Note: A scanning electron microscope sample holder for bidirectional characterization of atomic force microscope probe tips

A novel sample holder that enables atomic force microscopy (AFM) tips to be mounted inside a scanning electron microscopy (SEM) for the purpose of characterizing the AFM tips is described. The holder provides quick and easy handling of tips by using a spring clip to hold them in place. The holder can accommodate two tips simultaneously in two perpendicular orientations, allowing both top and side view imaging of the tips by the SEM.     

The direct measurement of temperature changes within freeze-fracture specimens during rapid quenching in liquid coolants

We have evaluated the cooling rates of specimens mounted in a variety of freeze-fracture holders when plunged into a series of liquid coolants. These rates were measured using miniature thermocouples placed within the mounted specimens. The most rapid cooling rates were obtained using propane at 83 K as the coolant. When mounted on a newly devised ‘copper sandwich’ holder, specimen cooling rates in excess of 4500 K/s have been recorded. A simple guillotine-like device for quenching freeze-fracture specimens under reproducible conditions is presented.     

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.     

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.     

Increasing the versatility of an EMscope SP2000A sputter cryo system on a Hitachi S-570 scanning electron microscope

The EMscope SP2000A Sputter Cryo System provides biologists with a quick and reproducible procedure to freeze, fracture, etch, and sputter coat biological samples, which are then transferred to a cold stage for scanning electron microscopic (SEM) observations. The standard specimen holder, which is supplied with the unit and referred to as a “stub,” will accommodate most specimens; however, fabricating a variety of additional holders to perform more specialized functions considerably increases the versatility of the cryounit and the resolution that can be achieved. For example, the adaptation of small hinged or hingeless gold holders, which are used in the freeze-fracture technique, produces clean fractures, allows for storage of samples in liquid nitrogen and permits samples to be frozen much more rapidly than the large standard holder. A short working distance holder has been designed, which allows samples to be inserted into the final objective lens, thereby resulting in a negative working distance up to ?3 mm. Use of this holder with the upper detector in the Hitachi S-570 SEM, enhances the secondary electron signal and thereby increases resolution of frozen samples severalfold. Another specimen holder has been designed, which allows conventional SEM stubs to be observed without removal of the cryostage. This modification permits use of the EMscope stage and specimen transfer device at room temperature without any further alteration or adjustment. These and other types of modified holders allow investigators to store, manipulate, fracture, and observe biological samples at resolutions not normally attainable with a standard SEM cryostage.     

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 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.     

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.     

The preparation of small, particulate specimens by critical point drying: application for scanning electron microscopy

A procedure is described using round glass cover-slips as supports for preparing specimens by critical point drying for the scanning electron microscope. Also described is the construction of a plastic Teflon holder to facilitate handling of the cover-slips.     

Heating of TEM specimens during ion milling

Sample heating during preparation of electron-thin specimens for observation in transmission electron microscopy (TEM) can produce artefacts which invalidate observations. This is particularly true of two-phase materials such as metal matrix composites, for which sample cooling with liquid nitrogen cannot be used to preserve the substructure during milling. A series of experiments is conducted using an age-hardenable aluminium alloy which produces a trace of peak temperature attained by TEM specimens during ion milling. It is shown that peak temperatures of the order of 650 K can be attained using conventional milling parameters; the technique must therefore be used with caution with materials such as metal matrix composites. A simplified one-dimensional heat transfer analysis of the problem is conducted to show that the most critical parameter is heat transfer along the sample holder legs and across interfaces along the heat path. Temperature differentials within the TEM specimen are shown to be less significant, yet these alone are capable of reaching 60 K within a dimpled specimen under usual milling conditions.     

Artefacts arising during preparation of hydrated paper pulp samples for low-temperature SEM

Beating, a pulp treatment widely used in the paper industry, causes disruption of cell wall layers and fibrillation. Previous studies of the effects of beating on fibre morphology have used conventional methods of specimen preparation, with all the attendant problems of shrinkage and distortion during dehydration. Low-temperature scanning electron microscopy (LTSEM) therefore seemed to offer an ideal method for examining fully hydrated wood pulp fibres. Cryofixation of pulp followed by sublimation of superficial ice, however, is shown to generate artefacts indistinguishable from structures present in the samples. Fibrillar and membranous structures were generated in LTSEM-prepared sugar solutions; their presence in pulp samples was therefore attributed to the dissolved carbohydrates inherent in pulp suspensions. Since artefact and fact are currently impossible to distinguish in LTSEM-prepared pulp samples, it seems that the technique should be applied to wet paper or pulp samples with considerable circumspection.     

Bean-induced loss of organic mass under electronmicroprobe conditions

Techniques for obtaining complementary replicas have already been shown to be valuable in aiding the interpretation of electron micrographs of replicas of specimens prepared by freeze-etching and freeze-fracturing techniques, and in the recognition of artefacts. This paper describes a simple and efficient method of obtaining complementary pairs of replicas of all types of specimen. Ordinary hollow brass rivets are used as specimen holders and are frozen in an end-to-end position using a special pair of forceps. Up to six rivets are placed in a device consisting of a hinged plate held together with clamps against the force of small springs. When the clamps are released, the pair of rivets are separated, fracturing the specimens. The device is easily adapted for use in any type of freeze-etching or high vacuum apparatus. On the example of Saccharomyces cerevisiae the application of the technique to the detection of artefacts in complementary replicas are described. It was shown that the fibrils observed on cross-fractured cell walls are produced by plastic deformation of cell wall components.     

