Structural changes in samples cryofixed by contact with a cold metal block

A common method of cryofixation is to bring a specimen rapidly in contact with a cold metal block. It is usually thought that during this process the surface of the specimen suffers little distortion since it freezes rapidly. Whether this is likely depends on the rate at which samples freeze compared with the speed at which the sample hits the cold block. There is some discrepancy between the published experimentally and theoretically determined freezing rates. As a contribution to this debate the distortion in cryofixed, freeze-substituted, striated muscle fibres has been investigated. In transverse sections, compression can be detected by deviations of the filament lattice from the hexagonal and used to estimate the time of freezing. Some specimens were frozen using a Gatan Cryosnapper, which freezes by catching the specimen between two nitrogen-cooled copper jaws. In addition, the speed with which the jaws close has also been determined. The results suggest that freezing of the well-preserved areas occurs in substantially less than 1 ms. This conclusion is supported by results obtained using metal-mirror apparatus in which the cushioned specimen was dropped onto a nitrogen- or helium-cooled copper block. All the specimens frozen against a cold block have a flat edge whereas muscle fibres are round. At the very edge there is evidence of structural damage as well as the more general lattice distortion.     

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.     

Influence of Ca content and oxygen partial pressure on microstructural evolution of (Co,Ca)O at elevated temperatures

Ca‐doped (1, 1.7, 5 and 10 mol% CaO) cobalt oxide single‐crystal samples, with an [001] orientation, were annealed at elevated temperatures of 1000–1200 °C for different times and at different oxygen partial pressures. The microstructure was examined by means of transmission light and electron microscopy. High‐temperature X‐ray diffractometry was used, with the aim of determining the temperature of the CoO ? Co₃O₄ transition in these materials. Extensive precipitation of Ca‐free Co₃O₄ spinel crystals was observed with increasing Ca content and oxygen activity. It is suggested that the electrical conductivity changes in this material may be related to this precipitation, because it changes the electronic state of cobalt cations.     

Returning to the same area of hair surfaces before and after treatment: a longitudinal AFM technique

We report the use of longitudinal (aspect ratio > 1 : 1) scanning atomic force microscopy as an aid in returning to the same area of hair fibres after bleaching, treatment with a commercial shampoo or the application of a ‘leave‐on’ conditioner product. The bleaching treatment used in this study was not found to affect the cuticular architecture and lateral force microscopy (LFM) also showed little difference after treatment, reflecting the homogeneity of the newly revealed surfaces. After treatment with a commercial shampoo, the hair sample again showed very little difference in topography or lateral force characteristics. Hair treated with the leave‐on conditioner product also showed no major topographical changes. LFM traces, however, showed regions between the ghost edge, marking the original position of the scale edge before cuticular erosion, and the existing scale edge, to have higher frictional properties than distal regions of the cuticle. A thin film of the leave‐on product thus seems to form in this region and extends from the foot of the scale edge.     

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.     

Ice crystal growth in skeletal muscle fibres

Ice crystal growth was studied in rapidly frozen skeletal muscle fibres which were treated with cryo-protective additives (glycerol, DMSO, sucrose) or which were untreated. Freeze cleaving and etching was the basic method, with conventional plastic embedding and cryo-ultramicrotomy as complementary techniques. Extensive crystal growth occurred during freezing in all unprotected fibres. Just below the fibre surface the crystals were numerous but small, while deeper in the fibre they were fewer but larger. The deeper within the specimen a fibre was located, the larger, in general, was the crystal size. The crystal volume density was about 55%, irrespective of crystal size. Ice recrystallization was practically absent at the temperature normally used in cryo-sectioning (–70°C). Anti-freeze treatment eliminated crystal growth. If the anti-freeze agents were used in non-toxic concentrations, however, their effect on crystal growth was very limited. ‘Dry’-cut, freeze-dried ultra-thin cryosections of protected and unprotected fibres confirmed these observations, while sections obtained by ‘wet’ cryo-cutting showed no apparent signs of crystal growth. In plastic sections of frozen and thawed fibres a previous occurrence of crystals was only slightly indicated. In interpreting the ultrastructure in ‘wet’-cut cryo-sections of unprotected frozen mucle fibres, the distorting effects of ice crystals through mechanical compression and alterations in sectioning conditions, must be taken into consideration. Crystal growth also strongly limits the possibilities of using ‘dry’-cut sections of untreated frozen tissue for analytical electron microscopy; only the most superficial parts of the fibres seem to be suitable for microanalysis.     

