Measurements of critical point shrinkage of glutaraldehyde fixed mouse liver

The dimensional changes of small cubes of glutaraldehyde fixed mouse liver tissue were measured using a light microscope image projected into the Quantimet 720 Image Analysing computer system. The dimensional changes occurring in the critical drying bomb could be followed at all stages when violent turbulence was not occurring. The results show that liver tissue blocks shrink in four stages whilst in the critical point drying bomb: (1) during substitution of the intermediate solvent with the transitional fluid; (2) when the transitional fluid is warmed above the critical temperature; (3) when the transitional fluid, now a gas, is allowed to escape from the CPD bomb – the rate of shrinkage increasing as atmospheric pressure is approached; (4) at atmospheric pressure when all the gas has been allowed to escape from the bomb. Taken together with the authors' previous findings, it would seem that substantial shrinkage of animal soft tissue specimens must occur whilst they are undergoing “critical point drying”. This fact should be taken into account when interpreting SEM images of CPD tissues.     

Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes

Critical-point drying and freeze drying were compared both quantitatively and qualitatively as preparative procedures for scanning electron microscopy. Isolated hepatocytes were used as model cells. Nomarski differential interference contrast microscopy was used for light microscopic measurements of the hepatocytes in the unfixed, the glutaraldehyde fixed, the glutaraldehyde + OsO₄ fixed, the critical-point dried and the freeze dried states. Critical-point dried hepatocytes were found to shrink to 38% of glutaraldehyde + OsO₄ fixed volume, whereas optimal freeze dried hepatocytes (frozen in water saturated with chloroform and freeze dried at 183 K for 84 h) were found to shrink to 51% of glutaraldehyde + OsO₄ fixed volume. Transmission and scanning electron micrographs of the critical-point dried cells showed well-preserved ultrastructure and surface structure. Micrographs of the freeze dried cells showed ultrastructure destroyed by internal ice crystals and surface structure destroyed by external ice crystals. Double-fixed isolated hepatocytes were shown to swell during storage in buffer and to shrink during storage after critical-point drying. For low magnification scanning electron microscopy (up to about 3000 times) both critical-point drying and freeze drying can be used. However, for high magnification scanning electron microscopy, critical-point drying is superior to freeze drying.     

Improvement to critical point drying technique for SEM

We have developed an improved method for dehydration and critical point drying which leads to a marked reduction in morphological artefact in at least two classes of problematical specimen: Foetal enamel and avian embryonic heads. Water is replaced by ethanol and ethanol by C₂C1₃F₃ by refluxing in a Soxhlet apparatus. Containers are designed to prevent air drying on transfer to the CPD bomb. We concluded that the thorough removal of both water and ethanol prior to CPD can reduce the types of artefact associated with post-CPD shrinkage (superdrying).     

The effects of glycol methacrylate as a dehydrating agent on the dimensional changes of liver tissue

The dimensional changes of liver sections during the course of processing with glycol methacrylate (GMA) or with ethanol are described. Tissue processing with ethanol served as a control. During prolonged processing steps (24 h each), linear shrinkage of tissue specimens dehydrated with GMA at room temperature was 13.2%. Subsequent infiltration with GMA resulted in trivial swelling, and polymerization in slight shrinkage (2.3%). In comparison, processing with cold GMA resulted in shrinkage during dehydration (about 10.8%), a slight swelling in pure GMA, followed by shrinkage during polymerization (2.2%). Short routine processing schedules resulted in similar shrinkage/swelling patterns, although precise values differed slightly. In all experiments, ethanolic dehydration resulted in smaller dimensional tissue changes than did GMA dehydration. The dimensional changes of tissue sections during stretching on water, mounting and drying compensated for the major part of the shrinkage manifested during processing.     

Air-drying of human leucocytes for scanning electron microscopy using the GTGO procedure

The utilization of tannic acid and guanidine hydrochloride as mordants for better osmium binding has been shown to serve as an excellent alternative to metal coating of organ tissue specimens for scanning electron microscopy (SEM). The present report describes the GTGO procedure, a modification of the TAO technique introduced by Murakami et al. (1977, 1978), which we have found successful for the preparation of air dried peripheral blood leucocytes for SEM studies. Air dried, GTGO-treated leucocytes show excellent preservation of surface features with minimal cell shrinkage. When critical point dried, GTGO-treated cells are examined, they also show less shrinkage than cells prepared with standard glutaraldehyde fixation and critical point drying. The potential application of this air drying procedure (GTGO-AD) to other soft biological specimens is currently under investigation. This technique is recommended as a new and effective air drying procedure for the successful preparation of cells for SEM.     

