Cryosputtering—a combined freeze-drying and sputtering method for high-resolution electron microscopy

Preparing cellular structures for visualization by high-resolution scanning electron microscopy (SEM) is a multi-step process which includes fixation, dehydration, drying and metal coating. Drying and metal coating are limiting for high-resolution work. Commonly, the dried samples are exposed to the air before they are inserted into a metal coating apparatus, thereby exposing them to moisture and the accompanying risk of rehydration, which may cause changes in the supramolecular structure. We have modified a freeze-dryer to accommodate a magnetron sputtering head, in order to sputter-coat the frozen-dried samples while still in the drying chamber in the cold, a process we call cryosputtering. A layer of 1·5 nm of tungsten was cryosputtered onto whole mounts of cytoskeletons from detergent-extracted human glioma cells or fibroblasts and the specimens were examined by high-resolution SEM and transmission electron microscopy (TEM). To reduce the effects of backstreaming oil from the vacuum system, a turbomolecular pump backed by a two-stage rotary vane pump was connected to the drying-coating chamber. This pump system provides a high vacuum, making it possible to dry the specimens at — 90°C/183 K, thus reducing the risk for recrystallization of water. Furthermore, the high vacuum minimizes the negative effects of contaminants, which can be deposited onto the specimen surface and affect the quality of the metal coat formed during sputtering.     

Scanning electron microscopy of piliated Neisseria gonorrhoeae processed with hexamethyldisilazane

Piliated Neisseria gonorrhoeae are virulent and attach readily to some human mucosal cells. The study of interactions between piliated Neisseria gonorrhoeae and surface structures of eukaryotic cells in tissue culture requires consistent high resolution imaging in scanning electron microscopy (SEM). The combination of the fixatives glutaraldehyde, osmium, tannic acid, and uranyl acetate improves preservation of pili and other delicate structures. Following the critical point drying (CPD) process, pili bundles remained intact, but charging produced image distortion in most of the specimens. The use of hexamethyldisilazane (HMDS) with air drying substantially reduced charging and image distortion. Less contrast and greater resolution of pili bundles and surface structures of bacteria or tissue culture cells were obtained at magnifications of 10,000 or higher. As an alternative to CPD, HMDS processing of cell culture monolayers was simple and was more efficient when a large number of samples was processed.     

The fixation,dehydration, drying and coating of cultured cells for SEM

Cultivated cells form a valuable model system for studies on the effects of various preparative protocols for scanning electron microscopy (SEM). The various effects of each preparative step can be followed in detail in the light microscope and no diffusion gradients complicate the fixation and other procedures as in the case of solid tissues. Studies on cultivated cells indicate that the glutaraldehyde component of a glutaraldehyde-based fixative does not contribute to the effective osmotic pressure of the fixative and thus the osmolarity of the buffer, and other components, must be equalized to that of the medium in which the cells grow. Even small deviations from this ideal effective osmotic pressure will result in osmotically induced artefacts. Disturbances of pH and temperature of the cultures prior to and during fixation will result in changes in the appearance of many cellular structures such as microspikes and ruffles. We find that osmium fixation is advisable in most instances for best possible membrane preservation and that even long periods of glutaraldehyde fixation do not compensate for osmium fixation. Dehydration always results in shrinkage. Freeze drying (FD) and critical point drying (CPD) also give rise to shrinkage, the former to a lesser degree than the latter. A gold-palladium alloy gives a less granular coating that does gold alone. When cultured cells are studied, a metal thickness of between 5 and 15 nm is usually sufficient to give rise to an adequate secondary electron production and to avoid charging even at accelerating voltages of 30–40 kV. Without treatment with OsO₄ a thicker metal coating is required.     

Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy

Structure of bacterial biofilms may be investigated using several variants of scanning electron microscopy (SEM). We apply lyophilization to prepare nitrifying bacterial biofilm for conventional SEM imaging in high-vacuum mode (CSEM). We therefore replace standard biofilm fixation in glutaraldehyde cross-linking, ethanol dehydration, and critical-point drying (CPD) with less-invasive low-temperature drying by sublimation in vacuum. We compare this approach with: (1) standard preparation with glutaraldehyde fixation, ethanol dehydration, and CPD before CSEM, (2) cryo-sputter preparation of rapidly frozen biofilm in hydrated state (cryo-SEM), and (3) in situ observation without any sample pretreatment in environmental SEM. Combined imaging with these modalities revealed two distinct immobilization patterns on the polyurethane foam: (1) large irregular aggregates (flocs) of bacterial biofilm that exist as irregular biofilm fragments, rope-like structures, or biofilm layers on the foam surface; (2) biofilm threads adherent to the surface of polyurethane foam. Our results indicate that lyophilization was suitable for preservation of bacterial cells and many forms of structure of extracellular matrix. The lyophilized material could be imaged with high resolution (using CSEM) to generate structural information complementary to that obtained with other SEM techniques.     