Imaging with low-voltage scanning transmission electron microscopy: a quantitative analysis

A dedicated specimen holder has been designed to perform low-voltage scanning transmission electron microscopy in dark field mode. Different test samples, namely InGaAs/GaAs quantum wells, InGaAs nanowires and thick InGaAs layers, have been analysed to test the reliability of the model based on the proportionality to the specimen mass-thickness, generally used for image intensity interpretation of scattering contrast processes. We found that size of the probe, absorption and channelling must be taken into account to give a quantitative interpretation of image intensity. We develop a simple procedure to evaluate the probe-size effect and to obtain a quantitative indication of the absorption coefficient. Possible artefacts induced by channelling are pointed out. With the developed procedure, the low voltage approach can be successfully applied for quantitative compositional analysis. The method is then applied to the estimation of the In content in the core of InGaAs/GaAs core-shell nanowires.     

A simple technique for examining frozen hydrated specimens in the scanning electron microscope.

The use of a wide angle backscattered electron detector in a scanning electron microscope, which has the capability of the specimen chamber pressure being controlled independently of the column pressure, provides a simple technique for examining frozen hydrated specimens. Large specimens have been examined within 1 min of being placed on the stub and have been examined for many hours without charging artefacts or distortion due to dehydration.     

A gripping stub* for the examination of multilaminous biological specimens in the scanning electron microscope

A simple specimen holder has been developed which expedites study of multilaminous biological specimens in the scanning electron microscope. It has the interesting additional property of allowing examination of the top and bottom of a single sample.     

Methods for additive hydration allowing observation of fully hydrated state of wet samples in environmental SEM

Methods for additive hydration are presented that enable longtime observation of very wet biological specimens in an environmental scanning electron microscope. The changes of structure due to dehydration for specimens placed on a Peltier-cooled holder, put on a special agar base or embedded in it or blown over by water vapor are compared. The degree of dehydration damage of the observed specimen structures is evaluated and compared with the structure of a nondestructively dried specimen, prepared by the critical point drying method.     

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 container for processing small volumes of cell suspensions for critical point drying

The attachment of lymphocytes to glass or filters, in order to facilitate handling for processing prior to scanning electron microscopy, may introduce artefacts in surface topography. A container has therefore been adapted, from an embedding capsule, for the preparation of small volumes of cell suspensions.     

A simple method for correlative light,scanning electron microscopic and X-ray microanalytical examination of the same section

Thin paraffin sections, mounted on scanning specimen holders previously coated with polyester film tape (Minnesota Mining and MFG Co., Scotch film tape No. 850 gold), were processed for light microscopy (LM) in the conventional way, then covered with celloxin shellac and examined in the LM by using the upper illuminating source. After removal of the shellac from the surface of the sample by immersion in acetone, the sections were air-dried, coated with a copper layer in a vacuum evaporator and examined in a scanning electron microscope (SEM). The method allows: (i) high-quality LM possibilities for establishment of the diagnosis in pathological cases; (ii) SEM examination of the same area as observed in LM; and (iii) EPMA measurements of insoluble precipitates embedded in the tissue. The usefulness of the proposed method is obvious in cases where the composition of a precipitate on LM scale is to be compared with the LM appearance of the surrounding tissue.     

Specimen thickness dependence of hydrogen evolution during cryo‐transmission electron microscopy of hydrated soft materials

The evolution of hydrogen from many hydrated cryo‐preserved soft materials under electron irradiation in the transmission electron microscope can be observed at doses of the order of 1000 e nm^?2 and above. Such hydrogen causes artefacts in conventional transmission electron microscope or scanning transmission electron microscopy (STEM) imaging as well as in analyses by electron energy‐loss spectroscopy. Here we show that the evolution of hydrogen depends on specimen thickness. Using wedge‐shaped specimens of frozen‐hydrated Nafion, a perfluorinated ionomer, saturated with the organic solvent DMMP together with both thin and thick sections of frozen‐hydrated porcine skin, we show that there is a thickness below which hydrogen evolution is not detected either by bubble observation in transmission electron microscope image mode or by spectroscopic analysis in STEM electron energy‐loss spectroscopy mode. We suggest that this effect is due to the diffusion of hydrogen, whose diffusivity remains significant even at liquid nitrogen temperature over the length scales and time scales relevant to transmission electron microscopy analysis of thin specimens. In short, we speculate that sufficient hydrogen can diffuse to the specimen surface in thin sections so that concentrations are too low for bubbling or for spectroscopic detection. Significantly, this finding indicates that higher electron doses can be used during the imaging of radiation‐sensitive hydrated soft materials and, consequently, higher spatial resolution can be achieved, if sufficiently thin specimens are used in order to avoid the evolution of hydrogen‐based artefacts.