Ultra‐thin resin embedding method for scanning electron microscopy of individual cells on high and low aspect ratio 3D nanostructures

The preparation of biological cells for either scanning or transmission electron microscopy requires a complex process of fixation, dehydration and drying. Critical point drying is commonly used for samples investigated with a scanning electron beam, whereas resin‐infiltration is typically used for transmission electron microscopy. Critical point drying may cause cracks at the cellular surface and a sponge‐like morphology of nondistinguishable intracellular compartments. Resin‐infiltrated biological samples result in a solid block of resin, which can be further processed by mechanical sectioning, however that does not allow a top view examination of small cell–cell and cell–surface contacts. Here, we propose a method for removing resin excess on biological samples before effective polymerization. In this way the cells result to be embedded in an ultra‐thin layer of epoxy resin. This novel method highlights in contrast to standard methods the imaging of individual cells not only on nanostructured planar surfaces but also on topologically challenging substrates with high aspect ratio three‐dimensional features by scanning electron microscopy.     

A new method for cell culture on an electron-transparent melamine foil suitable for successive LM,TEM and SEM studies of whole cells

A new cell culture technique is described which is based on the observation that foils cast from the melamine resin hexamethylol-melamine-ether are suitable for the cultivation of beating heart muscle cells and fibroblasts of the rat. This foil can be flamed for sterilization, is about 80 nm in thickness, homogeneous and smooth, withstands dehydration and critical point-drying, can be removed from glass and permits the imaging of whole cells successively by light microscopy, transmission and scanning electron microscopy. The method is capable of narrowing the gap between light and electron microscopy, yielding excellent whole cell preparations in various kinds of microscopic studies to be performed on one and the same cell.     

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.     

Use of microwave fixation in the preparation of cell cultures for observation with the scanning electron microscope

This paper describes a method for primary fixation of cultured cells for scanning (SEM) and transmission (TEM) electron microscopy using microwaves alone. This method of fixation takes 8 seconds and is therefore quicker and less expensive than conventional fixation techniques. The preservation of cell morphology is excellent and cultures of mammalian immune system cells and peripheral nervous tissue have been examined using this fixation.     

Morphologic and temporal analysis of vascular smooth muscle cell apoptosis induced by c-Myc and E1A

Apoptosis is a physiologic form of cell death present in many disease conditions. When the balance of mitosis versus apoptosis is altered, tumor-like growth or degeneration of tissues may ensue. This appears to occur in several diseases, including those of the cardiovascular system, where apoptosis plays a key role in atherosclerosis and restenosis following angioplasty. Since c-myc is upregulated in the pathogenesis of these diseases, we chose to study the sequential morphologic features of programmed cell death in vascular smooth muscle cells induced by c-myc and by the adenovirus early gene E1A. Morphology and timed events in apoptotic cell cultures were analyzed by scanning electron microscopy, transmission electron microscopy, and time-lapse videomicroscopy. We observed that both c-myc-and E1A-induced apoptosis (in serum-free medium) resulted in numerous, tightly packed clusters of apoptotic blebs, as well as in one or two asymmetrically larger blebs. Transmission electron microscopy analysis revealed the larger blebs contained mostly nuclear chromatin, whereas the many smaller fragments often had little or no chromatin. Time-lapse studies showed that apoptosis was induced at a slower rate in cells stably transfected with c-myc versus those stably transfected with E1A. The early changes of apoptosis, including cell shrinkage and intense blebbing, occurred in under 5 min in both cells. Slight alterations such as cell size and further rounding occurred up to 8 h following the initial changes of apoptosis. Rather than being a part of the apoptotic response, release from the culture floor almost entirely resulted from movement of the culture flask. These studies provide a framework of timed morphologic events for future mechanistic investigation into the key aspects of myc-and E1A-induced apoptosis in vascular smooth muscle.     

