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.     

Cooling bath for rapid freezing in electron microscopy

A cryogenic device, freely accessible from the top, is described which allows rapid cooling of specimens for electron microscopy by immersion into melting nitrogen.     

A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues

In this paper, we review some published studies using correlative light and electron microscopy methods. We further refined our criteria to include only those studies using live cells for light microscope and where high-pressure freezing was the method of specimen preparation for electron microscopy. High-pressure freezing is especially important for some difficult-to-fix samples, and for optimal preservation of ultrastructure in samples larger than a few micrometres. How the light microscope observations are done is completely sample dependent, but the choice of high-pressure freezer depends on the speed required to capture (freeze) the biological event of interest. For events requiring high time resolution (in the 4–5 s range) the Leica EM PACT2 with rapid transfer system works well. For correlative work on structures of interest that are either non-motile or moving slowly (minutes rather than seconds), any make of high-pressure freezer will work. We also report on some efforts to improve the capabilities of the Leica EM PACT2 rapid transfer system.     

Special specimen preparation methods for image processing in transmission electron microscopy: a review.

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.     

Gallium: a new supporting medium for specimen preparation

Gallium offers a useful alternative to conventional plastic and paraffin embedding media. Samples can be inserted into or removed from gallium cleanly and intact in minutes. Gallium can be polished, has good sectioning properties, is electrically conductive, tolerates high beam currents, is usually not a detectable sample component, and probably would not be a solvent for sample constituents. Samples can be immobilized in gallium within a few degrees of room temperature. Gallium has low toxicity, can be obtained in ultrapure form, is not expensive, and is reusable. Gallium has been used to prepare thin and thick sections and to expose bulk sample cross sections for STEM, SEM, microprobe and light microscope analysis. Gallium does not infiltrate; consequently it is not likely to be useful for soft biological samples.     

Transmission electron microscope specimen preparation for exploring the buried interfaces in plan view

A relatively easy and convenient process for the preparation of transmission electron microscope specimens of buried interfaces is described. The method is based on the alignment and realignment of the specimen rotation centre during ion milling. The ion‐milling time interval in which good samples are obtained is substantially extended in this way.     

A method for preparing powdered specimens for transmission electron microscopy

A new method of preparing TEM samples from powders is described. The method consists of compacting epoxy-powder composite mixtures in special molds by centrifuging prior to polymerization. The compact is then ion milled by standard methods. Samples prepared in this way are currently being used to study dislocation structures developed during processing of ceramic powders.     

A rapid preparation method for ultrastructural investigations of conifer needles

The method discussed here for preparing conifer needles for transmission electron microscopy combines three important components in a new way: chilling (273 K) during fixation to prevent autolysis, HEPES buffer to protect cytoplasm against washing out and VCD/HXSA as an ultralow viscid and therefore rapidly infiltrating medium. Ultrastructural analysis of Picea abies and Pinus silvestris shows very satisfactory tissue preservation.     

A scanning electron microscopy specimen holder for viewing different angles of a single specimen

The specimen holder for scanning electron microscopy described herein allows a single specimen to be examined in any possible view and significantly improves object illumination. The specimen is glued to a fine pin and flexibly mounted on a double‐sided adhesive conductive pad on a rotatable pivot. A milled pot placed beneath the specimen acts as an electron trap. This provides a homogeneous black image background by minimizing noisy signals from the specimen's surroundings. Microsc. Res. Tech. 73:1073–1076, 2010. © 2010 Wiley‐Liss, Inc.     

Use of membrane filters and osmium tetroxide etching in the preparation of sperm for scanning electron microscopy

A new method was developed which is suitable for the preparation of mammalian sperm for scanning electron microscopy under either laboratory or field conditions. Samples of ejaculates from humans, two ferret species, and epididymal sperm from the African elephant were diluted in Millonig phosphate buffer and then fixed in glutaraldehyde solution. A small sample of the fixed sperm suspension was diluted in the same buffer, withdrawn with a syringe, and injected very slowly onto either a cellulose acetate or a polycarbonate membrane filter. This step was essential to concentrate the dilute sperm samples. During the various dilution steps most of the granular prostatic secretions were lost. However, a protein-like sheath, which remained attached to most sperm, obscured the surface features and had to be removed for SEM studies. It was removed by prolonged fixation/etching in 1% osmium tetroxide. Membrane filters containing sperm on their surfaces then were dehydrated, dried by the critical point drying method, and sputter coated with gold. Polycarbonate filters were superior to cellulose acetate filters in producing a flat and homogeneous background.     

An application of rapid freezing,freeze substitution–fixation for scanning electron microscopy

A combined technique of the rapid freezing, freeze substitution–fixation method and the osmium–DMSO-osmium method was devised. By this combined method we clearly observed the architecture of intracellular components in three dimensions. Morphological characteristics were generally similar to those of tissue prepared by the osmium–DMSO-osmium method but different in some respects. Mucigen droplets in intestinal goblet cells, for example, appeared as separated spheres, while in specimens prepared by chemical fixation they were observed as a mass of fused droplets. In the Golgi complex, all cisternae were extremely flat, although they usually dilated on the cis side after chemical fixation. Particles on the mitochondrial tubules of liver cells were well distinguished. They were mushroom shaped, as are those observed by negative staining. The combined method, that is, the rapid freezing, osmium–DMSO-osmium method, is thought to be effective for studying the true structure of intracellular components by scanning electron microscopy.     

