A technique for the examination of polar ice using the scanning electron microscope

The microstructure and location of impurities in polar ice are of great relevance to ice core studies. We describe a reliable method to examine ice in the scanning electron microscope (SEM). Specimens were cut in a cold room and could have their surfaces altered by sublimation either before (pre‐etching) or after (etching) introduction to the cryo‐chamber of the SEM. Pre‐etching was used to smooth surfaces, whilst etching stripped away layers from the specimen surface, aiding the location of particles in situ, and allowing embedded structures to be revealed. X‐ray analysis was used to determine the composition of localized impurities, which in some cases had been concentrated on the surface by etching. Examining uncoated surfaces was found to be advantageous and did not detract from qualitative X‐ray analysis. Imaging uncoated was performed at low accelerating voltages and probe currents to avoid problems of surface charging.     

Quantitative x‐ray microanalysis of model biological samples in the SEM using remote standards and the XPP analytical model

It is shown that accurate x‐ray microanalysis of frozen‐hydrated and dry organic compounds, such as model biological samples, is possible with a silicon drift detector in combination with XPP (exponential model of Pouchou and Pichoir matrix correction) software using ‘remote standards’. This type of analysis is also referred to as ‘standardless analysis’. Analyses from selected areas or elemental images (maps) were identical. Improvements in x‐ray microanalytical hardware and software, together with developments in cryotechniques, have made the quantitative analysis of cryoplaned frozen‐hydrated biological samples in the scanning electron microscope a much simpler procedure. The increased effectiveness of pulse pile‐up rejection renders the analysis of Na, with ultrathin window detectors, in the presence of very high concentrations of O, from ice, more accurate. The accurate analysis of Ca (2 mmol kg^?1) in the presence of high concentrations of K is possible. Careful sublimation of surface frost from frozen‐hydrated samples resulted in a small increase in analysed elemental concentrations. A more prolonged sublimation from the same resurfaced sample and other similar samples resulted in higher element concentrations.     

Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy of frozen-hydrated biological samples

The feasibility of using a focused ion beam (FIB) for the purpose of thinning vitreously frozen biological specimens for transmission electron microscopy (TEM) was explored. A concern was whether heat transfer beyond the direct ion interaction layer might devitrify the ice. To test this possibility, we milled vitreously frozen water on a standard TEM grid with a 30‐keV Ga⁺ beam, and cryo‐transferred the grid to a TEM for examination. Following FIB milling of the vitreous ice from a thickness of approximately 1200 nm to 200–150 nm, changes characteristic of heat‐induced devitrification were not observed by TEM, in either images or diffraction patterns. Although numerous technical challenges remain, it is anticipated that ‘cryo‐FIB thinning’ of bulk frozen‐hydratred material will be capable of producing specimens for TEM cryo‐tomography with much greater efficiency than cryo‐ultramicrotomy, and without the specimen distortions and handling difficulties of the latter.     

Thermal stability of Ti and Pt nanowires manufactured by Ga+ focused ion beam

Ti and Pt nanowires have been produced by ultra high‐vacuum molecular beam epitaxy deposition of Ti thin films and focused ion beam (FIB) deposition of Pt thin films, followed by cross‐sectional FIB sputtering to form electron‐transparent nanowires. The thermal stability of the nanowires has been investigated by in situ thermal cycling in a transmission electron microscope. Epitaxial single crystal Ti nanowires on (0001)Al₂O₃ substrates are microstructurally stable up to 550–600 °C, above which limited dislocation motion is activated shortly before the Ti‐wires oxidize. The amorphous FIB‐deposited Pt wires are stable up to 580–650 °C where partial crystallization is observed in vacuum. Faceted nanoparticles grow on the wire surface, growing into free space by surface diffusion and minimizing contact area with the underlying wire. The particles are face‐centred cubic (fcc) Pt with some dissolved Ga. Continued heating results in particle spheroidization, coalescence and growth, retaining the fcc structure.     

