Image comparisons of snow and ice crystals photographed by light (video) microscopy and low-temperature scanning electron microscopy

Light (video) microscopy and low-temperature scanning electron microscopy (SEM) were used to examine and record images of identical precipitated and metamorphosed snow crystals as well as glacial ice grains. Collection procedures enabled numerous samples from distant locations to be shipped to a laboratory for storage and/or observation. The frozen samples could be imaged with a video microscope in the laboratory at ambient temperatures or with the low-temperature SEM. Stereo images obtained by video microscopy or low-temperature SEM greatly increased the ease of structural interpretations. The preparation procedures that were used for low-temperature SEM did not result in sublimation or melting. However, this technique did provide far greater resolution and depth of focus over that of the video microscope. The advantage of resolution was especially evident when examining the small particles associated with rime and graupel (snow crystals encumbered with frozen water droplets), whereas the greater depth of focus provided clearer photographs of large crystals such as depth hoar, and ice. Because the SEM images contained only surface information while the video images were frequently confounded by surface and internal information, the SEM images also clarified the structural features of depth hoar crystals and ice grains. Low-temperature SEM appears to have considerable promise for future investigations of snow and ice.     

Analysis of submicron particles by scanning electron microscopy-energy-dispersive X-ray spectrometry-accuracy of size measurement

Scanning electron microscopy combined with energy-dispersive x-ray spectrometry (SEM-EDXS) is widely used for particle analysis. In the case of submicron particles, especially for particles that are smaller than 300 nm, the measured particle size is influenced by specimen preparation, SEM operating parameters, the mean atomic number of the particles, and the threshold value used for binarization. The use of uncoated particles on a conductive substrate and image acquisition using an in-lens detector are recommended for precise morphologic results in this size range.     

Low-temperature scanning electron microscopy of particle-exposed mouse lung

Inflated frozen mouse lungs were examined using low-temperature scanning electron microscopy (LTSEM) following bulk fracture under vacuum. Various aspects of pulmonary architecture were identified and correlated with structures revealed by SEM following conventional fixation and preparation techniques. Surface etching of selected samples was performed by radiant heating, revealing characteristic cytoplasmic, nuclear and extracellular lattice patterns resulting from ice crystal formation during freezing. These patterns aided in distinguishing between intra- and extracellular spaces. Pulmonary fluids such as mucus and surfactant were identified. Iron oxide particles were introduced into the lungs of some animals by intratracheal instillation and were subsequently identified in frozen-hydrated lung tissue using characteristic X-ray identification and mapping techniques. Particles were observed both intra-and extracellularly and were commonly found in large deposits. These observations confirm the utility of LTSEM techniques for examination of particles within pulmonary tissue. Particle exposure by intratracheal instillation was found to result in a non-uniform distributional pattern.     

The application of electron spectroscopic imaging for quantification of the area fractions of calcium-containing precipitates in nervous tissue

Energy-filtering transmission electron microscopy has been applied to the quantification of area fractions of calcium-containing cytochemical reaction products in central nervous tissue and the retina of fish. The method of electron spectroscopic imaging using electrons with an energy loss of 250 eV produces images with a very high, structure-sensitive contrast. This is a suitable imaging condition for the reliable detection of reaction products and structural details in unstained ultrathin sections. The images were recorded with a sensitive TV camera and evaluated with the integrated digital image-analysis system of the Zeiss CEM 902 energy-filtering electron microscope. An empirical procedure was developed which objectively detects reaction products and calculates characteristic values, taking into account different staining intensities. This new and sensitive method enabled an assessment to be made of the influence of temperature and light adaptation on cytochemically detectable calcium in nervous tissue of fish. Higher amounts of calcium-containing reaction product were detected in synaptic clefts of the optic tectum in warm-adapted fish than in cold-adapted fish. In synaptic vesicles of photoreceptor cells in the fish retina, higher amounts of reaction product were found in dark-adapted fish than in light-adapted fish.     

High-resolution scanning electron microscopy of frozen-hydrated and freeze-substituted kidney tissue

Inner surfaces and fracture faces of rabbit kidney tissue were investigated with high-resolution scanning electron microscopy using two different cryopreparation techniques: (i) for the observation of fracture faces, cryofixed tissue was fractured and coated in a cryopreparation chamber dedicated to SEM, vacuum transferred onto a cold stage and observed in the frozen-hydrated state; (ii) for the observation of inner surfaces of the nephron, water was removed after freezing and fracturing by freeze substitution and critical-point drying of the tissue. By both methods, macromolecular structures such as intramembranous particles on fracture faces and particles on inner surfaces were imaged. The latter method was used to investigate in more detail surface structures of cells in the cortical collecting duct. These studies revealed a heterogeneity of intercalated cells not described thus far.     

