Rapid preparation of plant tissues for electron microscopy

Various plant tissues were prepared for electron microscopy using a rapid process similar to that of Hayat & Giaquinta (1970) for animal tissue. The entire preparation took less than 4 h instead of the several days taken for the usual method and gave very good results. Root, leaf and fruit tissues were used.     

Preparation of biomolecule gel matrices for electron microscopy

We report a new sample preparation method that allows the direct transmission electron microscopy evaluation of the architectural characteristics of biomolecules entrapped in gel matrices. We demonstrate that this sample preparation technique can be used for the identification and ultrastructural characterization of liposomes, collagen I and collagen III embedded in gel matrices, and has the potential to be useful for transmission electron microscopy (TEM) characterization of other biomolecule-gel matrix systems.     

Freeze-fracture for scanning electron microscopy

Two different freeze-fracture methods are explored for preparation of biological material for scanning electron microscopy. In the simpler method the tissues are first fixed and dehydrated. They are then frozen and fractured, and after thawing, critical-point dried. This method has already been used in a number of studies of animal tissues (heart, liver, kidney). It is applied here to the examination of plant material (leaf mesophyll cells). In the second method tissues, or cells, are first infiltrated with cryoprotectant (dimethylsulphoxide) then frozen and fractured, and not fixed until after thawing. The fixed tissues are finally dehydrated and critical-point dried. This method also has previously been used in the study of animal tissues, and is applied here to carrot protoplasts, chicken erythrocytes, and leaf mesophyll cells.     

Methods for characterizing plant fibers

The effectiveness of different microscopy techniques for measuring the dimensions of ultimate fibers from harakeke (Phormium tenax, New Zealand flax) was investigated using a factorial experimental design. Constant variables were geographical location, location of specimens along the leaf, season (winter), individual plant, a fourth leaf from a north-facing fan, age of plant, and cultivars (two). Experimental variables were microscopy techniques and measurement axis. Measurements of width and length of harakeke ultimate fibers depended on the microscopic preparation/technique used as well as the cultivar examined. The best methods were (i) transverse sections of leaf specimens 4 microm thick, embedded in Paraplast and observed using light microscopy, and (ii) nonfixed ultimate fibers observed using scanning electron microscopy.     

CTEM versus STEM versus HVEM: Evaluation of the three-dimensional morphology of arsenic inclusions

Electron-dense arsenic inclusions appeared in the nucleus of parenchymal hepatocytes from fish exposed to arsenate, but were absent in fish exposed under identical conditions to solutions lacking arsenic. Images of these inclusions were compared using conventional transmission electron microscopy (CTEM), scanning transmission electron microscopy (STEM), and high-voltage electron microscopy (HVEM). Stereo pairs from the same individual inclusions were examined using each of these methods to provide a more complete understanding of their three-dimensional organization and to evaluate the relative merits of each technique in the study of similar electron-dense structures. Comparable results were obtained with the three types of instrumentation. Although HVEM is the technique of choice for the analysis of three-dimensional images of such high electron-dense structures, STEM proved to be a good alternate technique for the selection and general evaluation of samples in preparation for HVEM.     

The current status of fixation for electron microscopy: A review

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.     

The use of filter membranes for high-pressure freezing of cell monolayers

Rapid freezing of cells and tissues, followed by freeze‐substitution fixation and plastic embedding, has become a highly reliable method for preparing samples for imaging in the electron microscope. High‐pressure freezing is an efficient means of immobilizing suspensions of yeasts, thick pellets of mammalian cells, or small (< 0.5 mm) pieces of plant or animal tissue. Monolayers of cultured mammalian cells that are too thick for efficient immobilization by other modes of rapid freezing have also been successfully preserved by this method. Monolayer cultures are often important because they can be imaged by light microscopy (LM) both before and after their preparation for electron microscopy (EM). Additionally, some monolayer cultures serve as model systems for physiological processes, so it is important that cells under study can grow on a substrate that is both physiologically appropriate and convenient for EM processing. Here we describe a reliable method for preparing mammalian cell monolayers (PtK₁ and polarized MDCK) for EM. Our protocol results in good preservation of cellular ultrastructure, it is a useful companion to studies of cell physioloy and, with some limitation, is suitable for correlative LM and EM.     

