Oolong tea extract as a substitute for uranyl acetate in staining of ultrathin sections

In conventional transmission electron microscopy, uranyl acetate staining is used to enhance the cellular components. However, uranyl acetate is considered a radioactive material that is very toxic if ingested or inhaled and subject to restrictions in many countries. In an attempt to introduce a substitute for uranyl acetate, we evaluated oolong tea extract (OTE) for staining of ultrathin sections. Tissue sections from normal rat liver representing an ideal model organ were processed according to a routine electron microscopic fixation and embedding procedure. Serial ultrathin sections were cut and processed with either routine double electron staining or 0.2% OTE staining for 30–40 min at room temperature followed by lead citrate staining (OTE staining method). Transmission electron microscopy observations revealed that all sub‐cellular structures in hepatocytes were clearly visible with OTE staining and the quality of staining was highly compatible with those of routine double staining methods. It is suggested that OTE could be used as a non‐radioactive and hazard‐free substitute for uranyl acetate in transmission electron microscopy staining.     

LR White sections as slot grid support films for transmission electron microscopy

The utility of LR White sections as slot grid support films for the examination of thin resin‐embedded tissue sections by transmission electron microscopy was investigated and compared with traditional formvar‐carbon films. Throughout a variety of staining procedures, which involved the use of organic solvent, oxidizing agents, strong acid and prolonged incubation, LR White support films remained intact and the attached tissue sections remained adherent. By contrast, complete loss of formvar‐carbon support films occurred in 25% of preparations during routine staining with aqueous reagents. This loss increased to 62% following staining with either alcoholic or oxidizing and acidic stains, and to 66% following prolonged (immunohistochemical) staining. Tissue contrast, ultrastructural detail and immunohistochemical staining intensity were comparable between sections on the two types of support film. The use of LR White sections as support films for slot grids represents a quick, cheap, simple and robust alternative to traditional support films and, furthermore, requires no carbon coating     

Microwaves and tea: new tools to process plant tissue for transmission electron microscopy

Optimizing sample processing, reducing the duration of the preparation of specimen, and adjusting procedures to adhere to new health and safety regulations, are the current challenges of plant electron microscopists. To address these issues, plant processing protocols for TEM, combining the use of polyphenolic compounds as substitute for uranyl acetate with microwave technology are being developed. In the present work, we optimized microwave-assisted processing of different types of plant tissue for ultrastuctural and immunocytochemical studies. We also explored Oolong tea extract as alternative for uranyl acetate for the staining of plant samples. We obtained excellent preservation of cell ultrastructure when samples were embedded in epoxy resin, and of cell antigenicity, when embedded in LR-White resin. Furthermore, Oolong tea extract successfully replaced uranyl acetate as a counterstain on ultrathin sections, and for in block staining. These novel protocols reduce the time spent at the bench, and improve safety conditions for the investigator. The preservation of the cell components when following these approaches is of high quality. Altogether, they offer significant simplification of the procedures required for electron microscopy of plant ultrastructure.     

Microwave-assisted rapid plant sample preparation for transmission electron microscopy

The preparation of plant leaf material for transmission electron microscopical investigations can be a very time- and labour-consuming task as the reagents infiltrate the samples quite slowly and as usually most steps have to be performed manually. Fixation, buffer washes, dehydration, resin infiltration and polymerization of the resin-infiltrated leaf samples can take several days before the specimen can be cut ultrathin and used for ultrastructural investigations. In this study, we present a microwave-assisted automated sample preparation procedure that reduces preparation time from at least 3 days to about 5 h – with only a few steps that have to be performed manually – until the plant sample can be ultrathin sectioned and observed with the transmission electron microscope. For studying the efficiency of this method we have compared the ultrastructure of different leaf material ( Arabidopsis thaliana , Nicotiana tabacum and Picea abies ) which was prepared with a conventional, well-established chemical fixation and embedding protocol and a commercially available automated microwave tissue processor. Despite the massive reduction in sample preparation time no negative effects on cutting properties of the blocks, stability of the sections in the electron beam, contrast and ultrastructure of the cells were observed under the transmission electron microscope when samples were prepared with the microwave-assisted protocol. Additionally, no negative effects were detected on the dimensions of fine structures of grana stacks (including membranes, inter- and intrathylakoidal spaces), the nuclear envelope and the plasma membrane as the diameter of these structural components did not differ between leaf samples (of the same species) that were processed with the automated microwave tissue processor or by conventional fixation and embedding at room temperature.     