Cell and organelle shrinkage during preparation for scanning electron microscopy: effects of fixation, dehydration and critical point drying.

The critical point drying method of preparing samples for scanning electron microscopy is associated with a variable amount of specimen shrinkage. We studied the causes of this phenomenon is isolated mouse hepatocyte nuclei and in human erythrocytes and found that the critical point drying process itself caused most of the shrinkage that we observed (a 25-30% reduction in diameter in both specimens). Glutaraldehyde fixation and ethanol dehydration caused only minimal size reduction, prior to critical point drying. Substitution of an inert (ethylene glycol-ethylene glycol monethyl ether) dehydration technique did not alter the final result. Previous studies in our laboratory using high resolution SEM and correlative transmission microscopy of isolated nuclei have demonstrated that the shrinkage represents a miniaturization of the organelles in which all structural components retain their usual relationships.     

Three‐dimensional analysis of intermediate filament networks using SEM tomography

We identified tomographic reconstruction of a scanning electron microscopy tilt series recording the secondary electron signal as a well‐suited method to generate high‐contrast three‐dimensional data of intermediate filament (IF) networks in pancreatic cancer cells. Although the tilt series does not strictly conform to the projection requirement of tomographic reconstruction, this approach is possible due to specific properties of the detergent‐extracted samples. We introduce an algorithm to extract the graph structure of the IF networks from the tomograms based on image analysis tools. This allows a high‐resolution analysis of network morphology, which is known to control the mechanical response of the cells to large‐scale deformations. Statistical analysis of the extracted network graphs is used to investigate principles of structural network organization which can be linked to the regulation of cell elasticity.     

A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues

In this paper, we review some published studies using correlative light and electron microscopy methods. We further refined our criteria to include only those studies using live cells for light microscope and where high-pressure freezing was the method of specimen preparation for electron microscopy. High-pressure freezing is especially important for some difficult-to-fix samples, and for optimal preservation of ultrastructure in samples larger than a few micrometres. How the light microscope observations are done is completely sample dependent, but the choice of high-pressure freezer depends on the speed required to capture (freeze) the biological event of interest. For events requiring high time resolution (in the 4–5 s range) the Leica EM PACT2 with rapid transfer system works well. For correlative work on structures of interest that are either non-motile or moving slowly (minutes rather than seconds), any make of high-pressure freezer will work. We also report on some efforts to improve the capabilities of the Leica EM PACT2 rapid transfer system.     

SEM of the tubules of the hepatopancreas and the stomach gland of the scorpion (buthus quinquestriatus): A methodological study

Different procedures were tested for the purpose of circumventing the technical obstacles encountered in SEM of the scorpion hepatopancreas and stomach gland. These problems are caused by the relatively high osmotic pressure of the body fluid of this animal, the irregular surface contour, and the very high content of fat in the organs under investigation. The best results were obtained following glutaraldehyde fixation at an effective osmotic pressure approaching that of scorpion body fluid, ensuring stabilization of fat by post-fixation in osmium tetroxide, and overcoming the charge effects via the OTOTO (Osmium-Thiocarbohydrozide) method in combination with metal coating. Dehydration by CPD or FD both gave rise to inevitable artefacts (shrinkage or cracking). Used together, however, CPD and FD were complementary. The surfaces of hepatopancreatic and stomach gland tubules showed a distinct similarity. Tubules of both organs were lined mostly with cells equipped with parallel arrays of microvilli on the apical surface and containing large amounts of fat.     

Applications of medium-voltage STEM for the 3-D study of organelles within very thick sections

Scanning transmission electron microscopy at 300 kV enables the visualization of nucleolar silver-stained structures within thick sections (3–8 μm) of Epon-embedded cells at high tilt angles (–50°; + 50°). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-μm-thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e^—/Å₂/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4-μm-thick sections with a spot-size of 5·6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 μm. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.     

Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells

Critical point drying (CPD) is a common method of drying biological specimens for scanning electron microscopy (SEM). Drying by evaporation of hexamethyldisilazane (HMDS) has been described as a good alternative. This method, however, is infrequently used. Therefore, we reassessed HMDS drying. Cultured rat hepatic sinusoidal endothelial cells (LEC), possessing fragile fenestrae and sieve plates, were subjected to CPD and HMDS drying and evaluated in the scanning electron microscope, atomic force microscope (AFM) and transmission electron microscope (TEM). We observed no differences between the two methods regarding cellular ultrastructure. In contrast with CPD, HMDS drying takes only a few minutes, less effort, low costs for chemicals and requires no equipment. We conclude that HMDS-dried specimens have equal quality to CPD ones. Furthermore, the method also proved useful for drying whole-mount cells for TEM and AFM.     