Freeze-substitution and low-temperature embedding of dairy products for transmission electron microscopy

Dairy products are comprised largely of fat, air and water, which makes it difficult to preserve their ultrastructure for electron microscopy. Keeping the samples frozen throughout fixation and embedding protects the structure and distribution of the components of emulsions and foams. Therefore, dairy products were freeze‐substituted and embedded at low temperature (?20 °C) to prepare them for transmission electron microscopy. Whipped cream, ice cream mix and dairy/non‐dairy mixed systems were frozen by plunging in propane, at its boiling point (?187 °C). Ice cream, because it is already frozen, was fractured into 1‐mm³ pieces in liquid nitrogen and then added to frozen fixative (?196 °C). Fixative solution consisted of glutaraldehyde, osmium tetroxide and uranyl acetate dissolved in either methanol or acetone. When material was to be stained after sectioning the fixative was limited to glutaraldehyde in methanol. The temperature was increased step‐wise from ?80 to ?20 °C. Solvent was replaced with resin; the polar resin Lowicryl HM4, the non‐polar resin Lowicryl HM20, LR White and LR Gold were tested. Samples were embedded and polymerized at ?20 °C using ultraviolet light to cross‐link the resin. Methanol proved to be the most effective solvent for substituting the ice; the hydrophobic resin Lowicryl HM20 was the most effective resin for retaining fat structure following osmium fixation.     

Improvements for imaging ceramics sintering in situ in ESEM

Sintering of green samples of alumina produced by ice‐templating was followed in situ in an environmental scanning electron microscope (ESEM) up to temperatures as high as 1375°C. These alumina samples with well‐defined architectures are of great interest in the field of materials science due to their high specific strength (especially in compression), low density and adaptable porosity. For the present study, they also have the advantage to exhibit an important topography, inducing interesting contrast when imaged in an ESEM. Improvements of the imaging conditions in the ESEM were essential to really follow the sintering process involving formation of necks between grains or shift of the centre of grains. This paper describes the improvements made and the results observed on the sintering process of alumina green samples processed by ice‐templating.     

Recent progress in freeze-fracturing of high-pressure frozen samples

Pancreatic tissue, bacteria and lipid vesicles were high‐pressure frozen and freeze‐fractured. In addition to the normal holder, a new type of high‐pressure freezing holder was used that is particularly suitable for suspensions. This holder can take up an EM grid that has been dipped in the suspension and clamped in between two low‐mass copper platelets, as used for propane‐jet freezing. Both the standard and the new suspension holder allowed us to make cryo‐fractures without visible ice crystal damage. High‐pressure frozen rat pancreas tissue samples were cryo‐fractured and cryo‐sectioned with a new type diamond knife in the microtome of a freeze‐etching device. The bulk fracture faces and blockfaces were investigated in the frozen‐hydrated state by use of a cryo‐stage in an in‐lens SEM. Additional structures can be made visible by controlled sublimation of ice (‘etching’), leading to a better understanding of the three‐dimensional organization of organelles, such as the endoplasmic reticulum. With this approach, relevant biological structures can be investigated with a few nanometre resolution in a near life‐like state, preventing the artefacts associated with conventional fixation techniques.     

Surface structure imaging by electron microscopy

Reconstructed structures at monolayer level on ‘clean and well-defined’ surfaces can be imaged by transmission electron microscopy in fixed beam illumination mode. The specimens are cleaned in-situ in the electron microscope in ultra high vacuum. Transmission electron diffraction pattern intensities can give useful information for determining the surface unit cell size of the structure, and the atom positions (geometric arrangement of atoms in the unit cell) especially those with a large unit cell, since the diffraction intensities are interpreted kinematically. High resolution surface imaging which gives directly the atom positions is tested here for a single monolayer terrace on Ag (111) surface. The result shows the value of HREM for studies of surface crystallography.     

