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.     

Application of polystyrene disk substrates in cellular cultivation methods: generalized specimen preparation protocol for scanning electron microscopy

Sample preparation for scanning electron microscopy (SEM) may vary by cellular type, composition and method of cultivation. It has been proposed here that a generalized method of sample preparation may be applied for the visualization of bacteria, fungi, and human cellular tissue without modification of protocol between cell types. The following protocol was developed to incorporate polystyrene disk substrates in the simplification of sample preparation for SEM and reduce the possibility of processing artefacts. The proposed method of preparation may be applied to samples grown in either liquid or solid cultural medium regardless of cell type. With the proposed protocol, centrifugation, isolation and critical point drying are not required, therefore increasing specimen integrity. The incorporation of polystyrene disks showed positive cellular adhesion and applications in SEM for bacterial, fungal and human neuronal tissue. In addition, the simplicity of the proposed protocol is highly adaptable and may be further incorporated to visually analyse the effects of antifungals, antibiotics and disease pathogenesis through pathogen–host interactions. The proposed method of specimen preparation was incorporated in either liquid or solid state growth mediums during the cultivation of the selected cellular samples and revealed great promise in the preservation and visualization under the scanning electron microscope.     

An improved specimen loader for cryo-scanning electron microscopy

Cryo-electron microscopy of cryofixed samples is a well-established and accepted technique for imaging liquid-containing specimens without removing water and other volatile components. There are many steps between cryofixation and cryo-observation in the microscope, during which the sample and sample holder need to be handled. One such major step is the loading of the specimen onto the sample holder and the fixing of the sample holder onto the transfer mechanism. During this handling, the specimen is often exposed (mostly inadvertently) to moisture in the atmosphere, which results in frost deposition. The new specimen loader described here is designed to overcome the traditional tedious handling and to achieve ease in specimen loading. The modifications made are mainly towards allowing movement of the liquid freon cup, eliminating the need for a lock-screw and improving the shape of the stage holder, which makes the mounting of the specimen holder easy, thereby permitting smooth specimen loading without too much handling and with consequent reduced frost deposition.     

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 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.     

A new HPF specimen carrier adapter for the use of high‐pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium

Cryogenic transmission electron microscopy of high‐pressure freezing (HPF) samples is a well‐established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic‐scanning electronic microscope (Cryo‐SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo‐SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM‐studies using HPF.     

Revealing local variations in nanoparticle size distributions in supported catalysts: a generic TEM specimen preparation method

The specimen preparation method is crucial for how much information can be gained from transmission electron microscopy (TEM) studies of supported nanoparticle catalysts. The aim of this work is to develop a method that allows for observation of size and location of nanoparticles deposited on a porous oxide support material. A bimetallic Pt‐Pd/Al₂O₃ catalyst in powder form was embedded in acrylic resin and lift‐out specimens were extracted using combined focused ion beam/scanning electron microscopy (FIB/SEM). These specimens allow for a cross‐section view across individual oxide support particles, including the unaltered near surface region of these particles. A site‐dependent size distribution of Pt‐Pd nanoparticles was revealed along the radial direction of the support particles by scanning transmission electron microscopy (STEM) imaging. The developed specimen preparation method enables obtaining information about the spatial distribution of nanoparticles in complex support structures which commonly is a challenge in heterogeneous catalysis.     

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 balanced technique for preparation of specimens from pathogenicity studies for scanning electron microscopy

This paper reports our experiences with preparing delicate biological specimens for scanning electron microscopy. Three different washing methods were evaluated: One method allowed the analysis of the location of the bacterium Mycoplasma mobile on piscine gill epithelium and the optimal evaluation of histopathologic changes caused by this microbe. These results were achieved when specimens were washed three times in a cacodylic acid buffer after completion of the in vitro infection experiment in gill explant cultures. We also found that of three different concentrations of glutaraldehyde, a fixation with a 1.5% solution was sufficient to achieve excellent structural preservation, even without using post fixation in osmium tetroxide. Furthermore, this study showed that the use of acetone-carbon dioxide in the critical point drying procedure resulted in well-preserved piscine gill epithelium and mycoplasmas. Finally, long-term storage of tissue specimens in 0.1 M cacodylic acid buffer is possible if the buffer is changed on a monthly basis to avoid growth of unwanted microorganisms, such as fungi.     

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.     

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.