Ultrastructure of Trypanosoma cruzi revisited by atomic force microscopy

Most advances in atomic force microscopy (AFM) have been accomplished in recent years. Previous attempts to use AFM to analyze the organization of pathogenic protozoa did not significantly contribute with new structural information. In this work, we introduce a new perspective to the study of the ultrastructure of the epimastigote form of Trypanosoma cruzi by AFM. Images were compared with those obtained using field emission scanning electron microscopy of critical point dried cells and transmission electron microscopy of negative stained detergent-extracted and air-dried cells. AFM images of epimastigote forms showed a flagellum furrow separating the axoneme from the paraflagellar rod (PFR) present from the emergence of the flagellar pocket to the tip of the flagellum. At high magnification, a row of periodically organized structures, which probably correspond to the link between the axoneme, the PFR and the flagellar membrane were seen along the furrow. In the origin of the flagellum, two basal bodies were identified. Beyond the basal bodies, small periodically arranged protrusions, positioned at 400 nm from the flagellar basis were seen. This structure was formed by nine substructures distributed around the flagellar circumference and may correspond to the flagellar necklace. Altogether, our results demonstrate the importance of the application of AFM in the structural characterization of the surface components and cytoskeleton on protozoan parasites.     

Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching

Fluorescence light microscopy (LM) has many advantages for the study of cell organization. Specimen preparation is easy and relatively inexpensive, and the use of appropriate tags gives scientists the ability to visualize specific proteins of interest. LM is, however, limited in resolution, so when one is interested in ultrastructure, one must turn to electron microscopy (EM), even though this method presents problems of its own. The biggest difficulty with cellular EM is its limited utility in localizing macromolecules of interest while retaining good structural preservation. We have built a cryo-light microscope stage that allows us to generate LM images of vitreous samples prepared for cryo-EM. Correlative LM and EM allows one to find areas of particular interest by using fluorescent proteins or vital dyes as markers within vitrified samples. Once located, the sample can be placed in the EM for further study at higher resolution. An additional benefit of the cryo-LM stage is that photobleaching is slower at cryogenic temperatures (−140°C) than at room temperature.     

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy

This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1–2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.     

Scanning force microscopy of chromatin fibers in air and in liquid

We have adapted specimen preparation techniques of conventional electron microscopy for visualizing chromatin structures in the scanning force microscope (SFM) in air and in liquid. The beaded substructure of the nucleoprotein filament was obtained after hypotonic lysis of chicken erythrocytes and air drying, whereas supranucleosomal structures were preserved after treatment of cell nuclei with detergent. In the latter case, the nucleosomes were still distinct but appeared more condensed. A modified droplet diffusion-spreading technique of chromatin from Namalwa cells (a human B-lymphoid line) yielded a uniform filamentous morphology and similar fiber appearance. A reversible swelling of spread chromatin was observed upon exposure of air-dried samples to solutions differing in salt concentrations.     

Some experiences in the use of a polymeric cryoprotectant in the freezing of plant tissue

Recently it has been suggested that polymeric cryoprotectants might be usefully employed for reducing ice crystal size during ultrastructural and analytical studies of frozen biological tissues. Furthermore, it was reported that they have little physiological effect and cause negligible structural changes in the tissue. Our experiences with one such polymer, polyvinyl pyrrolidone (PVP), in the cryopreservation of mature plant roots prepared for electron microscopy, have led us to conclude that preservation deep into the structure of this tissue is not improved. Even short periods of exposure of tissue to polymer cause rapid withdrawal of water from vacuolated cells of plant roots, resulting in shrinkage and collapse. Low temperature techniques have confirmed that little if any improvement in the reduction of ice crystal size results if the root is first treated with PVP.     

High-resolution imaging and nano-manipulation of biological structures on surface

In this mini-review we discuss our recent findings on imaging and manipulation of biological macromolecular structures by atomic force microscopy (AFM). In the first part of this review, we focus on high-resolution imaging of selected biological samples. AFM images of membrane proteins have revealed detailed conformational features related to identifiable biological functions. Different self-assembling behaviors of short peptides into supramolecular structures on various substrates under controlled environmental conditions have been systematically studied with AFM imaging. In the second part, we present a novel nano-manipulation technique for manipulating, isolating, amplifying, and sequencing of individual DNA molecules, which may find unique applications in the analysis of difficult sequence structures. Finally, we discuss how to characterize the elasticity of individual biomolecules and live cells. These results demonstrate that not only the high resolution capacity of the AFM is suited to resolve certain biological questions, but can also be applied to single molecule isolation and biomechanical analysis with its unique advantages.     