Reflection contrast microscopy for high resolution detection of (3)H-estradiol in ultrathin sections of human stratum corneum

A single autoradiographical method for light and electron microscopy (LM and EM) is presented. Human skin, containing (3)H-estradiol ((3)H-E2) after an in vitro permeation experiment, was processed via a non-extractive tissue preparation protocol, comprising cryo-fixation, freeze-drying, osmium tetroxide vapor fixation, and Spurr resin embedding. Semithin sections were processed for LM autoradiography, while ultrathin sections were processed both for high-resolution LM and EM autoradiography. The autoradiographs were visualized by bright-field microscopy (BFM), reflection contrast microscopy (RCM), and transmission electron microscopy to evaluate the potentials of RCM visualization in high-resolution LM autoradiography. RCM visualization of ultrathin vs. semithin resin sections showed an improved stratum corneum morphology. Histological staining was superfluous. The localization of (3)H-E2 in human stratum corneum using high-resolution LM autoradiography and RCM was as accurate as with high-resolution EM autoradiography.     

Flat embedding and immunolabelling of SW 1116 colon carcinoma cells in LR white: an improved technique in light and electron microscopy.

Human SW 1116 colon carcinoma cells were grown on matrix-covered coverslips and flat embedded in specially prepared gelatin capsules in the hydrophylic resin LR White. Dehydration and polymerization were carried out so as to maximize preservation of antigenicity. Sections were cut perpendicular to the substratum. To visualize mucin, semithin sections of SW 1116 cells were stained with periodic acid Schiff (PAS) reagent for light microscopy, and ultrathin sections were labelled with a monoclonal mucin antibody (Mab 19-9) and immunogold for electron microscopy. Immunofluorescence was carried out on whole cultured cells using Mab 19-9. The morphological preservation of SW 1116 cells embedded in LR White was comparable to that of Epon-embedded cells. Mucin was localized on the microvillar surface of the apical plasma membrane and occasionally in intercellular spaces between adjacent cells. Mucin was also present in vesicles in the apical and lateral part, and to a lesser extent in the basal part of the cells. We conclude that this new technology significantly improves the morphological preservation of cells and tissues in LR White, while also serving to sustain the antigenicity of cellular antigens.     

Pure optical contrast in scattering-type scanning near‐field microscopy

A simple contrast enhancement method is presented for Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution. The sections were treated with an acidified potassium permanganate oxidizing solution followed by uranyl acetate and lead citrate staining. The method, designated KMnO₄–UA/Pb staining, provided a much greater contrast in electron microscopy than conventional UA/Pb staining. In detail, the visibility of plasma membrane was especially improved and the nuclear heterochromatin, mitochondria and cytoplasmic ribosomes showed an adequate increase in electron density. In the mucous cells of rat Brunner's glands, the Golgi cisternae were well defined with the KMnO₄–UA/Pb staining. Interestingly, the membranes of the intermediate compartments were moderately reactive to the KMnO₄–UA/Pb staining, whereas the cis and the trans compartments were only faintly stained. It should be emphasized that the KMnO₄ oxidation following colloidal gold labelling did not cause a remarkable reduction of immunogold labelling and the enhanced contrast helped us to examine the gold particles with high accuracy. This contrast enhancement method is highly promising, with the potential to become a useful tool for histochemical investigation, including immunocytochemistry with the Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution techniques.     

