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Beginners guide to sample preparation techniques for transmission electron microscopy
Abstract
Background purpose: The revolution in microscopy came in 1930 with the invention of electron microscope. Since then, we can study specimens on ultrastructural and even atomic level. Besides transmission electron microscopy (TEM), for which specimen preparation techniques will be described in this article, there are also other types of electron microscopes that are not discussed in this review. Materials and methods: Here, we have described basic procedures for TEM sample preparation, which include tissue sample preparation, chemical fixation of tissue with fixatives, cryo-fixation performed by quick freezing, dehydration with ethanol, infiltration with transitional solvents, resin embedding and polymerization, processing of embedded specimens, sectioning of samples with ultramicrotome, positive and negative contrasting of samples, immunolabeling, and imaging. Conclusion: Such collection of methods can be useful for novices in transmission electron microscopy.
... The second part provides an overview of the current research of Croatian scientists and members of the Croatian Microscopy Society, covering a whole range of topics and focusing on plant biology (4,5), zoology (6,7), neuroscience (8) and chemistry (9). The third thematic unit presents a series of methodological review papers (10)(11)(12)(13)(14) that provide a comprehensive overview of various microscopic methods and their applications in different fields of biology and medicine. The volume ends with an exploration of the education methodology in the field of biology (15). ...
We are delighted to present a special issue of Periodicum biologorum,dedicated to the 30th anniversary of the Croatian Microscopy Societyas an independent Society and the 40th anniversary of its foundation.
The post-embedding immunogold (PI) technique for immunolabeling of neuronal tissues utilizing standard thin-section transmission electron microscopy (TEM) continues to be a prime method for understanding the functional localization of key proteins in neuronal function. Its main advantages over other immunolabeling methods for thin-section TEM are (1) fairly accurate and quantifiable localization of proteins in cells; (2) double-labeling of sections using two gold particle sizes; and (3) the ability to perform multiple labeling for different proteins by using adjacent sections. Here we first review in detail a common method for PI of neuronal tissues. This method has two major parts. First, we describe the freeze-substitution embedding method: cryoprotected tissue is frozen in liquid propane via plunge-freezing, and is placed in a freeze-substitution instrument in which the tissue is embedded in Lowicryl at low temperatures. We highlight important aspects of freeze-substitution embedding. Then we outline how thin sections of embedded tissue on grids are labeled with a primary antibody and a secondary gold particle-conjugated antibody, and the particular problems encountered in TEM of PI-labeled sections. In the Discussion, we compare our method both to earlier PI methods and to more recent PI methods used by other laboratories. We also compare TEM immunolabeling using PI vs. various pre-embedding immunolabeling methods, especially relating to neuronal tissue.
Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.
Transmission electron microscopy (TEM) is an ideal device to study the internal structure of cells and different types of biological materials, but adverse conditions inside electron microscopes such as damage induced by electron bombardment and vacuum evaporation of structural water necessitates complex preparation methods to survive this environment. In order to introduce the sample into the evacuated microscope column, it should be stabilized and altered to small enough (about 3 mm in diameter) and thin enough parts to permit the transmission of electrons. Depending on applications different thicknesses are required; for example, in biological research studies usually 300–500 nm thickness is indicated. To stabilize the specimen and preserve the sample structures, different preparation methods are used involving different steps based on the type of study and the specimen, although the ultimate goal of all these preparation technics is to maintain the native structure of the sample. In this chapter, we try to explain the series of steps that involve in preparation. Virtually every step can affect the quality of sample, and therefore it is important to execute each step in detail.
Researchers have used transmission electron microscopy (TEM) to make contributions to
cell biology for well over 50 years, and TEM continues to be an important technology in
our field. We briefly present for the neophyte the components of a TEM-based study,
beginning with sample preparation through imaging of the samples. We point out the
limitations of TEM and issues to be considered during experimental design. Advanced
electron microscopy techniques are listed as well. Finally, we point potential new users
of TEM to resources to help launch their project.
