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.     

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.     

Biological ultrastructure as revealed by high resolution cryo-SEM of block faces after cryo-sectioning

Ultrastructural information was obtained by imaging the block face of high-pressure-frozen cryo-sectioned biological samples in a high-resolution cryo-SEM. Cryo-sectioning leads to a well-defined flat artificial surface in contrast to cryo-fracturing. Typical artefacts of cryo-sections such as compression and crevasses were not visible on the block face. The ultrastructural features known from resin sections and from freeze-fractures could also be found on the block faces. The cytoplasms show particles of different size which most likely represent proteins. The effects of radiation damage could be reduced considerably by applying the double layer coating technique and backscattered electron imaging.
High quality cryo-sections are only obtained from vitrified material. Reasonably flat block faces were, however, also obtained from adequately frozen microcrystalline samples, thereby facilitating ultrastructural studies in the frozen hydrated state.     

Cryo‐scanning x‐ray diffraction microscopy of frozen‐hydrated yeast

We developed cryo‐scanning x‐ray diffraction microscopy, utilizing hard x‐ray ptychography at cryogenic temperature, for the noninvasive, high‐resolution imaging of wet, extended biological samples and report its first frozen‐hydrated imaging. Utilizing phase contrast at hard x‐rays, cryo‐scanning x‐ray diffraction microscopy provides the penetration power suitable for thick samples while retaining sensitivity to minute density changes within unstained samples. It is dose‐efficient and further minimizes radiation damage by keeping the wet samples at cryogenic temperature. We demonstrate these capabilities in two dimensions by imaging unstained frozen‐hydrated budding yeast cells, achieving a spatial resolution of 85 nm with a phase sensitivity of 0.0053 radians. The current work presents the feasibility of cryo‐scanning x‐ray diffraction microscopy for quantitative, high‐resolution imaging of unmodified biological samples extending to tens of micrometres.     

Visualization of biological specimens by cryoHVEM

This article describes the operation and the characteristics of cryoHVEM imaging of biological specimens using a top-entry cryostage. The procedure for inserting frozen specimens into the microscope column is also presented. Whole mounts were thus observed under optimal imaging conditions by combining: (i) fixation by fast freezing for structure preservation without exposure to chemicals, (ii) observation in the hydrated (frozen) state or in the dried state without exposure to the atmosphere after the initial fixation by freezing, and (iii) ultrastructural visualization with the key imaging factors of resolution, penetration and beam-induced damage at their best by high-voltage electron microscopy.     

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.     

Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.     

Ice crystal damage in frozen thin sections: freezing effects and their restoration

Thin sections of unfixed kidney, fast frozen without cryoprotectants, were fixed in osmium tetroxide vapour directly after freeze drying or after 30 min in a moist atmosphere. Dry sections fixed in vapour showed ice crystal damage characteristic for the freezing procedure. This was demonstrated with freeze fracture replicas from the same preparation. Ice crystal holes were obscured in serial sections which were freeze dried and allowed to rehydrate in a moist atmosphere. The same ultrastructural appearance was observed in frozen sections brought to room temperature immediately after cutting. Frozen thin sections from unfixed tissue, if freeze dried, are very sensitive to atmospheric conditions and need some form of stabilization (e.g. osmium vapour fixation, sealing with an evaporated carbon film) before electron microscope images can be interpreted as representative for the frozen state. Restoration of ice crystal damage can occur by melting frozen sections or by rehydration of freeze dried frozen sections. Restoration phenomena will impair studies aimed at the localization of diffusible substances by autoradiography or X-ray microanalysis.     

Direct observation of liquid crystals using cryo‐TEM: Specimen preparation and low‐dose imaging

Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo‐sectioning approaches, as a comprehensive toolset enabling high‐resolution direct cryo‐TEM observation of a broad range of LCs. We also present comparative analysis using cryo‐TEM and replica freeze‐fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high‐pressure freezing and cryo‐sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo‐TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent‐core thermotropic LCs, comprehensive understanding of the twist‐bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies. Microsc. Res. Tech. 77:754–772, 2014. © 2014 Wiley Periodicals, Inc.     

