A specimen holder for low-temperature scanning electron microscopy

A miniature vise built into a copper stub is described that holds bulk, pre-frozen, hydrated biological specimens during examination under the electron beam of the scanning electron microscope.     

A combined environmental straining specimen holder for high-voltage electron microscopy

A novel specimen holder that enables in situ observation of crack-tip deformation and/or fracture under a controlled environment is developed for a high-voltage electron microscope (HVEM). A window-type environmental cell (EC) that incorporates a uniaxial straining apparatus is built into a side-entry-type single-tilt specimen holder. The gas control in EC, straining apparatus design, limited field of view for crack-tip observation, and specimen preparation for the specimen holder are presented in detail. Experimental results successfully demonstrate that the developed specimen holder is quite useful for the dynamic observation of crack-tip deformation and/or fracture subjected to a hostile environment, such as hydrogen gas.     

Improved specimen holders for the scanning electron microscopy of insects and other small objects

Two specimen holders for use in scanning electron microscopy (SEM) of insect and other specimens glued to triangular cardboard points are described. They have important advantages over standard metal stub mounts. Diverse, precisely orientated, viewing angles are possible using single specimens, which can afterwards be re-pinned for return to the collection.     

A simple cryo-holder facilitates specimen observation under a conventional scanning electron microscope

A pre-cryogenic holder (cryo-holder) facilitating cryo-specimen observation under a conventional scanning electron microscope (SEM) is described. This cryo-holder includes a specimen-holding unit (the stub) and a cryogenic energy-storing unit (a composite of three cylinders assembled with a screw). After cooling, the cryo-holder can continue supplying cryogenic energy to extend the observation time for the specimen in a conventional SEM. Moreover, the cryogenic energy-storing unit could retain appropriate liquid nitrogen that can evaporate to prevent frost deposition on the surface of the specimen. This device is proved feasible for various tissues and cells, and can be applied to the fields of both biology and material science. We have employed this novel cryo-holder for observation of yeast cells, trichome, and epidermal cells in the leaf of Arabidopsis thaliana, compound eyes of insects, red blood cells, filiform papillae on the surface of rat tongue, agar medium, water molecules, penicillium, etc. All results suggested that the newly designed cryo-holder is applicable for cryo-specimen observation under a conventional SEM without cooling system. Most importantly, the design of this cryo-holder is simple and easy to operate and could adapt a conventional SEM to a plain type cryo-SEM affordable for most laboratories.     

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.     

High-angle triple-axis specimen holder for three-dimensional diffraction contrast imaging in transmission electron microscopy

Electron tomography requires a wide angular range of specimen-tilt for a reliable three-dimensional (3D) reconstruction. Although specimen holders are commercially available for tomography, they have several limitations, including tilting capability in only one or two axes at most, e.g. tilt-rotate. For amorphous specimens, the image contrast depends on mass and thickness only and the single-tilt holder is adequate for most tomographic image acquisitions. On the other hand, for crystalline materials where image contrast is strongly dependent on diffraction conditions, current commercially available tomography holders are inadequate, because they lack tilt capability in all three orthogonal axes needed to maintain a constant diffraction condition over the whole tilt range. We have developed a high-angle triple-axis (HATA) tomography specimen holder capable of high-angle tilting for the primary horizontal axis with tilting capability in the other (orthogonal) horizontal and vertical axes. This allows the user to trim the specimen tilt to obtain the desired diffraction condition over the whole tilt range of the tomography series. To demonstrate its capabilities, we have used this triple-axis tomography holder with a dual-axis tilt series (the specimen was rotated by 90° ex-situ between series) to obtain tomographic reconstructions of dislocation arrangements in plastically deformed austenitic steel foils.     

A controlled atmosphere specimen holder for transmission electron microscopy

A controlled atmosphere specimen holder (CASH) has been developed for transmission electron microscopy (TEM) experiments. It is designed for studying the specimen's microstructure before and after treatments in various gases (H₂, O₂, N₂, Ar, etc.) at temperatures up to 600°C. The experiments are carried out without exposing the specimen to the ambient atmosphere. No modification of the electron microscope itself is needed. The same area of the specimen can be easily located after each gas treatment, thus the changes in the microstructure can be studied directly. Preliminary results on the cyclic oxidation and reduction of a model cobalt catalyst are presented.     