Distribution and three‐dimensional appearance of the interstitial cells of Cajal in the rat stomach and duodenum

The relationship between the interstitial cells of Cajal (ICC) and enteric nerves or smooth muscles cells is not fully defined. Presently, distribution and appearance of ICC in the rat stomach and duodenum was studied by immunohistochemistry, electron microscopy, and three‐dimensional reconstruction. c‐kit expressing ICC were regularly observed in the Auerbach's myenteric plexus (AP) of the stomach and duodenum. ICC in stomach and duodenum muscle layers was dissimilarly distributed. c‐kit immunoreactive cells were sparsely distributed in the stomach circular muscle layer but were abundant in the duodenum deep muscular plexus (DMP). Electron microscopy revealed that stomach ICC‐AP were irregular ovals with few cytoplasmic processes, and possessed an electron‐dense cytoplasm, numerous mitochondria, intermediate filaments, and caveolae. Duodenum and stomach ICC‐AP were similar in appearance. Ultrastructure observations and three‐dimensional reconstructions revealed ICC‐AP processes wrapping the nerve fibers and projecting into the space between smooth muscle cells. While ICC‐AP was occasionally close to enteric nerves or smooth muscle cells, no connections were observed. ICC‐DMP in duodenum was elongated and adopted the same cell axis orientation as the circular muscle cells. Unlike ICC‐AP, ICC‐DMP formed gap junctions with smooth muscle cells and had close contact with nerves. These results indicate that ICC‐AP is regularly distributed in stomach and duodenum, while ICC‐DMP is exclusively located in the duodenum. ICC‐DMP, which possess gap junctions and closely contacts nerves, may participate in neuromuscular transmission. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.     

Comparing different methods to fix and to dehydrate cells on alginate hydrogel scaffolds using scanning electron microscopy

Scanning electron microscopy (SEM) is commonly used in the analysis of scaffolds morphology, as well as cell attachment, morphology and spreading on to the scaffolds. However, so far a specific methodology to prepare the alginate hydrogel (AH) scaffolds for SEM analysis has not been evaluated. This study compared different methods to fix/dehydrate cells in AH scaffolds for SEM analysis. AH scaffolds were prepared and seeded with NIH/3T3 cell line; fixed with glutaraldehyde, osmium tetroxide, or the freeze drying method and analyzed by SEM. Results demonstrated that the freeze dried method interferes less with cell morphology and density, and preserves the scaffolds structure. The fixation with glutaraldehyde did not affect cells morphology and density; however, the scaffolds morphology was affected in some level. The fixation with osmium tetroxide interfered in the natural structure of cells and scaffold. In conclusion the freeze drying and glutaraldehyde are suitable methods for cell fixation in AH scaffold for SEM, although scaffolds structure seems to be affected by glutaraldehyde. Microsc. Res. Tech. 78:553–561, 2015. © 2015 Wiley Periodicals, Inc.     

Gridded Aclar: preparation methods and use for correlative light and electron microscopy of cell monolayers,by TEM and FIB–SEM

Aclar, a copolymer film with properties very similar to those of tissue culture plastic, is a versatile substrate to grow cells for light (including fluorescence) and electron microscopic applications in combination with both chemical fixation and cryoimmobilization. In this paper, we describe complete procedures to perform correlative light and electron microscopy using Aclar as substrate for the culture of cell monolayers to be finally embedded in plastic. First, we developed straightforward, efficient and flexible ways to mark the surface of the Aclar to create substrates to locate cells first at the light microscopy and then the electron microscopy level. All the methods enable the user to self‐design gridded Aclar pieces, according to the purpose of the experiments, and create a large number of substrates in a short time. Second, we confirmed that marked Aclar supports the normal growth and morphology of cells. Third, we validated the correlative light and electron microscopy procedure using Aclar. This validation was done for the high‐resolution analysis of endothelial cells using transmission electron microscopy and focused ion beam–scanning electron microscopy in combination with the use of fluorescence, phase contrast and/or bright field microscopy to map areas of interest at low resolution. The methods that we present are diverse, easy to implement and highly reproducible, and emphasize the versatility of Aclar as a cell growth substrate for diverse microscopic applications.     

Membrane blisters: A fixation artifact a study in fixation for scanning electron microscopy

It is been shown by scanning electron microscopy that fixation in glutaraldehyde followed by fixation in osmium tetroxide results in the presence of membrane blisters on the surface of a variety of cells. Fixation in glutaraldehyde alone or osmium tetroxide alone does not result in such extensive artifacts. The blisters, usually 0.2–0.6 μm in diameter, are seen by transmission electron microscopy to be membrane-bound, virtually empty vesicles. It is concluded that the optimum preservation of the cell surface for scanning electron microscopy is provided by fixation in glutaraldehyde alone.     

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.     