Comparison of manual and automatic processing of biological samples for electron microscopy

The ultrastructure of a sample can be observed by electron microscopy (EM), which has become an indispensable research tool in morphological studies. However, EM sample preparation techniques are complicated and time‐consuming, with a high labor cost. The current study was conducted to compare the conventional manual and automated methods for sample processing and post‐staining for electron microscopy. Automated sample processing reduces OsO₄ contamination, improves the efficiency of sample preparation and is easy to use. Therefore, the results of their study provide a practical and feasible method for the preparation of biological samples for electron microscopy.     

Immunohistochemical myofiber typing and high-resolution myofibrillar lesion detection in LR white embedded muscle

We have developed a method of fixing, embedding, sectioning, and staining that allows high-resolution detection of myofibrillar structure and myosin immunocytochemical muscle fiber typing in serial semithin sections of LR White plastic embedded muscle at the light microscopic level. Traditional approaches, such as cryostat sections, permit fiber typing, but small myofibrillar lesions (1-3 sarcomeres) are difficult to detect because of section thickness. Semithin sections of hydrophobic resins do not stain well either histochemically or immunocytochemically. Electron microscopy can resolve lesions and discriminate fiber types based on morphology, but the sampling area is small. Our goal was to develop a rapid method for defining both fiber type and high-resolution primary myofibrillar lesion damage. Mild fixation (1-4% paraformaldehyde, 0. 05-0.1% glutaraldehyde) and embedment in a hydrophilic resin (LR White) were used. Myofibrillar structure was extremely well preserved at the light microscopic (LM) level, and lesions could be readily resolved in Toluidine blue stained 500-nm sections. Fiber type was defined by LM immunomyosin staining of serial plastic semithin sections, which demonstrated reciprocal staining patterns for "fast (Sigma M4276) and "total" (skeletal muscle) myosins (Sigma M7523).     

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.     

A novel technique for flat-embedding cryofixed plant specimens in LR white resin

Acrylic LR White is used preferentially for many research applications because it possesses unique advantages over other commercially available resin types. LR White has low toxicity; its low viscosity and hydrophilic properties enable it to infiltrate specimens with comparative ease, and specimens embedded in LR White take up stains for light microscopy well and tend to retain superior antigenicity relative to other resins. These qualities make LR White a good choice for immunohistochemistry, light microscopy, and transmission electron microscopy. However, LR White does not heat-polymerize in the presence of oxygen, which precludes its use with many commercially available embedding molds. Furthermore, flat-embedding specimens in LR White is often an arduous task. Therefore, in this paper, we report on the development of novel flat-embedding chambers for use with LR White. Another goal of our studies was the rapid fixation of larger specimens. Chemical fixation was inadequate because of the time required for tissue infiltration, but cryofixation quickly and effectively immobilized intracellular organelles. Mean amyloplast positions differed in vertically oriented versus control specimens after chemical fixation, whereas no significant difference was observed after cryofixation. Furthermore, cryofixation provided acceptable preservation at the light microscopy level, even though our specimens were relatively larger. This underscores the utility of cryofixation for light microscopy determination of organelle positioning within plant cells.     

Atomic force microscopy of histological sections using a chemical etching method

Physiology and pathology have a big deal on tissue morphology, and the intrinsic spatial resolution of an atomic force microscope (AFM) is able to observe ultrastructural details. In order to investigate cellular and subcellular structures in histological sections with the AFM, we used a new simple method for sample preparation, i.e. chemical etching of semithin sections from epoxy resin-embedded specimens: such treatment appears to melt the upper layers of the embedding resin; thus, removing the superficial roughness caused by the edge of the microtome knife and bringing into high relief the biological structures hidden in the bulk. Consecutive ultrathin sections embedded in epoxy resin were observed with a transmission electron microscope (TEM) to compare the different imaging properties on the same specimen sample. In this paper we report, as an example, our AFM and TEM images of two different tissue specimens, rat pancreas and skeletal muscle fibres, showing that most of the inner details are visible with the AFM. These results suggest that chemical etching of histological sections may be a simple, fast and cost-effective method for AFM imaging with ultrastructural resolution.     