Making EBSD on water ice routine

Electron backscatter diffraction (EBSD) on ice is a decade old. We have built upon previous work to select and develop methods of sample preparation and analysis that give >90% success rate in obtaining high‐quality EBSD maps, for the whole surface area (potentially) of low porosity (<15%) water ice samples, including very fine‐grained (<10 μm) and very large (up to 70 mm by 30 mm) samples. We present and explain two new methods of removing frost and providing a damage‐free surface for EBSD: pressure cycle sublimation and ‘ironing’. In general, the pressure cycle sublimation method is preferred as it is easier, faster and does not generate significant artefacts. We measure the thermal effects of sample preparation, transfer and storage procedures and model the likelihood of these modifying sample microstructures. We show results from laboratory ice samples, with a wide range of microstructures, to illustrate effectiveness and limitations of EBSD on ice and its potential applications. The methods we present can be implemented, with a modest investment, on any scanning electron microscope system with EBSD, a cryostage and a variable pressure capability.     

Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr

We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60‐kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween‐80 display hexagonally close‐packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge‐freezing, and (ii) frozen‐hydrated sections obtained from cells frozen under high pressure. The frozen‐hydrated sections were generated by sectioning at ?150 °C using a diamond knife with a 25° knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen‐hydrated sections and the arrangements found in negatively stained membrane extracts and plunge‐frozen cells, highlighting the potential of using frozen‐hydrated sections for the study of macromolecular assemblies within cells under near‐native conditions.     

Freeze-fracturing: a review of methods and results

Freeze-fracturing may be accomplished either under vacuum, or at atmospheric pressure. The devices available for freeze-cleaving are discussed in relation to the vacuum and temperature conditions prevailing during cleaving, etching and replication. It is concluded that most specimens can be satisfactorily cleaved (even at 4 K), and processed using simple cleavage devices and systems that provide ample cold-trapping protection for the specimen. Only for special purposes are microtome assemblies or ultra-high vacuum units essential. The fracturing process may produce artefacts by plastic deformation. Such artefacts have been noted in both non-biological and biological polymers cleaved at temperatures as low as 4 K. Contamination of the frozen surface need not be a problem provided that the specimen is transferred under ‘safe’ conditions, and is protected by well-designed cold traps. Freeze-cleavage and freeze-sectioning are compared. It is considered that there will be a temperature range for most heterogeneous specimens within which both cleavage and fracturing may occur, depending upon the nature of the molecules in the cleavage/sectioning plane. Local heating during freeze-sectioning will produce deformation artifacts as in freeze-cleavage, and may also lead to more general surface ‘flow’. The relationships between sectioning and fracturing biological specimens at low temperature require further clarification.     

Charging effect in electron-irradiated ice

To maintain the original distribution pattern of diffusible elements in biological samples, electron probe microanalysis is carried out with frozen hydrated bulk specimens and cryosections, analysed at temperatures below 130 K. Ice has a very low intrinsic conductivity at this working temperature and surface- and space-charging appears, when uncoated specimens are irradiated with non-penetrating electrons. Although coating with a grounded conductor abolishes the surface potential, the build-up of an internal space-charge field is possible, depending on the sample thickness and beam voltage used. Consequently, the geometry of the X-ray source volume and the spectral distribution of the emitted continuous and characteristic X-rays are affected. To simulate the situation for microanalysis of frozen hydrated specimens the charging process in electron irradiated ice is studied by recording simultaneous specimen currents from the top and bottom of ice layer preparations. The external currents yield information on the build-up of internal space-charge fields which result from the balance of charge injection, storage, and transport. Irradiation of uncoated bulk specimens with a finely focused beam results in the build-up of a space-charge field close to the surface, which causes a reduction of the depth of microprobe analysis. In coated bulk specimens the induced conductivity renders possible a current flow to the front electrode, thereby limiting the space-charge field. Sections with an effective rear electrode will not charge appreciably if the electron range is larger than about half the section thickness.     