Morphometric analysis of cryofixed muscular tissue for intraoperative consultation

For diagnostic purposes, cryofixation of tissues is a daily routine technique to investigate rapidly about the presence of tumours during a surgical procedure in patients. We performed morphometric analysis of cryofixed muscular tissues according to different techniques. About 1,000 muscle fibers and 1,493 nuclei, were automatically examined. After freezing, ice tissue interfaces shrinkage of the cells were present. Liquid isopentane or liquid nitrogen produced a statistical increase of fractal dimension, D, of the ice‐tissue interfaces, P < 0.001 respect to the formalin‐fixed samples, cryofixation performed inside the cryostat chamber at t = ?20°C produced a D value close to the formalin‐fixed samples. Shrinkage of the muscle fibers was higher in the samples cryofixed inside the cryostat chamber (P < 0.001). Cryofixation inside cryostat or by liquid nitrogen caused decreases of the nuclei dimensions and altered nuclear morphology (P < 0.01), liquid isopentane appeared not affecting the nuclei of the fibers. Cryofixation inside the cryostat chamber produced the highest shrinkage but it was reduced performing cryofixation in liquid nitrogen or isopentane. Freezing damage inside the muscle cells was absent in the samples cryofixed inside the cryostat, it was present after cryofixation by liquid nitrogen or isopentane. Subcellular components like the nuclei were preserved by isopentane. This paper present, for the first time, an objective method able to quantify and characterize the damages produced by cryofixation in biological sample for intraoperative consultation. Microsc. Res. Tech. 79:155–161, 2016. © 2016 Wiley Periodicals, Inc.     

SiCf–SiBC composites: microstructural investigations of the as-received material and creep tested composites under an oxidative environment

SiC_f–SiBC composites fabricated by Snecma Propulsion Solide (St Médard en Jalles, France) were investigated by SEM and HRTEM in the as‐received state and after creep tests performed in air, in a temperature range 1423–1573 K, under 170 and 200 MPa. These composites are reinforced by Hi‐Nicalon fibres (Nippon Carbon). A pyrocarbon interphase was first deposited on the fibres. The matrix was then deposited on the fibrous preform by several chemical vapour infiltrations (CVI). As a result the matrix is multilayered and based on the Si–B–C ternary system. This matrix is self‐sealing: this is due to the presence of boron inducing the formation of a sealant glass if the material is heated in an oxidative environment. This glass will protect fibres and fibre/matrix interphases against oxidation. Hi‐Nicalon fibres as well as the different matrix layers were studied by HRTEM and EDX. Some investigations were carried out on the creep‐tested specimens in order to characterize modifications observed in the different constituents of the composites, particularly at the interfaces between the matrix layers and at the fibre/matrix interface. It was shown that several matrix layers crystallized during the creep tests. Moreover, a thin silica layer was observed at the pyrocarbon/matrix interfaces. Differences between the behaviour of the same type of material creep tested under neutral atmosphere are discussed.     

Optical far-field microscopy of single molecules with 3.4 nm lateral resolution

Optical far‐field imaging of single molecules in a frozen solution at 1.2 K with a lateral resolution of 3.4 nm is reported. The mechanical stability of the fluorescence microscope, especially of the low‐temperature insert, allows for the localization of fluorescing molecules with a reproducibility of better than 5 nm within observation times up to 10 min. For observation times of 9 h the reproducibility of the lateral position is limited to about 20 nm due to mechanical drift. Lateral position and orientation of 314 single molecules, present within the confocal detection volume of ~10 µm³, are obtained. The possibility to correct for mechanical drift by monitoring the position of a spatial reference in the sample is demonstrated.     

