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.     

Scanning electron and light microscope correlation of individual human bone marrow cells before and after culture in nutrient agar.

This study was undertaken with the aim of identifying the different cell types found in human bone marrow by examining their surface morphology. In an attempt to obtain a homogeneous cell population, cells were both fractionated by discontinuous albumin density gradient centrifugation (DADGC) and selectively grown in nutrient agar. Both cell preparations underwent the critical point drying technique before examination under both the scanning electron microscope (SEM) and subsequently the light microscope (LM). When the SEM image of individual cells was compared with the corresponding LM image, it was not easy to identify the different cell types, because of the shrinkage and distortion that occurred during their preparation. The shrinkage observed under the SEM amounted to a 45% reduction in mean cell diameter. This shrinkage was confirmed by comparing the SEM and LM images of the same cell. Although shrinkage occurred throughout the dehydration sequence, critical point drying was responsible for a 25% reduction in mean cell diameter. Furthermore, direct observation under LM of fixed cells drying in air from ethanol, revealed visible contraction of the cell and distortion of the cell membrane. We assume that a similar morphological change occurred during critical point drying. We conclude that the shrinkage and distortion, caused by the dehydration process involved in SEM preparation, severely limit the value of a study of surface morphology by SEM in the identification of the different cell types found in human bone marrow.     

Tillandsia stricta Sol (Bromeliaceae) leaves as monitors of airborne particulate matter—A comparative SEM methods evaluation: Unveiling an accurate and odd HP‐SEM method

Airborne particulate matter (PM) has been included among the most important air pollutants by governmental environment agencies and academy researchers. The use of terrestrial plants for monitoring PM has been widely accepted, particularly when it is coupled with SEM/EDS. Herein, Tillandsia stricta leaves were used as monitors of PM, focusing on a comparative evaluation of Environmental SEM (ESEM) and High‐Pressure SEM (HPSEM). In addition, specimens air‐dried at formaldehyde atmosphere (AD/FA) were introduced as an SEM procedure. Hydrated specimen observation by ESEM was the best way to get information from T. stricta leaves. If any artifacts were introduced by AD/FA, they were indiscernible from those caused by CPD. Leaf anatomy was always well preserved. PM density was determined on adaxial and abaxial leaf epidermis for each of the SEM proceedings. When compared with ESEM, particle extraction varied from 0 to 20% in air‐dried leaves while 23–78% of particles deposited on leaves surfaces were extracted by CPD procedures. ESEM was obviously the best choice over other methods but morphological artifacts increased in function of operation time while HPSEM operation time was without limit. AD/FA avoided the shrinkage observed in the air‐dried leaves and particle extraction was low when compared with CPD. Structural and particle density results suggest AD/FA as an important methodological approach to air pollution biomonitoring that can be widely used in all electron microscopy labs. Otherwise, previous PM assessments using terrestrial plants as biomonitors and performed by conventional SEM could have underestimated airborne particulate matter concentration.     

The preparation of leaf surfaces for scanning electron microscopy: a comparative study

Cryopreservation is the superior technique for viewing leaf surfaces in the SEM. Epidermal cells become distorted when freeze dried and disrupt the orientation of epicuticular wax structures. The latter are largely lost during critical point drying. Nevertheless, the appearance of surface structures after subjecting them to each drying method is valuable in interpreting the features observed by cryopreservation.     

Scanning electron microscopic study of immunogold-labeled human leukocytes

For many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein is a simple, efficient, inexpensive, reproducible, and safe procedure using Peldri II, a proprietary fluorocarbon compound that is solid at room temperature and a liquid above 25°C, as a sublimation dehydrant for processing specimens for SEM. The utility of Peldri II was demonstrated in studies using leukocytes from the blood of healthy donors and patients with leukemia as well as from long-term lymphoblastoid cell lines. The application of the proposed Peldri II procedure was further documented in SEM studies in which the expression and distribution of the interleukin-2 receptor (IL-2R) on leukocyte surface membranes was imaged using colloidal gold-labeled antibodies (i.e., immunogold). When compared with current SEM preparation procedures using CPD, Peldri II is a useful alternative that is thought to offer several important advantages.     

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.     

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.     

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.     

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.     

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.     

Liquid substitution: A versatile procedure for SEM specimen preparation of biological materials without drying or coating

Certain liquids with a very low vapour pressure, such as glycerol or triethylene glycol, can be used to infiltrate biological specimens so that they may be observed in the scanning electron microscope (SEM) without drying. The conductive properties of the fluids allow specimens to be examined either uncoated or with very thin coatings. The advantages of liquid substitution include the retention of lipids, waxes, loose particles, and surface contaminants. Since the procedure does not require expensive equipment, it offers an alternative to critical point drying or cryo-preparation. For certain types of specimens, liquid substitution may represent the best preparation procedure. In addition, the fluids themselves may be imaged directly in the SEM, or indirectly by cathodoluminescence following labelling with fluorochromes.     