The use of a simple method to avoid cell shrinkage during SEM preparation

In the course of preparing specimens for scanning electron microscopy, both glutaraldehyde and OsO₄-fixed cells exhibit a considerable shrinkage with a reduction of the mean cellular diameter of about 45% after critical point drying. However, if cells are successively treated with glutaraldehyde, OsO₄, tannic acid and uranyl acetate solutions, cellular shrinkage of only 5% is observed.     

Comparing different methods to fix and to dehydrate cells on alginate hydrogel scaffolds using scanning electron microscopy

Scanning electron microscopy (SEM) is commonly used in the analysis of scaffolds morphology, as well as cell attachment, morphology and spreading on to the scaffolds. However, so far a specific methodology to prepare the alginate hydrogel (AH) scaffolds for SEM analysis has not been evaluated. This study compared different methods to fix/dehydrate cells in AH scaffolds for SEM analysis. AH scaffolds were prepared and seeded with NIH/3T3 cell line; fixed with glutaraldehyde, osmium tetroxide, or the freeze drying method and analyzed by SEM. Results demonstrated that the freeze dried method interferes less with cell morphology and density, and preserves the scaffolds structure. The fixation with glutaraldehyde did not affect cells morphology and density; however, the scaffolds morphology was affected in some level. The fixation with osmium tetroxide interfered in the natural structure of cells and scaffold. In conclusion the freeze drying and glutaraldehyde are suitable methods for cell fixation in AH scaffold for SEM, although scaffolds structure seems to be affected by glutaraldehyde. Microsc. Res. Tech. 78:553–561, 2015. © 2015 Wiley Periodicals, Inc.     

The effects of osmium tetroxide post‐fixation and drying steps on leafy liverwort ultrastructure study by scanning electron microscopy

Leafy liverwort is one of the most abundant and diverse plants in Indonesia. Their high variation and beneficial secondary metabolites contained in the oil bodies have attracted researchers' attention. The ultrastructural analysis of leafy liverworts is important as a means of species identification and also for further exploration of their oil bodies. However, the optimization of the preparation steps for observing leafy liverworts by SEM is necessary to avoid sample destruction. Fixation and drying play important roles in maintaining a sample's structure as close to its natural state as possible. Thus, in this study, we evaluated the effect of 4% Osmium tetroxide (OsO₄) and drying on leafy liverworts ultrastructure. Microlejeunea, Acrolejeunea, and Frullania were fixed with 2.5% glutaraldehyde. Some samples were then post‐fixed with 4% OsO₄, while the rest were directly dehydrated with an ethanol series and then subjected to different drying methods, i.e. air drying, freeze drying, and drying with hexamethyldisilazane (HMDS). According to the data obtained, post‐fixation with 4% OsO₄ could better maintain the integrity of the samples and enhance the contrast of leafy liverwort SEM images. In addition, samples dried with HMDS showed more detailed structures compared to those that were air dried. Different ultrastructure were found among the different leafy liverworts observed by SEM. Our data suggested the advantages of SEM in providing ultrastructure information on leafy liverworts as well as the optimum conditions to observe them with less deformation. OsO₄ post‐fixation could enhance the contrast of leafy liverwort SEM images and maintain the structure of the samples. Drying with HMDS provided a convenient way for rapid SEM preparation with less structural distortion.     

A comparative survey of techniques for preparing plant surfaces for the scanning electron microscope

A comparative investigation of techniques for the preparation of soft botanical tissue for the scanning electron microscope has been carried out using the leaves and petals of Pelargonium zonale as test specimens. Twelve different preparative procedures involving combinations of fixation, dehydration, air drying, freeze drying, critical point drying, coating methods, replicas and a temperature controlled specimen stage were tested.     

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.     

Dimensional stability in tissues dehydrated with 2,2-dimethoxypropane for electron microscopy

Dimensions of tissues fixed in glutaraldehyde-osmium tetroxide mixture, osmium tetroxide and uranyl acetate and then dehydrated in 2,2-dimethoxypropane (DMP) were measured using transmission and scanning electron microscopy. Rat cardiac muscle, kidney and other tissues were examined in this study. The mean dimensions of characteristic ultrastructural features of material prepared by this method are similar or larger than those reported in the literature for conventionally processed samples. Critical point drying of specimens dehydrated with DMP does not produce abnormal shrinkage. Simultaneous primary fixation of lipids and proteins in a glutaraldehyde osmium tetroxide mixture and omission of the water wash after uranyl acetate appear to be important in stabilizing the tissue for rapid dehydration. The rapid reaction of DMP and water yielding the products acetone and methanol does not appear to denature tissue components to a greater extent than conventional solvent exchange dehydration.     