A preparation method for observing intracellular structures by scanning electron microscopy

In order to observe intracellular structures by scanning electron microscopy, excess cytoplasmic matrix must be removed from the fractured surface of cells. Previously we reported an Osmium-DMSO-Osmium method devised for this purpose. This method is very effective in revealing intracellular structures, but requires osmium tetroxide for initial fixation with some consequent disadvantages. In the present study, a revised Osmium-DMSO-Osmium method is reported, in which an aldehyde mixture is used as the initial fixative instead of osmium tetroxide. As fixation is carried out by perfusion in this revised method, better preservation of fine structures is achieved than by the original method, especially in the central nervous tissue which tends to suffer from post-mortem degeneration. Moreover this method can be applied to cytochemical studies of intracellular structures with a scanning electron microscope (SEM). In this study, acid phosphatase of lysosomes is demonstrated in a coloured SEM micrograph.     

Low-energy electron microscopy (LEEM) and mirror electron microscopy (MEM) of biological specimens: Preliminary results with a novel beam separating system

Low-energy electron microscopy (LEEM) and mirror electron microscopy (MEM) utilize a parallel beam of slow-moving electrons backscattered from the specimen surface to form an image. If the electrons strike the surface an LEEM image is produced and if they are turned back just before reaching the surface an MEM image results. The applications thus far have been in surface physics. In the present study, applications of LEEM and MEM in the biological sciences are discussed. The preliminary results demonstrate the feasibility of forming images of uncoated cultured cells and cellular components using electrons in the threshold region (i.e. 0–10 V). The results also constitute a successful test of a novel beam-separating system for LEEM and MEM.     

Unstained and in vivo fluorescently stained bacterial nucleoids and plasmolysis observed by a new specimen preparation method for high-power light microscopy of metabolically active cells

Microscope slides were coated with a layer of gelatin, the thickness of the gelatin increasing linearly along the long axis. The bacterial suspension is applied to the dried gelatin and covered by a coverslip. The medium is absorbed by the gelatin and thus the cells applied against the coverslip. By this method, cultures of concentrations below 10⁸ cells/ml provide statistically relevant numbers for observation without prior concentration steps. It is easier to apply than the existing methods for the observation of bacterial nucleoids by phase contrast imaging. Because the cells are maintained in growing conditions the method is useful for the vital fluorescence DAPI-staining of various bacterial species and for observations of plasmolysis and its reversal at different physiological conditions and extracellular osmolalities. The previously generally assumed view that the plasmolytic changes of the cell morphology are immediate upon the hyperosmotic shock and are rapidly repaired when the cell is able to metabolize actively was confirmed; this is in contrast to some recent claims.     

A new rapid method of air-drying for scanning electron microscopy using tetramethylsilane

A new rapid air-drying technique which gives results comparable to critical point-drying is described for scanning electron microscopy, using a trematode parasite, Homalogaster paloniae, as a test specimen.     

Focused ion beam sample preparation of continuous fibre-reinforced ceramic composite specimens for transmission electron microscopy

The microanalysis of interfaces in fibre-reinforced composite materials is dependent on the successful preparation of specimens suitable for transmission electron microscope (TEM) inspection. Ideal samples should possess large amounts of structurally intact and uniform thin area in the fibre/matrix interface regions of the samples. Because fibre/matrix interfaces in this class of materials are often designed to fail under mechanical stress, conventionally prepared samples are prone to interfacial failure and differential thinning, both of which preclude detailed TEM microanalysis. These effects were seen in a conventionally dimpled and ion-beam-thinned specimen prepared from a continuous fibre reinforced ceramic composite composed of CaWO₄-coated Nextel 610^TM fibres in an alumina matrix. The dimpled specimen showed large amounts of interfacial failure, with only thick regions of the specimen left intact. To overcome these limitations, a focused ion beam (FIB) technique was applied to this same material. The superiority of the FIB-produced sample is evident in both the morphology and scanning transmission electron microscopy analyses of the sample.     

Calibration methods for quantitative image processing in electron microscopy

An image can be represented digitally as a matrix of numbers. When those numbers are linearly related to a property of the object, such as mass per unit area, a simple integration of an image area leads to a total of that property, such as the mass of a particle that is represented in a selected area. Following techniques pioneered by Bahr and Zeitler, we illustrate the use of photographic densitometry of films exposed in an electron microscope to measure electron scattering. The transmission of an electron micrograph will be linear with respect to mass thickness for a particular value of background brightfield density, hence allowing determination of the mass of microscopic particles. We show here a digital computer method for conveniently establishing the linear condition by quantitative image processing using micrographs of polystyrene spheres. The method also serves to produce calibration curves for cases where the transfer from transmission to mass thickness is not linear. We also illustrate how an inexpensive computer is used to display and integrate regions of micrographs to determine particle mass.