A simple method for the handling and orientation of small specimens for electron microscopy

Cyanoacrylic glue (Eastman 910) was used to affix small pieces of nasal scrapings to lens paper immediately before fixation in the glutaraldehyde. The lens paper not only served to hold specimens together so that they were not lost during tissue processing, but also functioned as a ‘landmark’ for the specimens, so that specimens could be oriented in a specific manner during embedding and subsequent sectioning.     

Transmission electron microscopy method providing high resolution in the cell section without radiation damage

Several new features of mitochondrial nucleoid and its surroundings in mammalian cells were described previously (Pracha?, 2016). Very small details were observed using the improved transmission electron microscopy method, as described in the article. In the meantime, the method has again been improved to 2 Å resolutions in the cell section. The method described in detail in the present work is documented on the same records that were published in lower resolution in the work Pracha? (2016), enabling comparison of the achieved resolution with the previous one. New records are also presented, showing extremely high resolution and thus implying the importance of the method. Potential use of this method in different fields is suggested.     

A comparative study of colloidal particles as imaging standards for microscopy

Colloidal particles have long been used as imaging standards for electron microscopy and, more recently, for scanning probe microscopy. We have analysed gold, polystyrene and silica colloidal particles by both transmission electron microscopy and atomic/scanning force microscopy in an attempt to determine if any can be truly used as 'standards' of shape and/or size. From the transmission electron micrographs, we have obtained precise information of the particle circumference and mean diameter. By comparing the ratio of these to the value for π, we obtained a measure of the sphericity of the particles. We have also shadowed the particles with metal at a known angle and have analysed the shadow length to determine the particles' heights and shapes. The height information obtained from the shadow length data collected from the transmission electron micrographs was then compared with that obtained by atomic/scanning force microscopy. Our results show that cleaned (washed) silica or polystyrene particles closely approach true spheres. In the case of gold particles, height data obtained from shadow lengths analysed in transmission electron micrographs show good agreement with that obtained from the atomic/scanning force microscopy images even without washing. However, the gold particles often deviate from sphericity. Based upon both the shape and the physical properties of the colloidal particles, silica would be the best choice as a standard. We also have noticed that metal shadowing of colloidal particle samples used for atomic/scanning force microscopy offers an advantage which we call a 'nanoscale metric' visible in the image directly at each particle site. This information can be important if one wishes to use samples prepared from colloidal particles simply and reliably to determine the probe shape for scanning probe microscopy from image deconvolution/restoration methods or as a calibration sample.     

A simple method for whole-cell preparation in electron microscopy

A simple method for whole-cell preparation without using gold or platinum grids as substrata for culture is described. Cells were cultured on formvar film over round pores, each 3 mm in diameter, of Thermonox coverslips. The cells on the formvar coated coverslip are fixed, stained, dehydrated in situ, and introduced into a critical point drying apparatus. A small quantity of 0.2% mesh-cement is applied to slot grids, and they are laid onto the formvar film over the pores of the coverslip. After the grids are removed from the plastic substratum, they are ready for observation under the electron microscope.     

A method and devices of electron microtomography in scanning electron microscopy

A method of microtomography of layered microstructures during detection of backscattered electrons in a scanning electron microscope is described. This method is based on the formation of layer-by-layer images hidden under the surfaces of microstructures using reflected electrons filtered within a narrow energy window. An improved deflector-type spectrometer with toroidal electrostatic sector electrodes is applied for microtomography and spectroscopy. To improve the sharpness and accuracy of separation of individual buried heteroboundaries, the modulation principle of video-signal detection is implemented.     

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 method of freeze-drying and fixing spermatozoa for electron microscopy

A procedure is described whereby freshly ejaculated bull semen is freeze-dried, then treated by glutaraldehyde fixative and processed for electron microscope observation of the spermatozoa. Some of the structural features of bull spermatozoa are demonstrated in electron micrographs. In general the procedure may be found useful as an alternative technique for the examination of such cell-carrying fluids as whole blood, cerebrospinal fluid, as well as bacterial cultures.     

Sectioning spherical aluminum oxide particles for transmission electron microscopy

A technique was developed to aid in the interpretation of nucleation and growth characteristics of otherwise electron-opaque Al₂O₃ particles. The technique involved embedding a dense collection of particles in an Epon mixture and sectioning with a diamond knife on an ultramicrotome. Serial sections were utilized to obtain three-dimensional information. It was found that ultramicrotomy does not introduce artifacts in ceramics which would otherwise affect measurements of the crystallite size or phase fraction, nor does it affect the crystallographic data significantly. It was observed that α-Al₂O₃ nucleated at the surface of isolated particles and in the neck region of sintered particles. Other data, obtained from diffraction and in situ hot-stage studies, indicated that each polycrystalline α-Al₂O₃ particle transformed to a nearly single crystal of γ-Al₂O₃, which extended into adjacent particles under favorable circumstances.     

A method for the fabrication of thin-foil apertures for electron microscopy

A method is described for making thin foil apertures by piercing a plastic film with a heated microneedle and subsequently metallizing the plastic film by vacuum evaporation. Apertures can be made with diameters from 5 μm to 100 μm. They are round and very sharp-edged and can be made in various patterns and combinations of sizes.     

Rapid method for resectioning of light microscopy sections for electron microscopy

A rapid method is described for obtaining ultrathin sections from light microscopy sections. Five-micrometre epoxy sections, heat-flattened to slides, were affixed to the tips of plastic blocks by light-curable dental bond, and cured while still on the microscope stage by illumination with blue light for 2 min. Sections were detached from the slides by rapid cooling and then resectioned for electron microscopy.     

A simple and effective method for reducing wool for electron microscopy

A simple, yet effective, method for preparing wool for ultrastructural studies is described. Usually such procedures incorporate a reduction step to convert the disulphide bonds of keratin to thiol groups, which enhances subsequent staining. This reduction process generally involves exposing the wool fibres to severe chemical treatments, which may modify their ultrastructure. Additionally, many of the reagents employed are highly toxic and unpleasant to use. A method of reduction is reported that provides images of ultrastructure at least comparable to previous methods whilst exposing the wool fibres to much milder conditions. Tris(2-carboxyethyl)phosphine has been used to reduce both bulk samples and ultrathin sections of wool, prior to staining with silver methenamine.     

A comparative study of replication techniques for use in scanning electron microscopy of orthopaedic implants

Three replication techniques for use in scanning electron microscopy of orthopaedic implants are described and compared. The three consist of one type of silicone replica (Xantopren® Blue) and two types of plastic film replicas (collodion in amyl acetate and Formvar in ethylene dichloride). The clearest image with the highest resolution and contrast is obtained with a Formvar replica. Collodion produces almost as good an image and the replicas are easier to produce. However, neither Formvar nor collodion can be used on implant surfaces which are grossly curved or rough textured since the replicas are not rigid enough to retain the true shape of the surface. Xantopren Blue is considerably easier to use than either of the plastic films, gives a true reproduction of specimen shape and allows replication of rough surfaces. Its only deficiencies relative to the plastic films are resolution and contrast at high magnification. If the sample to be replicated is relatively flat and smooth, and high magnification is desired, plastic film replication materials are recommended.