Iso-butanol saturated water: a simple procedure for increasing staining intensity of resin sections for light and electron microscopy

The addition of the alcohol iso‐butanol (2‐methylpropan‐1‐ol) to water was found to improve the post‐staining procedures for semi‐thin and ultrathin resin sections, for both light and electron microscopy. Stain penetration was enhanced with samples embedded in both acrylic and epoxy resins and provided structural information not previously seen. These improvements were found with general (non‐specific) stains and a fluorescence stain for light microscopy, as well as for a range of heavy metal stains for electron microscopy. The use of this water/solvent medium also gave improved results when used in a variety of immunogold labelling procedures, resulting in a greater specific label density without affecting background gold levels. The use of this solvent/water medium may have wider applications for other types of staining.     

The biological reality of the interlacunar network in the embryonic,cartilaginous, skeleton: A thiazine dye/absolute ethanol/LR White resin protocol for visualizing the network with minimal tissue shrinkage

Third toe phalanges of chicks aged 8–13 days in ovo and 7-day post-natal rat femoral growth plate were examined to determine whether the interlacunar network (IN), a structure with no lipoprotein membrane component or cytoplasmic organelles, is a genuine component of young growth cartilage. In chick phalanges dehydrated by 70% (v/v) ethanol and LR White resin, variable metachromatic staining of the interlacunar network by toluidine blue and red staining by picro-Sirius red indicate the presence of glycosaminoglycans and collagen. The network in phalanges dehydrated by 80% (v/v) ethanol appears little different; however, the network is much less widely detectable in phalanges dehydrated by 90% (v/v) ethanol and, after dehydration by absolute ethanol, is almost completely undetectable. In contrast, when the young cartilage is permeated by a thiazine dye such as toluidine blue, using a solution of dye in the aldehyde fixative, the network is widely detectable, following dehydration by absolute ethanol, both in chick phalanges and in rat growth plate. Comparison of projected areas shows that the extent to which whole chick feet are found to have shrunk, by the time that they are photographed under LR White resin, is determined principally by the extent of dehydration, by 70% (v/v) or absolute ethanol; post-shrinkage areas are 33% or 35% of areas measured in buffer for 70% (v/v) ethanol/LR White resin and 71% or 75% for absolute ethanol/LR White resin (the higher value in each is for the toluidine blue treatment). The network is thus present in radically shrunk tissue, but, significantly, is also fully represented in tissue shrunk by only a conventional margin and is therefore not produced as an artefact by exceptional tissue shrinkage as has been suggested.     

Embedding in Spurr's resin is a good choice for immunolabelling after freeze drying as shown with chemically unfixed dendritic cells

Immunocytochemical reactions on biological specimens depend on many factors, the most crucial one being the maintenance of antigenicity. Antigens are vulnerable at each stage during preparation for electron microscopy. One of the least traumatic methods of preparing biological tissues for post‐embedding immunolabelling includes the following steps: (1) physical stabilization of the native biological material by rapid freezing (cryofixation) and keeping the immobilized biological sample at low temperature, thereby avoiding any movements of water, ions and macromolecules; (2) dehydrating the frozen biological material by freeze‐drying at low temperature; (3) embedding of the dehydrated specimen. Here we show that embedding of chemically unfixed dendritic cells in Spurr's resin after cryofixation and freeze‐drying enables the conservation of fine ultrastructure without cell distortion or shrinkage. Furthermore, we demonstrate the feasibility of protein localization in ultrathin sections by immunolabelling of the major histocompatibility class II molecules.     