Measurement of specimen charging in scanning electron microscopy with a kelvin probe

A vibrating Kelvin probe in form of a platinum wire loop is used to measure the surface potential U_s on electron-irradiated free-floating metal and insulator specimens as a function of electron energy E. This allows an accurate measurement of the critical electron energy E₂ for no charging. At energies below E₂, the positive charging increases with decreasing energy to U_s=2–5 eV at E=0.5 keV and switching off the collector bias of the Everhart-Thornley detector. A two-to threefold increase of U_s is observed when the bias is switched on. For E > E₂, the strong increase of a negative surface potential can be measured. Insulating films free-supported on a conductive substrate show a steep decrease to small positive and negative U_s when the film thickness becomes lower than the electron range at a critical energy E₃ > E₂. At insulating specimen the temporal decrease of charging can be measured when the electron beam is switched off.     

The usefulness of glutaraldehyde-carbohydrazide copolymerization in biological specimen stabilization for scanning electron microscopy

Biological specimens can be prepared for scanning electron microscopy by means of copolymerizing the fixing agent glutaraldehyde with carbohydrazide prior to air drying. Such preparations are more stable in the electron microscope, show less internal cellular disruption and retain more of their native elemental composition than specimens prepared by means of dehydration and critical-point drying. Specimens observed in the scanning electron microscope can often be recovered for thin sectioning with no additional embedment, and can then be observed by means of transmission elecltron microscopy. The preparation (termed GACH) can be performed in almost any laboratory with no specialized equipment and, for the most part, may be carried out at room temperature. The technique appears to provide the promise of further research applications in scanning electron microscopy which may employ conjugated procedures of immunocytochemistry and cathodoluminescence as well as X-ray microanalysis in limited situations.     

A versatile sample holder for single plane illumination microscopy

Single Plane Illumination Microscopy is an emerging and powerful technology for live imaging of whole living organisms. However, sample handling that relies on specimen embedding in agarose or gel is often a key limitation, especially for time‐lapse monitoring. To address this issue, we developed a new concept for a holder device allowing us to prepare a sample container made of hydrogel. The production process of this holder is based on 3D printing of both a frame and casting devices. The simplicity of production and the advantages of this versatile new sample holder are shown with time‐lapse recording of multicellular tumour spheroid growth. More importantly, we also show that cell division is not impaired in contrast to what is observed with gel embedding. The benefit of this new holder for other sample types, applications and experiments remains to be evaluated, but this innovative concept of fully customizable sample holder preparation potentially represents a major step forward to facilitate the large diffusion of single plane illumination microscopy technology.     

Digital imaging for scanning electron microscopy

The development and application of digital imaging technology has been one of the major advancements in scanning electron microscopy (SEM) during the past several years. This digital revolution has been brought about by significant progress in semiconductor technology, notably the availability of less expensive, high-density memory chips and the development of inexpensive, high-speed, analog-to-digital and digital-to-analog converters, mass storage, and high-performance central processing units. This paper reviews a number of the advantages presented by digital imaging as applied to the SEM and describes a system developed at the National Institute of Standards and Technology for this purpose.     

A natural color scanning electron microscopy image

A method for obtaining a scanning electron microscopy (SEM) image with natural color information (NCSEM image) has been developed by using an SEM operated under low vacuum or low voltage, a video microscope (VM), and a high-performance personal computer. In order to produce an NCSEM image with both the fine structures of the SEM image and the color information included in the VM image, an improvement of the depth of focus in VM image is required. This can be successfully performed by some digital image processing techniques. A difference in resolution between the SEM image and the VM image does not disturb our study as shown in the text. In addition, the well-known hue, saturation, intensity (HSI) color model, which is frequently utilized for color image manipulation, was originally altered for the present purpose.     