Pros and cons: cryo-electron microscopic evaluation of block faces versus cryo-sections from frozen-hydrated skin specimens prepared by different techniques

Over the last two decades, several different preparative techniques have been developed to investigate frozen‐hydrated biological samples by electron microscopy. In this article, we describe an alternative approach that allows either ultrastructural investigations of frozen human skin at a resolution better than 15 nm or sample throughput that is sufficiently high enough for quantitative morphological analysis. The specimen preparation method we describe is fast, reproducible, does not require much user experience or elaborate equipment. We compare high‐pressure freezing with plunge freezing, and block faces with frozen‐hydrated slices (sections), to study variations in cell thickness upon hydration changes. Plunge freezing is optimal for morphological and stereological investigations of structures with low water content. By contrast, high‐pressure freezing proved optimal for high‐resolution studies and provided the best ultrastructural preservation. A combination of these fast‐freezing techniques with cryo‐ultramicrotomy yielded well‐preserved block faces of the original biological material. Here we show that these block faces did not exhibit any of the artefacts normally associated with cryo‐sections, and – after evaporating a heavy metal and carbon onto the surface – are stable enough in the electron beam to provide high‐resolution images of large surface areas for statistical analysis in a cryo‐SEM (scanning electron microscope). Because the individual preparation steps use only standard equipment and do not require much experience from the experimenter, they are generally more usable, making this approach an interesting alternative to other methods for the ultrastructural investigation of frozen‐hydrated material.     

Preservation of high resolution protein structure by cryo-electron microscopy of vitreous sections

We have quantitated the degree of structural preservation in cryo-sections of a vitrified biological specimen. Previous studies have used sections of periodic specimens to assess the resolution present, but preservation before sectioning was not assessed and so the damage due particularly to cutting was not clear. In this study large single crystals of lysozyme were vitrified and from these X-ray diffraction patterns extending to better than 2.1 Å were obtained. The crystals were high pressure frozen in 30% dextran, and cryo-sectioned using a diamond knife. In the best case, preservation to a resolution of 7.9 Å was shown by electron diffraction, the first observation of sub-nanometre structural preservation in a vitreous section.     

Volume changes during preparation of mouse embryonic tissue for scanning electron microscopy

This paper describes the results of experiments in which the volume changes in mouse embryo limb samples were followed more or less continuously after fixation through dehydration and critical point drying, with in some instances data relating to post critical point drying shrinkage. 14 and 15 day p. c. mouse embryos were fixed in 3 % glutaraldehyde in cacodylate buffer and stored in this fixative until use. Single specimens were studied using a Quantimet image analysing computer to record the changes in projected area of the unmounted specimens as they were passed through the usual series of reagents according to various commonly used dehydration schedules. The area changes were converted to volume changes for the purposes of presentation in this paper. The Quantimet system could not be used to follow volume changes in the CPD bomb so that most experiments detail the volume in the intermediate fluid before CPD and the size of the specimen immediately after it was removed from the CPD bomb. A few experiments were conducted in which the specimens were measured whilst they were in the CPD bomb. The measurements relating to dehydration and CPD procedures were compared with measurements of air dried and freeze dried specimens. All three drying methods cause considerable shrinkage: freeze drying to 85 % of the glutaraldehyde fixed tissue volume; critical point drying to 41% (after 24 h); and air drying from a volatile solvent to about 18% of the fixed tissue volume. Air drying from water caused a shrinkage to about 12% of the original volume. There was no significant difference between the various commonly used CPD schedules or between GA only and GA + Os O₄ fixed tissue. CPD via cellosolve and CO₂ caused substantially more shrinkage than other methods. Dimensional changes during specimen preparation are probably associated with changes in shape and in relative relationships between organelles, cells and tissues having different compositions. This should be borne in mind by all those interpreting scanning electron micrographs of dried animal soft tissue specimens.     

Microtubule structure studied by quick freezing: Cryo-electron microscopy and freeze fracture

Microtubules have been quickly frozen and examined by electron microscopy using several techniques: (1) freezing of a thin layer of solution by plunging into cryogen, followed by cryo-electron microscopy of the unstained vitrified samples; (2) freezing by the propane-jet method, followed by freeze fracturing and metal replication. The unstained frozen-hydrated microtubules show a structure in agreement with X-ray diffraction data; they differ from negatively stained particles mainly by the better preservation of cylindrical shape. Secondly, they reveal a supertwist of the profilaments that is not detected reliably by other methods. This allows a determination of the number of protofilaments and the polarity. The structural resolution of unstained microtubules is similar to that of stained ones (about 2–3 nm); it is limited by low contrast and lack of crystalline order. Propane-jet or cryo-block freezing followed by freeze fracturing reveals the structures of the inner and outer surfaces of the microtubule wall at a resolution of 4 nm or better. The outside is dominated by the longitudinal protofilaments whereas on the inside one observes tilted cross-striations. Although the freezing temperatures of the two methods are different (liquid nitrogen or helium) they yield similar results for the case of thin layers of protein solution.