Microscopy and microanalysis of crystalline glazes

Crystalline glazes on ceramic plates produced commercially in the U.K. and on ceramic pots produced commercially in Taiwan and Spain have been examined by X‐ray diffraction, conventional and polarized light microscopy, and scanning electron microscopy in order to identify the crystalline phases present in the glazes and to ascertain through X‐ray microanalysis the partitioning behaviour of the transition metal ions used to colour the glazes and the crystals within them. In each case examined, the macroscopic two‐dimensional spherulites within the glazes clearly seen by the naked eye were found to consist of large numbers of radially orientated acicular crystals each 5 µm or less in width embedded within the silica‐rich glaze. Energy dispersive X‐ray microanalysis and X‐ray diffraction of these crystals identified these crystals as willemite, α‐Zn₂SiO₄. The strong [001] texture of these crystals within the glaze evident from the X‐ray diffraction patterns was consistent with polarized light microscopy observations of the willemite crystals. In addition to willemite, small iron‐doped gahnite (ZnAl₂O₄) crystals were found in a honey‐coloured crystalline glaze and acicular rutile (TiO₂) crystals were found in the Portmeirion Pottery plates examined. Transition metal ions with a preference for tetrahedral coordination were observed to substitute for Zn²⁺ ions in willemite and to partition preferentially to the willemite crystals, whereas ions preferring octahedral coordination preferred to remain in the glaze.     

A cryoclamp for the rapid cryofixation of the isolated blood-perfused rabbit cardiac papillary muscle preparation at predefined times during the contraction cycle

There is increasing evidence that the distribution of monovalent cations in cardiac cells may be non-uniform, particularly in the region immediately beneath the sarcolemma, and we have proposed that a build-up of sodium in this region could be an important factor in the development of ischaemia-reperfusion injury. Electron probe X-ray microanalysis is ideal for the study of such changes in distribution but the application of the technique to this problem imposes severe requirements on the specimen and on the method for cryofixation. The specimen must be perfused through its vasculature so that it can be made truly ischaemic and be successfully reperfused. It is necessary to be able to cryofix the specimen without disturbance of its blood supply, electrical stimulation or temperature. It is also important to know the time in the contraction cycle when cryofixation occurs. Here we describe the design of an automated cryofixation device which can be used to cryofix a blood perfused papillary muscle preparation at predetermined time points in the contraction cycle. Preliminary data obtained from the analysis of rabbit papillary muscles subjected to varying periods of ischaemia are included as an example of the use of the cryoclamp.     

Atomic force microscopy study of DNA deposited on poly l-ornithine-coated mica

Analyses of individual biomolecules, like DNA, or DNA–protein complexes, via atomic force microscopy, require ‘gentle’ methods to immobilize DNA on surfaces, which allow the ensemble of molecules to adopt conformations dictated primarily by their physical characteristics, and which possibly permit the use of a wide selection of buffers. We show that poly‐l ‐ornithine‐coated mica is a good substrate for fast, reliable deposition of DNA for wet or dry imaging. The surface firmly secures DNA, which retains the B‐form helical rise (0.34 nm bp^?1). The conformations of DNA that result are reminiscent of three‐dimensional random coils projected on to a plane. The contrast is good, especially in solution, and buffers with physiological concentrations of salt with or without divalent cations may be used. This is important for comparison of scanning probe microscopy results with those obtained by different techniques.     

Freeze-fracture electron microscopic and low temperature X-ray scattering studies of the effect of cryofixation upon serum low density lipoprotein structure