Utilization of 3D printing for an intravital microscopy platform to study the intestinal microcirculation

Intravital microscopy of the intestine is a sophisticated technique that allows qualitative and quantitative in vivo observation of dynamic cellular interactions and blood flow at a high resolution. Physiological conditions of the animal and in particular of the observed organ, such as temperature and moisture are crucial for intravital imaging. Often, the microscopy stage with the animal or the organ of interest imposes limitations on how well the animal can be maintained. In addition, the access for additional oxygen supply or drug administration during the procedure is rather restricted. To address these limitations, we developed a novel intravital microscopy platform, allowing us to have improved access to the animal during the intravital microscopy procedure, as well as improved microenvironmental maintenance. The production process of this prototype platform is based on 3D printing of device parts in a single‐step process. The simplicity of production and the advantages of this versatile and customizable design are shown and discussed in this paper. Our design potentially represents a major step forward in facilitating intestinal intravital imaging using fluorescent microscopy.     

A double embedding technique for the electron microscopic analysis of the liver acinus.

A double embedding technique allowing accurate and systematic sampling of zones of the hepatic acinus by electron microscopy was developed. The method involved the primary embedding of large (10 mm x 30 mm x 0.5 mm) liver slices and the preparation of 10 micrometers thick tissue sections by means of a rotary microtome with a steel knife. Veins 20--40 micrometers in diameter, the landmarks of the hepatic acinus, were localized by light microscopy, dissected from surrounding tissue and reembedded for ultramicrotomy. The method facilitated the systematic evaluation of hepatocyte ultrastructure in each zone of the microcirculatory unit of the liver, the hepatic acinus.     

Preparation and mechanical requirements for freeze-etching thick-walled plant tissues*

The freeze-etching technique is useful in studying cells from higher plant tissues even when thick cell walls are present. However, such cells require special consideration both in preparation for the freeze-etching process as well as during fracturing and subsequent stages. Modifications to the technique and to the apparatus are described which make it possible to freeze-etch thick-walled plant tissues more successfully than by employing other methods.     

Methods for Preparing Samples of Track Membranes for Scanning Electron Microscopy

Methods for preparing porous polymer samples to investigate their structure by scanning electron microscopy are described. The technique for cleavages preparation is complemented by preliminary treatment of a polymer with radiation, photo- or thermal-oxidation destruction to render it brittle. The advantages of this technique have been demonstrated with Mylar, polycarbonate, and polypropylene track membranes.     

Characterization of polymer blends by low voltage scanning electron microscopy

A technique for characterization of polymer blends by low voltage scanning electron microscopy is described. The method allows observation of the distribution of phases in a blend due to good topographical and compositional contrast. This is possible because of lower beam penetration and high secondary emission coefficient (δ ? 1) at low accelerating voltages. Uncoated polymer samples are imaged with no charging or beam damage problems. The technique has a great advantage over conventional transmission electron microscopy techniques because the sample preparation is minimal and larger areas can be prepared for viewing.     

Characterization of Alzheimer paired helical filaments by electron microscopy

We show how electron microscopy can be used to answer several critical issues in neurodegenerative disorders that course with the formation of aberrant filamentous structures. Thus, electron microscopy is a useful technique to study in vitro assembly of pathogenic proteins, to map the regions involved in filament formation, as well as to detect by immunoelectron microscopy which proteins bind to the filaments. Furthermore, electron microscopy is the main technique used to discover if an animal model develops fibrillar pathology and if those filaments are similar to those found in human patients. This review focuses on Alzheimer's disease and related tauopathies, although similar studies have been done with other neurodegenerative disorders as, for example, Huntington's disease.     

A method for preparing living plant cell walls for scanning electron microscopy

A method is described for the preparation for scanning electron microscopy of cell walls from living plant cells. The method involves digestion with a protease followed by freeze-drying, and enables wall details to be clearly observed.     