Modification of a TEM-goniometer specimen holder to enable beam current measurements in a (S)TEM for use in quantitative X-ray microanalysis

In order to have available a specimen holder suited to measure the beam current as is often required in quantitative electron probe X-ray microanalysis, the rod of a low background beryllium specimen holder of a transmission electron microscope was modified. The tip was electrically insulated from the mass of the microscope and connected electrically to the central contact of a BNC connector mounted on the specimen holder handle. With this modified specimen holder the current absorbed by the specimen and/or the specimen holder could be measured easily and accurately. The modified specimen holder has been used to measure the beam current stability of an analytical electron microscope under various conditions. Data were obtained for tungsten as well as lanthanum hexaboride cathodes. Small changes to other types of specimen tips made it possible to exchange these for the low background tip.     

A new specimen mounting system for scanning electron microscopy*

A system of specimen mounting discs and holders has been developed for use in high vacuum. Discs are held in place with a tightly fitting cap, thus avoiding the use of adhesives. The mounting discs are especially convenient for biological preparations, are much less expensive than conventional SEM ‘stubs’ and are easier to handle and store.     

Preparation of lithium specimens for transmission electron microscopy

A method of preparing undeformed thin lithium specimens for TEM is described. A solution of dehydrated methanol and toluene is used both for initial dishing of the foil by chemical polishing and also for final thinning. Under electron beam irradiation in the TEM, new pure Li crystals can grow out of the existing Li specimen. These in situ crystals can be used for the study of the microstructure and electronic structure of Li using TEM and electron energy loss spectrometry.     

Double-layer coating for high-resolution low-temperature scanning electron microscopy

Specimen damage caused by mass loss due to electron beam irradiation is a major limitation in low-temperature scanning electron microscopy of bulk specimens. At high primary magnifications (e.g. 100 000x) a hydrated sample is usually severely damaged after one slow scan (about 3000 e^— nm^—2). The consequences of this beam damage are significantly reduced by coating the frozen-hydrated sample with a 5–10-nm-thick carbon layer. Since this layer covers up surface details, the sample is first unidirectionally shadowed with a thin heavy metal layer (e.g. 2 nm of platinum) that is in close contact with the biological surface (double layer coating). This heavy metal layer can be visualized in field-emission scanning electron microscopy with the material-dependent backscattered electron signal. The method allows for routine observation of large frozen-hydrated samples. By use of an in-lens field-emission SEM and a sensitive backscattered electron detector, structural information comparable to that obtained with the transmission electron microscopy freeze-fracture replica technique can be achieved.     

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.     

A novel sample holder allowing atomic force microscopy on transmission electron microscopy specimen grids: repetitive, direct correlation between AFM and TEM images

A novel sample holder that allows atomic force microscopy (AFM) to be performed on transmission electron microscope (TEM) grids is described. Consequently, AFM and TEM images were repeatedly obtained on exactly the same sample area. For both techniques, a thin carbon film was used as the imaging substrate. Although these techniques have been previously used in conjunction, AFM and TEM images on exactly the same area have not been repeatedly obtained for any system. Correlation of AFM and TEM images is useful for work where the three‐dimensional topographical information provided by the AFM could be used to better interpret the two‐dimensional images provided by the TEM and vice versa. To demonstrate the applicability of such correlation, new results pertaining to a fibrillar collagen system are summarized.     

A cassette system for systematic cryostorage of low-temperature scanning electron microscopy stubs

A system is described for the storage of cylindrical (10 × 3.5 mm) stubs for low-temperature scanning electron microscopy. The system facilitates rapid retrieval of mounted specimens, maximizes the capacity of the low-temperature (liquid nitrogen) specimen store, locates each stub exactly in a protected well, and eliminates the possibility of specimen damage from conventional hazards during transport between the storage facility and microscope.     

Relocation of minute specimens for scanning electron microscopy

A relocatable stub-holder assembly for SEM that can record the position of small specimens by x-y-coordinates is described. It enables the accurate pin-pointing of objects for further study later.