This article presents the best current practices for preparation of biological samples for examination as thin sections in an electron microscope. The historical development of fixation, dehydration, and embedding procedures for biological materials are reviewed for both conventional and low temperature methods. Conventional procedures for processing cells and tissues are usually done over days and often produce distortions, extractions, and other artifacts that are not acceptable for today's structural biology standards. High-pressure freezing and freeze substitution can minimize some of these artifacts. New methods that reduce the times for freeze substitution and resin embedding to a few hours are discussed as well as a new rapid room temperature method for preparing cells for on-section immunolabeling without the use of aldehyde fixatives.
This study describes the subcellular distribution of glutathione in roots and leaves of different plant species (Arabidopsis, Cucurbita, and Nicotiana). Glutathione is an important antioxidant and redox buffer which is involved in many metabolic processes including plant defense. Thus information on the subcellular distribution in these model plants especially during stress situations provides a deeper insight into compartment specific defense reactions and reflects the occurrence of compartment specific oxidative stress. With immunogold cytochemistry and computer-supported transmission electron microscopy glutathione could be localized in highest contents in mitochondria, followed by nuclei, peroxisomes, the cytosol, and plastids. Within chloroplasts and mitochondria, glutathione was restricted to the stroma and matrix, respectively, and did not occur in the lumen of cristae and thylakoids. Glutathione was also found at the membrane and in the lumen of the endoplasmic reticulum. It was also associated with the trans and cis side of dictyosomes. None or only very little glutathione was detected in vacuoles and the apoplast of mesophyll and root cells. Additionally, glutathione was found in all cell compartments of phloem vessels, vascular parenchyma cells (including vacuoles) but was absent in xylem vessels. The specificity of this method was supported by the reduction of glutathione labeling in all cell compartments (up to 98%) of the glutathione-deficient Arabidopsis thaliana rml1 mutant. Additionally, we found a similar distribution of glutathione in samples after conventional fixation and rapid microwave-supported fixation. Thus, indicating that a redistribution of glutathione does not occur during sample preparation. Summing up, this study gives a detailed insight into the subcellular distribution of glutathione in plants and presents solid evidence for the accuracy and specificity of the applied method.
The aim of cytochemical techniques is to localize specific biochemical components in particular tissue and cell compartments. However, since preparation of tissues for structural observation results in major alterations of the properties of their components, a major problem is to retain an adequate degree of their biochemical properties as well as adequate structural preservation. In the present study, we describe results obtained using various colloidal gold cytochemical techniques on tissues processed through different approaches. We found that any manipulation of the tissue during its processing can result in modifications of tissue components, leading to problems in cytochemistry. Indeed, washing of the tissue before fixation, the nature of the fixative solution, the chemical basis of the resins, and the physical conditions of embedding can all introduce changes in tissue components which can be cytochemically demonstrated. This has been illustrated with application of the protein A-gold, lectin-gold, and enzyme-gold cytochemical techniques on tissues submitted to different processings: fixation by perfusion or by immersion; glutaraldehyde vs paraformaldehyde fixative solutions; cryo-ultramicrotomy; embedding in epoxy, GMA, Lowicryl, or LR resins. The results obtained have demonstrated that conditions for optimal labeling must be worked out for each class of binding sites, and that no single procedure can be recommended as THE best approach in cytochemistry.
Immunocytochemistry for transmission electron microscopy provides important information on the location and relative abundance of proteins inside cells. Gaining access to this information without extracting or disrupting the location of target proteins requires specialized preparation methods. Sectioning frozen blocks of chemically fixed and cryoprotected biological material is one method for obtaining immunocytochemical data. Once the cells or tissues are cut, the thawed cryosections can be labeled with specific antibodies and colloidal gold probes. They are then embedded in a thin film of plastic containing a contrasting agent. Subcellular morphology can be correlated with specific affinity labeling by examination in the transmission electron microscope. Modern technical advancements both in preparation protocols and equipment design make cryosectioning a routine and rapid approach for immunocytochemistry that may provide increased sensitivity for some antibodies.