Functionalized magnetic nanoparticles attenuate cancer cells proliferation: Transmission electron microscopy analysis

The penetration and transportation of nanoparticles (NPs) inside the cancer cells is critical to study. In this article, cancer cells (HCT‐116) were treated with functionalized magnetic NPs for the period of 48 hr and studied their ultrastructure by transmission electron microscopy (TEM). The NPs‐treated cells were prepared by chemical fixation and sliced into electron‐transparent arbitrary sections (200 × 200 μm²) by ultramicrotome. Major events of NPs–cell interaction, such as penetration of NPs, encapsulation of NPs into the intracellular compartments, transportation of NPs, and NPs exit, were examined by TEM to understand the mechanism of cell death. The NPs showed the uniform spherical shape with broad size distribution (100–400 nm), while cells displayed irregular morphology with average diameter ~5 μm. Our results showed the successful penetration of NPs deep into the cell, encapsulation, transportation, and exocytosis. Furthermore, we tested the different concentrations (0, 1.5, 12.5, and 50 μg/ml) of NPs on cancer cells and evaluated the cell viability. Laser confocal microscopy and colorimetric analysis together demonstrated that the cell viability is a dose‐dependent phenomenon, where 50 μg/ml specimen showed the highest killing of cancer cells compared to other dosages.     

Doxazosin treatment alters stromal cell behavior and increases elastic system fibers deposition in rat prostate

Doxazosin (DOX), an α‐adrenoceptor antagonist, induces the relaxation of smooth muscle cell tonus and reduces the clinical symptoms of benign prostatic hyperplasia (BPH). However, the effects of DOX in the prostate stromal microenvironment are not fully known. In a previous study, we showed that DOX treatment for 30 days increased deposition of collagen fibers in the three rat prostatic lobes. Herein, we investigated the effects of DOX on stromal cell ultrastructure and elastic fiber deposition. Adult Wistar rats were treated with DOX (25 mg/kg/day); and the ventral, dorsal, and anterior prostates were excised at 30 days of treatment. The prostatic lobes were submitted to histochemical and stereological‐morphometric analyze and transmission electron microscopy (TEM). Histochemical staining plus stereological analysis of the elastic fiber system showed that DOX‐treated prostatic lobes presented more elaunin and elastic fibers than controls, mainly in the ventral lobe. Ultrastructural analysis showed that fibroblasts and smooth muscle cells from DOX‐treated prostates presented active synthetic phenotypes, evidenced by enlarged rough endoplasmic reticulum and Golgi apparatus cisterns, and confirmed the observation of thickened elaunin fibers. Our findings suggest that, under α‐adrenergic blockade by DOX, the fibroblasts become more active and smooth muscle cells shift from a predominantly contractile to a more synthetic phenotype. The deposition of collagen and elastic system fibers in the prostatic stroma may counterbalance the absence of smooth muscle tone during α‐blockers treatment. Microsc. Res. Tech. 73:1036–1044, 2010. © 2010 Wiley‐Liss, Inc.     

Imaging analysis of the interface between osteoblasts and microrough surfaces of laser‐sintered titanium alloy constructs

Previous work using focused ion beam (FIB) analysis of osteoblasts on smooth and microrough Ti surfaces showed that the average cell aspect ratio and distance from the surface are greater on the rough surface. In order to better interrogate the relationship between individual cells and their substrate using multiple imaging modalities, we developed a method that tracks the same cell across confocal laser scanning microscopy (CLSM) to correlate surface microroughness with cell morphology and cytoskeleton; scanning electron microscopy (SEM) to provide higher resolution for observation of nanoroughness as well as chemical mapping via energy dispersive X‐ray spectroscopy; and transmission electron microscopy (TEM) for high‐resolution imaging. FIB was used to prepare thin sections of the cell‐material interface for TEM, or for three‐dimensional electron tomography. Cells were cultured on laser‐sintered Ti‐6Al‐4V substrates with polished or etched surfaces. Direct cell to surface attachments were observed across surfaces, though bridging across macroscale surface features occurred on rough substrates. Our results show that surface roughness, cell cytoskeleton and gross morphology can be correlated with the cell‐material cross‐sectional interface at the single cell level across multiple high‐resolution imaging modalities. This work provides a platform method for further investigating mechanisms of the cell‐material interface.     

