histological methods, especially dehydration, common methods used in studying connective tissue matrix should be evaluated to determine their effect on matrix morphology.     

Identification and ultrastructural imaging of photodynamic therapy-induced microfilaments by atomic force microscopy

Atomic force microscopy (AFM) is an emerging technique for imaging biological samples at subnanometer resolution; however, the method is not widely used for cell imaging because it is limited to analysis of surface topology. In this study, we demonstrate identification and ultrastructural imaging of microfilaments using new approaches based on AFM. Photodynamic therapy (PDT) with a new chlorin-based photosensitizer DH-II-24 induced cell shrinkage, membrane blebbing, and reorganization of cytoskeletons in bladder cancer J82 cells. We investigated cytoskeletal changes using confocal microscopy and atomic force microscopy. Extracellular filaments formed by PDT were analyzed with a tandem imaging approach based on confocal microscopy and atomic force microscopy. Ultrathin filaments that were not visible by confocal microscopy were identified as microfilaments by on-stage labeling/imaging using atomic force microscopy. Furthermore, ultrastructural imaging revealed that these microfilaments had a stranded helical structure. Thus, these new approaches were useful for ultrastructural imaging of microfilaments at the molecular level, and, moreover, they may help to overcome the current limitations of fluorescence-based microscopy and atomic force microscopy in cell imaging.