Observation particle morphology of colloidal system by conventional SEM with an improved specimen preparation technique

On the basis of our previous report that polymer emulsion with different viscosity can be investigated by conventional scanning electron microscopy (SEM), we have developed an improved specimen preparation technique for obtaining particle morphology and size of colloidal silver, collagen, glutin, and polymer microspheres. In this study, we expect to provide a means for charactering the three-dimensional surface microstructure of colloidal particles. Dilution of the samples with appropriate volatile solvent like ethanol is effective for SEM specimen preparation. At a proper ratio between sample and ethanol, the colloidal particles are dispersed uniformly in ethanol and then deposited evenly on the substrate. Different drying methods are studied to search a proper drying condition, in which the small molecule solvent is removed without destroying the natural particle morphology. And the effects of ethanol in the specimen preparation process are described by analyzing the physicochemical properties of ethanol. The specimen preparation technique is simple and can be achieved in common laboratory for charactering the particle morphology of colloidal system.     

3D volumes constructed from pixel-based images by digitally clearing plant and animal tissue

Construction of three-dimensional volumes from a series of two-dimensional images has been restricted by the limited capacity to decrease the opacity of tissue. The use of commercial software that allows colour-keying and manipulation of two-dimensional images in true three-dimensional space allowed us to construct three-dimensional volumes from pixel-based images of stained plant and animal tissue without generating vector information. We present three-dimensional volumes of (1) the crown of an oat plant showing internal responses to a freezing treatment, (2) a sample of a hepatocellular carcinoma from a woodchuck liver that had been heat-treated with computer-guided radiofrequency ablation to induce necrosis in the central portion of the tumour, and (3) several features of a sample of mouse lung. The technique is well suited to images from large sections (greater than 1 mm) generated from paraffin-embedded tissues. It is widely applicable, having potential to recover three-dimensional information at virtually any resolution inherent in images generated by light microscopy, computer tomography, magnetic resonance imaging or electron microscopy.     

Electron-microscope preparation techniques applied to the light microscopy of the cambium and its derivatives in Pinus radiataD. Don.

Epon 812 was used successfully as an embedding medium for the preparation of the secondary meristem and its derivatives in Pinus radiata for light microscopy. Specimens were fixed in glutaraldehyde followed by osmium tetroxide solution, using a schedule developed for the electron microscopy of these tissues. Sections from 2 to 5 μm thick were cut using an LKB Ultratome III and glass knives. Sections were evaluated without prior removal of the embedding medium using bright-field, phase-contrast and interference-contrast microscopy. Results for cambium were generally superior to those obtained by using the conventional fixatives for light microscopy and paraffin embedding. The advantages and disadvantages of this technique are discussed and it is concluded that the use of Epon for embedding this type of material is easier and gives better results than older established methods. It is suggested that methods of electron microscopy be considered when the preparation of a material for light microscopy has proved difficult or impossible by other means, or when particularly thin sections are required.     

Simple method for the preparation of inp based samples for TEM investigation

A novel, rapid, and simple method is described for the preparation of InP based samples for investigation by transmission electron microscopy (TEM). The key feature of the technique is Ar⁺ ion bombardment in an iodine ambient. Cross sectional micrographs of Au/InP samples are shown as an example. The technique developed produces a large area of transparent region.     

The use of freeze-substitution in the preparation of plant tissue for ion localization studies

A method, utilizing freeze substitution, is described for the preparation of plant tissue for analytical electron microscopy. The fine structure of the cytoplasm was adequately preserved after freezing leaf tissue in 2-methylbutane at ?170°C. Furthermore, following substitution in ether, losses of sodium and potassium from the tissue were less than 4% of the original ion content and the loss of chloride was less than 1%. The merits of the procedure as a means of tissue preparation for ion localization studies are discussed.     

Focused ion beam characterization of plasma-assisted deposition on polymer films at the nanoscale

In this paper, a novel technique is presented for the characterization at the nanoscale of plasma-assisted deposit on polyethylene-terephthalate (PET) polymer films. In previous studies, some microcharacterization and morphology analyses of plasma-assisted deposition were performed by atomic force microscopy (AFM). In the work presented here, we analysed the thickness and homogeneity of plasma-assisted deposits by focused ion beam (FIB). This technique with 5-7 nm resolution requires no sample preparation and relies on a sequence of operations on a relatively fast time scale, so that it is easy to make thorough investigations of the sample. We performed electron and ion imaging of the surface of the material, and a subsequent ionic cutting allowed the study of the morphology of the same sample. We developed a novel approach to the edge detection techniques (EDT) in images for a fast evaluation and monitoring of the deposited layer.