A simple method to 'point count' silt using scanning electron microscopy aided by image analysis

This study presents a simple method to ‘point count’ silt‐sized grains using backscattered scanning electron microscopy together with image analysis. The work materialized out of the need to determine the heavy mineral abundance within silt obtained from coastal dunes to aid in the interpretation of dune weathering. This technique allows two broad mineral groups to be quantified according to their modal abundance. The groups are characterized by their dominant atomic elements present; atomic numbers > 20 are classified as ‘high’ (metal oxides, zircon, monazite, carbonates, pyroxenes and amphiboles) and those < 20 as ‘low’ (quartz, feldspars and organics). As a check on this technique, X‐ray fluorescence was used. This showed a strong positive correlation (r² = 0.85) with the developed point counting technique.     

The observation of intact hepatic endothelial cells by cryo-electron microscopy

Liver sinusoidal endothelial cells (LSECs) can optimally be imaged by whole mount transmission electron microscopy (TEM). However, TEM allows only investigation of vacuum‐resistant specimens and this usually implies the study of chemically fixed and dried specimens. Cryo‐electron microscopy (cryo‐EM) can be used as a good alternative for imaging samples as whole mounts. Cryo‐EM offers the opportunity to study intact, living cells while avoiding fixation, dehydration and drying, at the same time preserving all solubles and water as vitrified ice. Therefore, we compared the different results obtained when LSECs were vitrified using different vitrification conditions. We collected evidence that manual blotting at ambient conditions and vitrification by the guided drop method results in the production of artefacts in LSECs, such as the loss of fenestrae, formation of gaps and lack of structural details in the cytoplasm. We attribute these artefacts to temperature and osmotic effects during sample preparation just prior to vitrification. By contrast, by using an environmentally controlled glove box and a vitrification robot (37 °C and 100% relative humidity), these specific structural artefacts were nearly absent, illustrating the importance of controlled sample preparation. Moreover, data on glutaraldehyde‐fixed cells and obtained by using different vitrification methods suggested that chemical prefixation is not essential when vitrification is performed under controlled conditions. Conditioned vitrification therefore equals chemical fixation in preserving and imaging cellular fine structure. Unfixed, vitrified LSECs show fenestrae and fenestrae‐associated cytoskeleton rings, indicating that these structures are not artefacts resulting from chemical fixation.     

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.     

Automated in‐chamber specimen coating for serial block‐face electron microscopy

When imaging insulating specimens in a scanning electron microscope, negative charge accumulates locally (‘sample charging’). The resulting electric fields distort signal amplitude, focus and image geometry, which can be avoided by coating the specimen with a conductive film prior to introducing it into the microscope chamber. This, however, is incompatible with serial block‐face electron microscopy (SBEM), where imaging and surface removal cycles (by diamond knife or focused ion beam) alternate, with the sample remaining in place. Here we show that coating the sample after each cutting cycle with a 1–2 nm metallic film, using an electron beam evaporator that is integrated into the microscope chamber, eliminates charging effects for both backscattered (BSE) and secondary electron (SE) imaging. The reduction in signal‐to‐noise ratio (SNR) caused by the film is smaller than that caused by the widely used low‐vacuum method. Sample surfaces as large as 12 mm across were coated and imaged without charging effects at beam currents as high as 25 nA. The coatings also enabled the use of beam deceleration for non‐conducting samples, leading to substantial SNR gains for BSE contrast. We modified and automated the evaporator to enable the acquisition of SBEM stacks, and demonstrated the acquisition of stacks of over 1000 successive cut/coat/image cycles and of stacks using beam deceleration or SE contrast.     