Transfer,observation and analysis of frozen hydrated specimens

Hitherto, the observation of frozen hydrated specimens in transmission electron microscopes has been inhibited due to the technical difficulties experienced in transferring the specimen to the microscope and maintaining it at a low temperature during observation. This has resulted in loss of the primary advantage of freezing since the frozen water had to be removed from the specimen before it could be introduced into the electron microscope. The cryo-transfer system overcomes these objections and provides a means to transfer frozen hydrated specimens from any preparation equipment into the microscope without ice condensation on the specimen. The cryo-transfer system consists of a cryo-transfer unit, a cryo-specimen holder and a temperature control unit.     

A new cryo‐STEM method for imaging food colloids in the scanning electron microscope

A new cryo‐scanning transmission electron microscopy (cryo‐STEM) technique for imaging casein micelles in a field emission scanning electron microscope is presented. Thin films of micellar casein suspensions on lacey carbon grids were prepared using a modified sample holder developed by Gatan UK. Bright and dark field images were obtained at ?135°C showing casein micelles in their frozen hydrated state and in the size range 30–500 nm. Results were compared favorably with published images of casein micelles obtained with conventional cryo‐transmission electron microscopy, suggesting that cryo‐STEM is a useful alternative technique for visualizing food colloids close to their native state. SCANNING 32: 150–154, 2010. © 2010 Wiley Periodicals, Inc.     

Quantitative digital X-ray imaging using frozen hydrated and frozen dried tissue sections

Application of quantitative X-ray imaging to frozen hydrated tissue sections has presented a number of major problems including lack of a suitable algorithm which could deal effectively with mass loss due to radiation damage, problems of low characteristic X-ray signal to background ratios, and provide a means of analysis of the same location in both hydrated and dried states. This paper presents details of the application of our algorithm for analysis of frozen hydrated, then dried cryosections applied to quantitative X-ray imaging, which provides relatively high precision quantitative measurement of elemental content (related to both wet and dry weight) and water content of each pixel. This algorithm largely circumvents many of the problems of analysis of frozen hydrated tissue sections. Our algorithm for X-ray imaging obtains reasonably precise quantitative measurements coupled with morphological information by trading speed and image resolution.     

Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II. Tomography

Using a cryo scanning transmission X-ray microscope ( Maser, et al . (2000 ) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy. J. Microsc . 197, 68–79), we have obtained tomographic data-sets of frozen hydrated mouse 3T3 fibroblasts. The ice thickess was several micrometres throughout the reconstruction volume, precluding cryo electron tomography. Projections were acquired within the depth of focus of the focusing optics, and the three-dimensional reconstruction was obtained using an algebraic reconstruction technique. In this first demonstration, 100 nm lateral and 250 nm longitudinal resolution was obtained in images of unlabelled cells, with potential for substantial further gains in resolution. Future efforts towards tomography of spectroscopically highlighted subcellular components in whole cells are discussed.     

Solutions to difficulties encountered in low-temperature SEM of some frozen hydrated specimens: Examination of Penicillium nalgiovense cultures

Penicillium nalgiovense cultures, which are used in the food industry, were found to be collapsed when prepared by standard procedures for scanning electron microscopy. Neither freeze-drying nor critical point-drying preserved the structure of cultures grown on agar media. Cryofixation and preparation of frozen hydrated samples using the Hexland Cryotrans CT 1000 attachment in conjunction with an AMR 1000A scanning electron microscope yielded micrographs of uncollapsed structures which could be used for morphological characterization. Several additional steps had to be used in sample preparation to achieve satisfactory results. Samples were held in a humid chamber prior to freezing; growth substrate was trimmed as thinly as possible (less than 1 mm above the support); the sides of samples were painted with a conductive cement to their upper edge; and frozen samples were coated intermittently with gold sputtered in several 2-min bursts.     