We report here a correlated X-ray diffraction and freeze-fracture electron microscope study of the effects of several cryofixation procedures upon human serum low density lipoprotein (LDL₂) structure. Only when the LDL₂ solutions contained 75%, by weight, glycerol were the room temperature and post cryofixation low temperature LDL₂ X-ray scattering curves indistinguishable from one another. Other cryofixation procedures, slow or rapid, with or without glycerol, resulted in differences between the room temperature and low temperature LDL₂ X-ray scattering curves, in part due to the effect of quenching upon the solvent. Freezeetching electron microscopy of the slowly cryofixed LDL₂ showed marked aggregation of the particles and an unusual morphological appearance. In contrast, after rapid cryofixation or cryofixation in the presence of glycerol, freeze-etch electron microscopy revealed well-isolated particles which had a knobby morphology. The results demonstrate that under certain conditions (in the presence of 75% glycerol) cryofixation results in minimal, if any, structural alteration of, at least, the LDL₂ lipid moiety. Further, this study underlines the more general conclusion that any high resolution structural study employing a cryofixation step must be interpreted with caution and the effect of cryofixation upon the sample structure need be evaluated by independent means.     

Thermostatic tissue platform for intravital microscopy: 'the hanging drop' model

Intravital microscopy imposes the particular problem of the combined control of the body temperature of the animal and the local temperature of the observed organ or tissues. We constructed and tested, in the rat ileum microcirculation preparation, a new organ‐support platform. The platform consisted of an organ bath filled with physiological solution, and contained a suction tube, a superfusion tube, an intestine‐support hand that was attached to a micromanipulator and a thermometer probe. To cover the intestine we used a cover glass plate with a plastic ring glued on its upper surface. After a routine procedure (anaesthesia, monitoring and surgery), the intestine segment (2–3 cm long) was gently exteriorized and placed on the ‘hand’ of the organ support. A small part of the intestine formed a small ‘island’ in the bath that was filled with physiological salt solution. The cover glass was secured in place. The physiological salt solution from the superfusion tube, which was pointed to the lower surface of the cover glass, formed a ‘hanging drop’. The objective of the microscope was then immersed into distilled water that was formed by the cover glass plastic ring. The ‘hanging drop’ technique prevented any tissue quenching, ensured undisturbed microcirculation, provided for stable temperature and humidity, and permitted a clear visual field.     

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.     

Direct observation of the asphaltene structure in paving-grade bitumen using confocal laser-scanning microscopy

The structure of the asphaltene phase in the bitumen is believed to have a significant effect on its rheological properties. It has traditionally been difficult to observe the asphaltene phase in unaltered samples of bitumen. The maltenes are thought to form a continuous phase in which the asphaltenes are ‘dispersed’. In this study, confocal laser‐scanning microscopy (CLSM) operating in fluorescence mode was used to examine the structure of paving‐grade Safaniya and San Joaquin bitumen. The asphaltene fraction fluoresces in the 515–545 nm wavelength range when irradiated with light with a wavelength of 488 nm. The major advantages of CLSM are that the bitumen sample requires little pretreatment or preparation that may affect the original dispersion of asphaltenes and the bitumen is observed at ambient temperature and pressure. This reduces the possibility of producing images that are not representative of the original material. CLSM was able to show the distribution of maltene and asphaltene components in bitumen. The asphaltene aggregates in the bitumen were observed to be 2–7 µm in size and formed a dispersed ‘sol’ structure in the continuous maltene matrix rather than a network ‘gel’ structure. Surprisingly, the structure and fluorescence of the asphaltene phase does not appear to alter radically upon oxidative ageing. The structure of the asphaltene phase of an AR4000 San Joaquin bitumen was found to be more homogeneous than that of Safaniya bitumen, illustrating the range of structures that can be observed in bitumens by this method.     

Lamellar subcomponents of the cuticular cell membrane complex of mammalian keratin fibres show friction and hardness contrast by AFM