Sectionable microcarrier beads: An improved method for the preparation of cell monolayers for electron microscopy

Cross-linked dextran beads provide an excellent surface for tissue-cultured cell monolayers, and can be processed for transmission (TEM) and scanning (SEM) electron microscopy, as well as light microscopy (LM). Cells are grown to confluency on the surface of the microcarriers, where at any point aliquots can be removed and experimentally treated as desired (e.g. immunocytochemistry) providing a representative sample. Sample preparation for TEM follows standard procedures for any cell monolayer, but infiltration times must be at least doubled to allow penetration of the beads. The polymerized blocks can then be sectioned for TEM or LM with no additional steps required. SEM sample preparation involves attaching the fixed bead/cell suspension to a glass coverslip with poly-1-lysine, dehydration, critical point drying, and coating for conductivity. The fixed and dried sample can also be attached directly to the SEM stub as free beads and subsequently gold coated. These beads provide (1) an increased surface area of cells visible per area of thin section, (2) eliminates the careful orientation required for flat substrate methods of embedding, (3) decreases the amount of sample manipulation in the forms of re-embedding and gluing, and (4) decreases the amount of drying artifact seen as cracking in SEM monolayer preparations.     

A procedure for rupture-free preparation of confluently grown monolayer cells for scanning electron microscopy

Monolayers of PtK-1 and HeLa cells grown on glass or plastic supports are extremely susceptible to lacerations, e.g., splits and cracks caused mainly by shrinkage when prepared for scanning electron microscopy (SEM). We find that a four-step fixation procedure including glutaraldehyde, OsO₄, tannic acid, and uranylacetate application, in combination with critical point drying, drastically reduces these structural damages. In addition, the conductivity of the specimens is enhanced, so that they can be investigated without gold coating. Transmission electron microscopy (TEM) investigation of perpendicular sections in the area of lacerations provides evidence that the subcortical cytoskeletal elements are of crucial importance in maintaining cell membrane stability during the preparations. Our relatively quick and simple procedure results in an improved structural appearance of the cells.     

SEM sample preparation for cells on 3D scaffolds by freeze-drying and HMDS

Common dehydration methods of cells on biomaterials for scanning electron microscopy (SEM) include air drying, hexamethyldisilazane (HMDS) or tetramethysilane (TMS) treatment and critical point drying (CPD). On the other side, freeze-drying has been widely employed in dehydrating biological samples and also in preparing porous biomaterial scaffolds but not in preparing cells on three-dimensional (3D) biomaterials for SEM examination. In this study, we compare cells on porous hydroxyapatite (HA) prepared by air drying, HMDS and freeze-drying. The effects of fixation and using phosphate buffered saline (PBS) in the fixation were also assessed on three porous calcium phosphate (CaP) materials, namely, HA, α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP) samples. There is no significant difference in samples prepared by HMDS treatment and freeze-drying viewed at low magnification. Besides, it is better not to use phosphate buffer in the fixation step for CaP materials to avoid undesirable spontaneous precipitation of CaPs. On the other hand, fewer exchanges of liquids are required for freeze-drying and hence chemical fixation may not be absolutely required for samples prepared by freeze-drying. Other technical details of the preparation were also investigated and discussed. This study suggests both HMDS and freeze-drying can be employed to dehydrate cells on 3D scaffolds for SEM examination.     

A quantitative study of the preparation of rabbit aortic endothelial cells for scanning electron microscopy

Quantitative studies were done with the scanning electron microscope (SEM) on aortic endothelial cells from ten rabbits. Of these, five were plastic casts and five were dehydrated with three different, but standard, techniques. The results indicated that all forms of dehydration caused significant shrinkage artefacts and that these were different in different directions in both the thoracic and abdominal aorta. The greatest shrinkage was found with the critical point drying technique, 45% in the abdominal aorta and 31% in the thoracic aorta. In the abdominal aorta this shrinkage was mainly due to a shrinkage in length (36%) rather than a shrinkage in width (15%). In comparison, in the thoracic aorta critical point drying resulted in a 15% shrinkage in length and a 19% shrinkage in width. Air drying and alcohol dehydration caused considerable shrinkage (29% and 18% respectively in the thoracic aorta, 29% and 36% respectively in the abdominal aorta). Directional differences were also found with these techniques, for instance alcohol dehydration in the thoracic aorta resulted in 0% shrinkage in length and 18% shrinkage in width.     

New application of air-drying techniques for studying Ephemeroptera and Plecoptera eggs by scanning electron microscopy

Hexamethyldisilizane (HMDS) and tetramethylsilane are organic compounds that are volatile at ambient temperature and which can therefore be used for air-drying biological samples for SEM studies. The techniques using these compounds provide results that are comparable with those obtained by critical point drying, but which involve a very simple process that saves time and money. Both techniques were applied to SEM studies of Ephemeroptera and Plecoptera eggs in order to assess their suitability as alternative methods to critical point drying for these kinds of biological material. The results show no morphological differences between eggs HMDS air-dried and critical point-dried.     

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.     

A multisample chamber for dehydration and critical point drying

The principles and methods for constructing an improved chamber for dehydration and critical point drying of multiple biological samples are described. The specimen chamber design is based on vertical positioning of the electron microscope grids or coverslips and permits minimal perturbation of laminar solvent flow past the specimens. This condition is requisite for optimal exposure of samples to solvents, which is necessary for complete dehydration and drying. Fragile samples, including chromosomes, critical point dried in the multisample chamber demonstrate crisp, well-preserved, three-dimensional morphology.     

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.     

发酵用压缩空气余热再生吸附干燥工艺

有氧发酵生产过程所需的低压压缩空气一般采用冷冻干燥,干燥后压缩空气的含水量较高且过程耗能高。提出一种使用余热再生吸附式干燥工艺,直接对低压高温压缩空气进行干燥,不需消耗其他能源。采用该工艺不仅可使干燥空气露点低于-10℃,而且还具有良好的节能效益。