Application of ion etching to immunoscanning electron microscopy

A method for achieving both the light and electron microscopic observations of the same immunolabeled semithin section is described. Mild ion etching (IE) was performed on the semithin LR white resin sections of rat pancreas to evaluate conditions for scanning electron microscopic secondary electron image observations. Before immunocytochemical staining, very mild, rapid etching was conducted as follows: ionization voltage 300 V, operating vacuum 35 Pa, and etching time 1 min, employing an ion coater above sections on glass slides. The sections were immunohistochemically stained with anti-insulin and immunogold in association with silver enhancement techniques for light microscopic observation, in which B cells in pancreatic islets were positively stained brown. Subsequently, essential mild IE was performed over the stained section as follows: 350 V, 38 Pa, 29 min. The samples were coated with platinum for scanning electron microscopic secondary electron images, in which the cores of secretory granules of the B cells were positively labeled with gold-silver particles. The present method is suitable for detection of substances involving immunogold labeling. It enables us to obtain high-resolution images at low magnification that can be correlated with light microscopic observations. Middle to high magnifications are applicable for detailed observations with secondary electron imaging scanning electron microscopy.     

An easy method for orientated embedding tissue culture cell monolayers in LR white resin for postembedding immunocytochemistry

Partial polymerization of LR White resin blocks is a frequently encountered problem if oxygen is present during the polymerization reaction. Instructions are given for a simple method to embed cell culture monolayer in LR White acrylic resin, which is suitable for immunocytochemistry. The advantage of this method is that it is easy and reliable. It also ensures accurate orientation of the cell monolayer in relation to the desired plane of sectioning.     

A new method for investigating the undersurface of cell monolayers by scanning electron microscopy

Glow discharge is commonly used for cleaning the inside of coating units and for cleaning hard surfaces before carbon or metal evaporation procedures. In this study it has been used to remove the embedding medium to reveal, for scanning electron microscope (SEM) study, the undersurfaces of Balb/c 3T3 fibroblastic cells that have been cultured on Thermanox discs and embedded in LR White resin. Ten to twenty-minute ionization times were found to reveal the largest area of the undersurface without causing damage to the cells. Chemical etching of the resin was also shown to reveal the undersurface of the cells, but caused some damage, preventing successful re-embedding for transmission electron microscopy, and at higher magnifications revealed less detail. A circular impression within the main outline of the cells was observed in many cells, which is considered to reflect the presence of a nucleus. The undersurfaces of most cells, after applying both methods of etching, displayed a number of very short processes. Subsequent transmission electron microscopy of ultrathin sectioned, re-embedded, areas of the gold sputter-coated blocks revealed the depth of ionization that had occurred and confirmed that the specimens observed in SEM were the undersurfaces of cells. This method can be modified to study the attaching surface of any organism to a substratum.     

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

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

Freeze-substitution and low-temperature embedding of dairy products for transmission electron microscopy

Dairy products are comprised largely of fat, air and water, which makes it difficult to preserve their ultrastructure for electron microscopy. Keeping the samples frozen throughout fixation and embedding protects the structure and distribution of the components of emulsions and foams. Therefore, dairy products were freeze‐substituted and embedded at low temperature (?20 °C) to prepare them for transmission electron microscopy. Whipped cream, ice cream mix and dairy/non‐dairy mixed systems were frozen by plunging in propane, at its boiling point (?187 °C). Ice cream, because it is already frozen, was fractured into 1‐mm³ pieces in liquid nitrogen and then added to frozen fixative (?196 °C). Fixative solution consisted of glutaraldehyde, osmium tetroxide and uranyl acetate dissolved in either methanol or acetone. When material was to be stained after sectioning the fixative was limited to glutaraldehyde in methanol. The temperature was increased step‐wise from ?80 to ?20 °C. Solvent was replaced with resin; the polar resin Lowicryl HM4, the non‐polar resin Lowicryl HM20, LR White and LR Gold were tested. Samples were embedded and polymerized at ?20 °C using ultraviolet light to cross‐link the resin. Methanol proved to be the most effective solvent for substituting the ice; the hydrophobic resin Lowicryl HM20 was the most effective resin for retaining fat structure following osmium fixation.