Microcarriers for high-pressure freezing and cryosectioning of adherent cells

A method is described employing microcarrier spheres of cross‐linked dextran for obtaining ultra‐ and semithin vitreous sections from high‐pressure frozen anchorage‐dependent (mammalian) cells. Avoiding trypsination or scraping cells off from the culture surface, the presented approach allows for cryoimmobilization, cryosectioning and cryoelectron microscopy/tomography of frozen‐hydrated cells in an unperturbed manner which is important to preserve the native state of, for instance, the cytoskeleton. Furthermore, our studies on the ‘life cycle’ of Herpes simplex virus in Vero cells demonstrate that cell monolayers on microcarrier beads are well suited for fluorescence microscopic characterization of the sample prior to high‐pressure freezing.     

High resolution cryo system designed for JEM 100CX electron microscope

A high resolution (3.5 A) cold stage operated at 123 K and an improved anti-contamination device have been constructed and operated in a top-entry JEM 100CX electron microscope. High resolution electron diffraction patterns and images of hydrated tobacco mosaic virus particles in vitrified ice have been recorded with the use of this cryo system.     

Argon broad ion beam tomography in a cryogenic scanning electron microscope: a novel tool for the investigation of representative microstructures in sedimentary rocks containing pore fluid

The contribution describes the implementation of a broad ion beam (BIB) polisher into a scanning electron microscope (SEM) functioning at cryogenic temperature (cryo). The whole system (BIB‐cryo‐SEM) provides a first generation of a novel multibeam electron microscope that combines broad ion beam with cryogenic facilities in a conventional SEM to produce large, high‐quality cross‐sections (up to 2 mm²) at cryogenic temperature to be imaged at the state‐of‐the‐art SEM resolution. Cryogenic method allows detecting fluids in their natural environment and preserves samples against desiccation and dehydration, which may damage natural microstructures. The investigation of microstructures in the third dimension is enabled by serial cross‐sectioning, providing broad ion beam tomography with slices down to 350 nm thick. The functionalities of the BIB‐cryo‐SEM are demonstrated by the investigation of rock salts (synthetic coarse‐grained sodium chloride synthesized from halite‐brine mush cold pressed at 150 MPa and 4.5 GPa, and natural rock salt mylonite from a salt glacier at Qom Kuh, central Iran). In addition, results from BIB‐cryo‐SEM on a gas shale and Boom Clay are also presented to show that the instrument is suitable for a large range of sedimentary rocks. For the first time, pore and grain fabrics of preserved host and reservoir rocks can be investigated at nm‐scale range over a representative elementary area. In comparison with the complementary and overlapping performances of the BIB‐SEM method with focused ion beam‐SEM and X‐ray tomography methods, the BIB cross‐sectioning enables detailed insights about morphologies of pores at greater resolution than X‐ray tomography and allows the production of large representative surfaces suitable for FIB‐SEM investigations of a specific representative site within the BIB cross‐section.     

Cryogenic‐temperature electron microscopy direct imaging of carbon nanotubes and graphene solutions in superacids

Cryogenic electron microscopy (cryo‐EM) is a powerful tool for imaging liquid and semiliquid systems. While cryogenic transmission electron microscopy (cryo‐TEM) is a standard technique in many fields, cryogenic scanning electron microscopy (cryo‐SEM) is still not that widely used and is far less developed. The vast majority of systems under investigation by cryo‐EM involve either water or organic components. In this paper, we introduce the use of novel cryo‐TEM and cryo‐SEM specimen preparation and imaging methodologies, suitable for highly acidic and very reactive systems. Both preserve the native nanostructure in the system, while not harming the expensive equipment or the user. We present examples of direct imaging of single‐walled, multiwalled carbon nanotubes and graphene, dissolved in chlorosulfonic acid and oleum. Moreover, we demonstrate the ability of these new cryo‐TEM and cryo‐SEM methodologies to follow phase transitions in carbon nanotube (CNT)/superacid systems, starting from dilute solutions up to the concentrated nematic liquid‐crystalline CNT phases, used as the ‘dope’ for all‐carbon‐fibre spinning. Originally developed for direct imaging of CNTs and graphene dissolution and self‐assembly in superacids, these methodologies can be implemented for a variety of highly acidic systems, paving a way for a new field of nonaqueous cryogenic electron microscopy.