There is a substantial body of information indicating that 18‐methyleicosanoic acid (18‐MEA) is covalently linked to the outer surface of all mammalian keratin fibres and also forms the outer β‐layer of the cuticular cell membrane complex (CCMC) which separates the cuticle cells from each other. Low cohesive forces are expected between the lipid‐containing outer β‐layer and the δ‐layer of the CCMC, thus providing a weak point for cuticular delamination and presenting a fresh layer of 18‐MEA to the newly exposed surface. We have used lateral force microscopy and force modulation atomic force microscopy (AFM) to examine human hair fibres in which the non‐covalently linked fatty acids have been removed. Examination of the lateral force images of new cuticle surfaces revealed by the attrition of overlying cuticle layers showed three separate zones of clearly defined frictional contrast. These are thought to correspond with the δ‐layer, the proteinaceous epicuticle and outer β‐layers of the CCMC. The δ‐layer was found to have a thickness of 16 nm (SD = 1 nm, n = 25), comparable to the 18.0 nm thickness measured from transverse cross‐sections of fibres with transmission electron microscopy. Force modulation AFM showed that the outer β‐layer was softer than the epicuticle and the δ‐layer. The frictional contrast was removed following treatment with methanolic KOH (0.1 mol dm^?3) at 25 °C for 30 min, suggesting the hydrolysis of the thioester linkage and removal of 18‐MEA from the surface.     

Static and dynamic experiments in cryo-electron microscopy: comparative observations using high-vacuum, low-voltage and low-vacuum SEM

We carried out a unique comparative study between three modes of cryo‐scanning electron imaging: high‐vacuum, low‐voltage and low‐vacuum, using ice cream as a model system. Specimens were investigated both with and without a conductive coating (Au/Pd) and at temperatures for which ice either remains fully frozen (< ?110 °C) or undergoes sublimation (?110 to ?90 °C). At high magnification, high‐vacuum imaging of coated specimens gave the best results for ‘static’ specimens (i.e. containing fully frozen ice). Low voltages, such as 1 kV, could be used for imaging uncoated specimens at high vacuum, although slight ‘classical’ charging artefacts remained an issue, and the reduced electron beam penetration tended to decrease the definition between different microstructural features. However, this mode was useful for observing in situ sublimation from uncoated specimens. Low‐vacuum mode, involving small partial pressures of nitrogen gas, was particularly suited to in situ sublimation work: when sublimation was carried out in low vacuum in the absence of an anti‐contaminator plate, sublimation rates were significantly reduced. This is attributed to a small partial pressure of sublimated water vapour remaining near the specimen surface, enhancing thermodynamic stability.     

X-ray omni microscopy

The science of wave‐field phase retrieval and phase measurement is sufficiently mature to permit the routine reconstruction, over a given plane, of the complex wave‐function associated with certain coherent forward‐propagating scalar wave‐fields. This reconstruction gives total knowledge of the information that has been encoded in the complex wave‐field by passage through a sample of interest. Such total knowledge is powerful, because it permits the emulation in software of the subsequent action of an infinite variety of coherent imaging systems. Such ‘virtual optics’, in which software forms a natural extension of the ‘hardware optics’ in an imaging system, may be useful in contexts such as quantitative atom and X‐ray imaging, in which optical elements such as beam‐splitters and lenses can be realized in software rather than optical hardware. Here, we develop the requisite theory to describe such hybrid virtual‐physical imaging systems, which we term ‘omni optics’ because of their infinite flexibility. We then give an experimental demonstration of these ideas by showing that a lensless X‐ray point projection microscope can, when equipped with the appropriate software, emulate an infinite variety of optical imaging systems including those which yield interferograms, Zernike phase contrast, Schlieren imaging and diffraction‐enhanced imaging.     

Embedding in Spurr's resin is a good choice for immunolabelling after freeze drying as shown with chemically unfixed dendritic cells

Immunocytochemical reactions on biological specimens depend on many factors, the most crucial one being the maintenance of antigenicity. Antigens are vulnerable at each stage during preparation for electron microscopy. One of the least traumatic methods of preparing biological tissues for post‐embedding immunolabelling includes the following steps: (1) physical stabilization of the native biological material by rapid freezing (cryofixation) and keeping the immobilized biological sample at low temperature, thereby avoiding any movements of water, ions and macromolecules; (2) dehydrating the frozen biological material by freeze‐drying at low temperature; (3) embedding of the dehydrated specimen. Here we show that embedding of chemically unfixed dendritic cells in Spurr's resin after cryofixation and freeze‐drying enables the conservation of fine ultrastructure without cell distortion or shrinkage. Furthermore, we demonstrate the feasibility of protein localization in ultrathin sections by immunolabelling of the major histocompatibility class II molecules.