Identification and study of the same cell in monolayer culture by different methods of light microscopy,scanning, and transmission electron microscopy. Application for cryodamaged cells examination

Cells were cultivated on transparent conductive substrates, glass slides coated with indium oxide; individual cells were marked with a diamond indentor. Cell cultures were frozen (–15°C), thawed, and then stained with fluorescent dyes to determine cell damage. The marked cells were examined by phase contrast, fluorescence, and Nomarski DIC microscopy. After aldehyde and osmium tetroxide fixation, the cell preparations were sequentially treated with tannic acid, uranyl acetate, and lead citrate. The same marked cell could be sequentially studied by light microscopy (LM; in water immersion conditions), scanning electron microscopy (SEM; after dehydration and critical point drying), and transmission electron microscopy (TEM; after embedding of cell samples in epoxy resin and laser marking of the cell previously marked with a diamond indentor). The method used ensures good preservation of cell morphology, cell surface relief, and intracellular structures. The treatment used renders the cells conductive and permitted SEM of uncoated culture cells on conductive substrates.     

Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.     

Specific labeling of connexin43 in NRK cells using tyramide-based signal amplification and fluorescence photooxidation

Imaging of gap junction proteins, the connexins, has been performed in tissue culture cells both by labeling of connexins with immunocytochemical tags and by cloning and expressing chimeras of connexins and fluorescent proteins such as Green Fluorescent Protein. These two approaches have been used to gain information about protein localization or trafficking at light microscopic resolution. Electron microscopy provides higher resolution; however, analysis of electron micrographs of unlabeled connexins has been generally limited to recognition of gap junction structures. Immunolabeling of gap junction proteins in whole cells at the electron microscopic level has been difficult to achieve because of the fixation sensitivity of most gap junction antibodies. To obtain reasonable sensitivity, immunoperoxidase procedures are typically employed, and these suffer from relatively poor resolution. Here we describe the combination of tyramide signal amplification techniques and fluorescence photooxidation for higher resolution immunolocalization studies for correlative light and electron microscopic imaging. By using correlative microscopy, we can not only localize connexin pools or structures, but also discover what other cellular substructures interact with gap junction proteins. The use of tyramide signal amplification techniques is necessary to increase fluorescence levels that have decreased due to increased specimen fixation required to maintain cell ultrastructure. The fluorescence photooxidation technique provides a high-resolution method for staining of proteins in cells. Unlike colloidal gold-based methods, fluorescence photooxidation allows for three-dimensional localization using high-voltage electron microscopy.     

A procedure for minimizing cellular shrinkage in electron microscope preparation: a quantitative study on frog gall bladder

Nomarski differential interference contrast microscopy of whole-mount preparations of frog gall bladders has been used for light microscopic measurement of epithelial cell luminal area. Measurements were performed: in buffer after fixation, and in cured Epon after step-by-step and after continuous dehydration and infiltration procedures. Sections cut perpendicularly to the luminal surface were used for the measurement of epithelial cell height. From these measurements epithelial cell mean volumes were calculated. The comparative measurements showed that a step procedure results in significantly smaller cellular dimensions than does a procedure of slow and continuous change of concentration of the preparative media, the difference in epithelial cell mean volume being about 40%. Electron microscopy of thin sections of continuously processed specimens showed superior ultrastructural preservation of cytoplasmic density and microplical arrangement compared to the step processed counterparts. The principle was successfully applied in processing for scanning electron microscopy of osmotically extremely fragile preparations of erythrocyte ghosts; similarly, formation of the luminal cobblestone appearance of gall bladder epithelium was prevented. The present studies support the view that at least part of the volume shrinkage through the procedures of embedding is caused by osmotic gradients set up in the tissue.     

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.     

Influence of tissue composition on the final volume of rat liver blocks prepared for electron microscopy

Methods for measuring and evaluating changes in volume of small tissue blocks prepared for transmission electron microscopy are presented. The results indicate: (1) that some blocks swell and others shrink, and (2) that changes in block volume can be explained by inhomogeneous changes occurring in three different tissue compartments. For stereological studies attempting to extrapolate changes in fixed and embedded tissue to those of fresh tissue, consequences of inhomogeneous volume changes in tissue compartments include a decrease in reliability and an increase in statistical variance of stereological density estimates. Since factors could not be found to correct for the changes in individual tissue compartments, alternative strategies are considered for dealing with the volume artifacts of fixation.