Methanol as a rapid fixative for the investigation of plant surfaces by SEM

A rapid fixation/dehydration method of plant specimens for scanning electron microscopy is presented. Prior to critical-point drying (CPD) the specimens are immersed in pure methanol. Methanol incubation instantly fixes the elastically extended cell walls. Owing to this instant fixation, shrinking of the specimens is prevented, resulting in an improved preservation of cell dimensions comparable to in vivo conditions. The method is most suitable for plant epidermal surfaces. It avoids the time-consuming fixation/dehydration in routine investigation of plant surfaces prior to CPD, especially for delicate specimens.     

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.     

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.     

"Large" and "small" nuclear pore complexes; the influence of glutaraldehyde.

Aliquots of lymphocyte cell suspensions were pretreated according to the following three schedules before freeze fracturing: (a) prefixed with 2% glutaraldehyde before infiltration with 25% glycerol in medium RPMI-1640; (b) frozen in medium RPMI-1640 without additional pretreatment; and (c) frozen after pretreatment with 25% glycerol in medium RPMI-1640. The diameters of the fractured nuclear pore complexes of cells prefixed with glutaraldehyde were normally distributed within the range 70-120 nm (median 90 nm). The nuclear envelopes of 66-75% of cells processed through schedules b and c, which omitted glutaraldehyde fixation, had 70-120 nm diameter pores, while the remainder had pores with diameters in the range 120-175 nm. The large pores were structurally similar to the smaller pores except for their dimensions. These results indicate that glutaraldehyde gives rise to shrinkage of the larger pores to the minimum, smaller, diameter. Apparent orifices of at least 30 nm diameter were sometimes observed at the centres of these large pore complexes. We propose that the variation in pore diameters may indicate opening and closure of this orifice, and that the widely reported "central granule" of the nuclear pore complex corresponds with the orifice in a closed configuration.     

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.     

Comparison of Two Digestive Techniques for Preparation of Vascular Elastic Networks for SEM Observation

Two digestion procedures are now available to expose and isolate networks of vascular elastic fibers for three-dimensional SEM observation. This study was designed to observe and elucidate the differences between the two types of digestion (sodium hydroxide vs. formic acid) and the differences between the two types of dehydration (ethanol-critical-point drying vs. freeze drying) used in each procedure. Canine venous valve segments, containing delicate networks of elastic fibers, and femoral arteries, containing large elastic lamellae, were used to compare the effects of the digestion and dehydration procedures on two types of vessels with different content and organization of elastic tissue. Results indicated there was no significant difference in the architecture of the elastic networks of either vessel based on the method of digestion. The major architectural changes in the elastic networks occurred as a result of the dehydration procedure used following digestion. Freeze drying is probably the best for arterial specimens due to their prominent lamellae, which give added support to maintain their normal architecture. It is suggested that both methods of dehydration be used on corresponding venous specimens containing delicate elastic networks. In this way, the investigator can benefit from the advantages of each method and overcome their respective disadvantages to get a more accurate picture of the three-dimensional architecture of these delicate networks.     

Quantitation of shrinkage during preparation for scanning electron microscopy: Human lung

Human lung tissue is found to shrink considerably with preparation for SEM. Fifty-one blocks of glutar-aldehyde-fixed and inflated lung, approximately 2.5 cm × 2.5 cm × 1 cm, shrank a mean of 19% (± 4.0% SD) linear dimension through post fixation, dehydration and critical point drying. Shrinkage with fixation was not measured. Blocks of lung were observed to shrink equally in length (L) and width (W), L = 19.4% ± 2.7 SD, W = 19.0% ± 4.0 SD. Final shrinkage was the same whether samples were dehydrated in acetone or ethanol, although with acetone more of the shrinkage occurred during the dehydration process and less occurred during critical point drying.     

Backscattered electron imaging of immunogold-labeled and silver-enhanced microtubules in cultured mammalian cells

This technique permits the visualization of microtubules in situ by employing silver-enhanced immunogold labeling and backscattered electron imagery. For best results, monolayer cultures of PtK₂ cells are lysed with Triton X-100 in a microtubule stabilizing buffer, fixed with 1% glutaraldehyde, reduced with NaBH₄, incubated with monoclonal antitubulin and 5-nm gold-labeled anti-IgG, silver enhanced, freeze dried, lightly coated with aluminum, and examined in an SEM equipped with a backscattered electron detector. A high contrast view of the entire microtubule complex of each cell is obtained. Microtubules in freeze-dried preparations have relatively smooth surfaces, whereas those in critical point dried preparations are more irregular or beaded. At high magnifications, an unstained inner core of each microtubule can be resolved. Backscattered electron imaging appears to be a promising technique for localizing cytoskeletal proteins and other intracellular antigens that can be labeled with immunogold and enhanced with silver.     

Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

Trace metals play important roles in biological function, and x‐ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side‐by‐side comparison shows that plunge‐freezing‐based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. When chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x‐ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.