Scanning electron microscopy and transmission electron microscopy in situ studies of grain boundary migration in cold-deformed aluminium bicrystals

The crystallography of recrystallization has been investigated in channel‐die deformed pure aluminium bicrystals with {100}<011>/{110}<001> orientations. The microstructural and microtextural changes during the early stages of recrystallization were followed by systematic local orientation measurements using scanning and transmission electron microscopes. In particular, orientation mapping combined with in situ sample heating was used to investigate the formation and growth of new grains at very early stages of recrystallization. Grain boundary migration and ‘consumption’ of the as‐deformed areas was always favoured along directions parallel to the traces of the {111} slip planes that had been most active during deformation.     

An ice lithography instrument

We describe the design of an instrument that can fully implement a new nanopatterning method called ice lithography, where ice is used as the resist. Water vapor is introduced into a scanning electron microscope (SEM) vacuum chamber above a sample cooled down to 110 K. The vapor condenses, covering the sample with an amorphous layer of ice. To form a lift-off mask, ice is removed by the SEM electron beam (e-beam) guided by an e-beam lithography system. Without breaking vacuum, the sample with the ice mask is then transferred into a metal deposition chamber where metals are deposited by sputtering. The cold sample is then unloaded from the vacuum system and immersed in isopropanol at room temperature. As the ice melts, metal deposited on the ice disperses while the metals deposited on the sample where the ice had been removed by the e-beam remains. The instrument combines a high beam-current thermal field emission SEM fitted with an e-beam lithography system, cryogenic systems, and a high vacuum metal deposition system in a design that optimizes ice lithography for high throughput nanodevice fabrication. The nanoscale capability of the instrument is demonstrated with the fabrication of nanoscale metal lines.     

Chromosome morphology after long-term storage investigated by scanning near-field optical microscopy

Fluorescence in situ hybridization coupled with far‐field fluorescence microscopy is a commonly used technique to visualize chromosomal aberrations in diseased cells. To obtain the best possible results, chromatin integrity must be preserved to ensure optimal hybridization of fluorescence in situ hybridization probes. However, biological samples are known to degrade and storage conditions can be critical. This study concentrates its investigation on chromatin stability as a function of time following fluorescence in situ hybridization type denaturing protocols. This issue is extremely important because chromatin integrity affects the fluorescence response of the chromosome. To investigate this, metaphase chromosome spreads of human lymphocytes were stored at both ?20 and ?80 °C, and were then imaged using scanning near‐field optical microscopy over a nine month period. Using the scanning near‐field optical microscope's topography mode, chromosome morphology was analysed before and after the application of fluorescence in situ hybridization type protocols, and then as a function of storage time. The findings revealed that human chromosome samples can be stored at ?20 °C for short periods of time (～ several weeks), but storage over 3 months compromises chromatin stability. Topography measurements clearly show the collapse of the stored chromatin, with variations as large as 60 nm across a chromosome. However, storage at ?80 °C considerably preserved the integrity with variations in topography significantly reduced. We report studies of the fluorescent response of stored chromosomes using scanning near‐field optical microscopy and their importance for gaining further understanding of chromosomal aberrations.     

Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells

We describe a method for high‐pressure freezing and rapid freeze‐substitution of cells in tissue culture which provides excellent preservation of membrane detail with negligible ice segregation artefacts. Cells grown on sapphire discs were placed ‘face to face’ without removal of tissue culture medium and frozen without the protection of aluminium planchettes. This reduction in thermal load of the sample/holder combination resulted in freezing of cells without visible ice‐crystal artefact. Freeze‐substitution at −90°C for 60 min in acetone containing 2% uranyl acetate, followed by warming to −50°C and embedding in Lowicryl HM20 gave consistent and clear membrane detail even when imaged without section contrasting. Preliminary data indicates that the high intrinsic contrast of samples prepared in this way will be valuable for tomographic studies. Immunolabelling sensitivity of sections of samples prepared by this rapid substitution technique was poor; however, reducing the uranyl acetate concentration in the substitution medium to 0.2% resulted in improved labelling. Samples substituted in this lower concentration of uranyl acetate also gave good membrane detail when imaged after section contrasting.