Currently, most cytologists “fix” cells with chemical fixatives for examination with light and electron microscopes. There is little doubt in examining such cells that we have gained enormous information pertaining to cell structure and function. Sadly, however, all but the most critical and astute cytologists are probably unaware that the cells they fix and examine represent little of the way in which the cell existed immediately prior to fixation. Most researchers probably have never examined the cell during the fixation process, and as a result have little appreciation of the violent and dramatic changes that occur over a period of seconds and minutes (frequently up to 30 min) before it is finally stabilized. Not only does the process of chemical fixation take time, but the organization of the cytoplasm is drastically altered during the time required for the fixative to diffuse into the cell (Fig. 1). Various organelles such as nuclei, for example, are frequently described as passing through the septal pore of a fungal hypha when, in fact, they probably were nowhere near the septum prior to fixation. In addition to the alteration in the position of organelles during fixation, considerable membrane blebbing and fusion occurs in the post-fixation process [cf. 23, 24]. Mersey and McCully [38] and others [2, 3, 14, 36, 41, 48] have documented the time-course of fixation as well as the concomitant cytoplasmic distortion for various cells. Most cytologists would gain a great appreciation for the dynamics of the fixation process by reading these and other references on the nature of chemical fixation.
Forssman antigen, a neutral glycosphingolipid carrying five monosaccharides, was localized in epithelial MDCK cells by the immunogold technique. Labeling with a well defined mAb and protein A-gold after freeze-substitution and low temperature embedding in Lowicryl HM20 of aldehyde-fixed and cryoprotected cells, resulted in high levels of specific labeling and excellent retention of cellular ultrastructure compared to ultra-thin cryosections. No Forssman glycolipid was lost from the cells during freeze-substitution as measured by radio-immunostaining of lipid extracts. Redistribution of the glycolipid between membranes did not occur. Forssman glycolipid, abundantly expressed on the surface of MDCK II cells, did not move to neighboring cell surfaces in cocultures with Forssman negative MDCK I cells, even though they were connected by tight junctions. The labeling density on the apical plasma membrane was 1.4-1.6 times higher than basolateral. Roughly two-thirds of the gold particles were found intracellularly. The Golgi complex was labeled for Forssman as were endosomes, identified by endocytosed albumin-gold, and lysosomes, defined by double labeling for cathepsin D. In most cases, the nuclear envelope was Forssman positive, but the labeling density was 10-fold less than on the plasma membrane. Mitochondria and peroxisomes, the latter identified by catalase, remained free of label, consistent with the notion that they do not receive transport vesicles carrying glycosphingolipids. The present method of lipid immunolabeling holds great potential for the localization of other antigenic lipids.
Chemical fixation of cells has been used extensively in different fields of electron microscopy. The type of fixation can be selected to maximize retention of different cell constituents where morphology is of prime interest. For techniques such as immunocytochemistry and autoradiography, a compromise between morphology and retention of antigenicity or radiolabel has to be reached. By careful choice of fixative, buffer and other physical parameters, optimum results can be obtained for both animal and plant tissues. Emphasis has been put on more recent developments in technique.
Electron Microscopy by John Bozzola and Lonnie Russell continues to be a text of choice for instruction in classes that cover beginning electron microscopy for biologists and should also be on the bookshelf of all technologists that work with biological specimens for transmission or scanning EM. The first chapter in the book presents a brief, but interesting, history of the development of instrumentation and techniques for specimen preparation and sets the stage for the wealth of practical information that is presented in the remaining twenty chapters. These chapters are laid out in a logical chronological fashion of exactly what one must do to start with an unfixed specimen and end with a publication quality micrograph.
Glutathione (g-glutamyl-cysteinyl-glycine) is an antioxidant that plays important protective roles during pathogen attack. Glutathione synthesis is a highly compartment-specific pathway and relies on the supply of its precursors cysteine, glutamate, and glycine. To study the importance of glutathione in the development of tolerance and resistance, glutathione and its precursors were quantified with cytohistochemical methods in a highly tolerant cultivar ('Quine') of Cucurbita pepo L. during zucchini yellow mosaic virus (ZYMV) infection. Glutathione concentrations were strongly increased in almost all cell compartments of younger leaves (up to 247% in peroxisomes), older leaves (up to 73% in the cytosol), and roots (up to 104% in nuclei) during ZYMV infection. In addition, glutathione precursor concentrations were generally unchanged or increased in all cell compartments of younger leaves and roots, indicating that glutathione synthesis in these organs was not limited by low levels of glutathione precursors. The lower increase of glutathione in older leaves could be caused by strongly decreased levels of glycine in plastids (42%) and the cytosol (53%) during ZYMV infection, as they are the main centers for glutathione synthesis. Nevertheless, as the increase in glutathione was in general much higher than that observed in a highly susceptible cultivar in previous studies, we can conclude that elevated glutathione concentrations can be strongly correlated with the development of tolerance during ZYMV infection.
Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryo-electron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.
One of the important developments in quantitative electron microscopy has been the application of optical and computer imaging methods to electron micrographs. In general these techniques of image analysis have been applied to electron micrographs from isolated biological structures prepared in the presence of various negative stains. To make full use of image processing techniques there are obvious advantages in preparing suitable specimens containing large areas of repeating features. However, the number of naturally occurring biological specimens exhibiting crystalline or paracrystalline features suitable for high resolution electron microscopy and subsequent image analysis is relatively small.
Some recent experiments on the in vitro formation of crystalline and paracrystalline arrays from highly concentrated and purified isometric, filamentous and rod-like viruses is reviewed. The problems associated with the preparative procedures for producing two-dimensional and three-dimensional crystalline arrays are discussed together with the possibility of extending the negative staining-carbon film method for studying the gradual dissociation or assembly of viral components.
Cryofixation and freeze substitution methods were developed for ultrastructural studies of cells in complex plant tissues. Leaf tissues and root tips of tobacco (Nicotiana tabacum L. var. Maryland Mammoth) were frozen with a RMC MF7200 propane jet freezer and freeze substituted sequentially with tannic acid and osmium tetroxide/uranyl acetate in acetone. High quality preservation was consistently obtained for epidermal and phloem cells of the leaf, and epidermal, cortical, meristematic, and cap cells of the root tip. Leaf mesophyll cells were also often well frozen. Organelles, including nuclei, endoplasmic reticulum, mitochondria, Golgi bodies, and plastids, showed excellent structural integrity and contrast. Most notable is the superior preservation of the cytoskeleton. Our results demonstrate that the propane jet freezer can be used routinely for high quality cryofixation of higher plant cells in certain complex tissues. This could have important implications for the use of cryofixation approach in a wide range of research in plant biology.
The key preparation steps in the Tokuyasu thawed frozen section technique for immunocytochemistry, namely freezing, sectioning, thawing, and drying, were studied. A spherical tissue culture cell was used as a model system. The frozen hydrated section technique indicated that glutaraldehyde-fixed, 2.1 M sucrose-infused pellets of cells were routinely vitrified by immersion in liquid nitrogen but water was crystallized when lower sucrose concentrations (0.6-1 M) were used. Quantitative mass measurements showed that the fixed cells are freely permeable to sucrose. The frozen hydrated sections were severely compressed but cell profiles regained their circular appearance upon thawing. The average section thickness of our frozen-hydrated sections was 110 nm: this was reduced to 30-50 nm upon thawing, washing, and air-drying. This change was accompanied by severe drying artifacts. By using the methyl cellulose drying technique, this collapse upon air-drying could be significantly reduced, but not completely prevented, giving an average thickness of 70 nm.
This chapter describes the practice and application of the thin frozen section technique to the biogenesis of the semliki forest virus (SFV) membrane glycoproteins, and presents a general procedure for making high-titered antiserum to amphiphilic membrane proteins. The results indicate that the spike proteins pass through the Golgi stacks, and antibodies to the spike proteins specifically label all the membrane compartments on the intracellular transport pathway. Parallel biochemical experiments show that the spike proteins acquired complex sugars in the Golgi stacks, but the precise location is not determined. The spike proteins move from cis to trans Golgi cisternae. The spike proteins pass through the Golgi stacks during intracellular transport and are present in all cisternae of the stack. The concentration of spike proteins in endoplasmic reticulum (ER) and Golgi membranes is extremely low. Radiolabeling infected cells with defined specific-activity amino acids show that a typical infected cell, 4-6 hr after infection, is synthesizing about 120,000 spike proteins per minute.
Ultrathin frozen sections can be cut smoothly from many fixed and appropriately treated specimens. To use such sections for immunochemical localization of intracellular antigens, fixation conditions must be selected to optimize at least three variables, namely, preservation of ultrastructure, preservation of antigenicity and retention of accessibility of the antigen to the antibody. Furthermore, staining of the sections must be such that both the immunolabels and structures are clearly recognized. Our efforts to attain these goals are described in relation to their historical background. Although there are still problems to be solved and improvements to be made, we now consider that cryoultramicrotomy has reached the stage of being useful in studying many questions which will not be easily approached otherwise.
Freeze-substitution is a physicochemical process in which biological specimens are immobilized and stabilized for microscopy. Water frozen within cells is replaced by organic solvents at subzero temperatures. Freeze-substitution is widely used for ultrastructural and immunocytochemical analyses of cells by transmission and scanning electron microscopy. Less well recognized is its superiority over conventional chemical fixation in preserving labile and rare tissue antigens for immunocytochemistry by light microscopy. In the postgenome era, the focus of molecular genetics will shift from analyzing DNA sequence structure to elucidating the function of gene networks, the intercellular effects of polygenetic diseases, and the conformational rearrangements of proteins in situ. Novel strategies will be needed to integrate knowledge of chemical structures of normal and abnormal macromolecules with the physiology and developmental biology of cells and tissues from whole organisms. This review summarizes the progress and future prospects of freeze-substitution for such explorations.
The intracellular effects of GSH (reduced glutathione) and BSO (buthionine sulfoximine) treatment on glutathione content were investigated with immunogold labeling in individual cellular compartments of Cucurbita pepo L. seedlings. Generally, GSH treatment led to increased levels of glutathione in roots and leaves (up to 3.5-fold in nuclei), whereas BSO treatment significantly decreased glutathione content in all organs. Transmission electron microscopy revealed that glutathione levels in mitochondria, which showed the highest glutathione labeling density of all compartments, remained generally unaffected by both treatments. Since glutathione within mitochondria is involved in the regulation of cell death, these results indicate that high and stable levels of glutathione in mitochondria play an important role in cell survival strategies. BSO treatment significantly decreased glutathione levels (1) in roots by about 78% in plastids and 60.8% in the cytosol and (2) in cotyledons by about 55% in the cytosol and 38.6% in plastids. After a short recovery period, glutathione levels were significantly increased in plastids and the cytosol of root tip cells (up to 3.7-fold) and back to control values in cotyledons. These results indicate that plastids, either alone or together with the cytosol, are the main center of glutathione synthesis in leaves as well as in roots. After GSH treatment for 24 h, severe ultrastructural damage related to increased levels of glutathione was found in roots, in all organelles except mitochondria. Possible negative effects of GSH treatment leading to the observed ultrastructural damage are discussed.
Transmission electron microscopy (TEM) provides a powerful set of methods to investigate cellular and subcellular structures using thin sections. In this article we summarize some of the different approaches available for researchers interested in using these methods. The essential details involved in specimen preparation for immunolabelling are covered. The best sectioning approach for preserving specimens for structural analysis is Cryo EM of Vitrified Sections (CEMOVIS), a method where still frozen sections are examined in the transmission electron microscope. Because the specimens are kept at low temperature during sectioning and examination, this method is not amenable for immunolabelling, where antibodies are applied to sections at ambient temperature. To combine structural analysis with immunocytochemical analysis of antigens, the approach of freeze-substitution without chemical fixative is the method of choice, at least from a theoretical point of view. In practice, however, the vast majority of electron microscopic (EM) immunocytochemical analyses are carried out using chemically-fixed specimens that have been embedded in specialized resins (such as the Lowicryls) using freeze-substitution or ambient temperature methods. Antibody labelling of thawed cryosections through chemically-fixed specimens (the Tokuyasu method) is also a popular method for preparing cells and tissues for TEM analysis. Here, we provide an overview of all these sectioning methods for EM, focusing mostly on the practical details. Given the space limitation, the fine details necessary to apply these methods have been successfully omitted and will have to be obtained from the technical references we provide.
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