Imaging thin and thick sections of biological tissue with the secondary electron detector in a field-emission scanning electron microscope

A field-emission scanning electron microscope (FESEM) equipped with the standard secondary electron (SE) detector was used to image thin (70–90 nm) and thick (1–3 μm) sections of biological materials that were chemically fixed, dehydrated, and embedded in resin. The preparation procedures, as well as subsequent staining of the sections, were identical to those commonly used to prepare thin sections of biological material for observation with the transmission electron microscope (TEM). The results suggested that the heavy metals, namely, osmium, uranium, and lead, that were used for postfixation and staining of the tissue provided an adequate SE signal that enabled imaging of the cells and organelles present in the sections. The FESEM was also used to image sections of tissues that were selectively stained using cytochemical and immunocytochemical techniques. Furthermore, thick sections could also be imaged in the SE mode. Stereo pairs of thick sections were easily recorded and provided images that approached those normally associated with high-voltage TEM.     

Quantitative and reliable in vitro method combining scanning electron microscopy and image analysis for the screening of osteotropic modulators

The increased generation and up-regulated activity of bone resorbing cells (osteoclasts) play a part in the impairment of bone remodeling in many bone diseases. Numerous drugs (bisphosphonates, calcitonin, selective estrogen receptor modulators) have been proposed to inhibit this increased osteoclastic activity. In this report, we describe a pit resorption assay quantified by scanning electron microscopy coupled with image analysis. Total rabbit bone cells with large numbers of osteoclasts were cultured on dentin slices. The whole surface of the dentin slice was scanned and both the number of resorption pits and the total resorbed surface area were measured. Resorption pits appeared at 48 h and increased gradually up to 96 h. Despite the observation of a strong correlation between the total resorption area and the number of pits, we suggest that area measurement is the most relevant marker for osteoclastic activity. Osteotropic factors stimulating or inhibiting osteoclastic activity were used to test the variations in resorption activity as measured with our method. This reproducible and sensitive quantitative method is a valuable tool for screening for osteoclastic inhibitors and, more generally, for investigating bone modulators.     

The effect of freezing method and frozen storage conditions on the microstructure of wild blueberries as observed by cold-stage scanning electron microscopy

Microstructural information about fresh fruit is difficult to obtain using standard electron microscopy methods. Frozen fruit presents additional temperature-related challenges. However, the food industry would benefit from data that show structure/function relationships in fruit and changes that occur during freezing and frozen storage. The feasibility of using cold stage scanning electron microscopy (cryo-SEM) to describe and define the structure/function relationships of quality attributes of frozen wild blueberries was tested. Cryo-SEM was used to evaluate the structure of wild blueberries that had been frozen using three different methods, stored at three different temperatures, and held in frozen storage for 5 months. Micro-structural data were then compared with quality measures, which included changes in percentage drip, damaged berries, and anthocyanin leakage, to explore relationships between structural changes and observed quality changes. Cryo-SEM results indicated that microstructural changes observed in frozen blueberry samples are characteristic of their temperature history. Quality measurements and microstructural data are in agreement that improvement of frozen blueberry quality resulted from faster freezing rates and lower temperatures of storage than are used presently in standard production methods. This study indicates that cryo-SEM can be a valuable tool in assessing frozen blueberry quality and developing optimal conditions for freezing wild blueberries.     

Examination of mucosal surfaces by scanning electron microscopy before and after removal of debris

A rapid method is described whereby gastrointestinal biopsy specimen surfaces can be examined by scanning electron microscopy with overlying tract contents (debris) intact, and also re-examined after cleaning to determine the structure of the underlying mucosal surface. The conductive coating of gold is removed using mercury, a non-wetting agent. The specimen surface is suitably cleaned of debris after a brief ultrasonication in absolute ethanol which mixes with the transitional fluid (CO₂) used for critical point drying.     

A method of direct particle deposition on a carbon planchet to study mineral dust in human lung by scanning electron microscopy

Following Na-hypochlorite digestion of lung tissue, mineral particles extracted in the chloroform layer were deposited directly on a pre-smoothed carbon planchet for combined scanning electron microscopy and X-ray energy dispersive spectrometry (SEM and XEDS). Total mineral particle counts were obtained, and detailed physical characteristics of the fibrous particles were documented at 600, 1,500, 4,500 and 9,000 x in three lungs without, and one lung with, histories of occupational exposure. This preparation method was simple, collected more than 99% of identifiable mineral particles in the chloroform layer, gave excellent object to background contrast without heavy metal coatings, and was suitable for XEDS. Comparable fibrous particles from the chloroform layer could also be studied by selected-area electron diffraction to complement the results of XEDS. By this method, we found particles or fibers larger than 0.1 μm were readily counted and measured at 4,500 x. At 600 x, ferruginous bodies were found to be more than twice in number than when sought for by light microscopy. It was determined that 4,500 x is the most efficient magnification to examine and diagnose this type of specimen. The present study illustrates the importance of determining the most efficient magnification to be utilized in particle counts.     

Immunogold localization of plant surface arabinogalactan-proteins using glycerol liquid substitution and scanning electron microscopy

We have studied the spatial distributions of arabinogalactan-protein (AGP) epitopes on the surface of maize embryogenic calli and roots using monoclonal antibodies JIM4 and MAC207. For this purpose, a new immunogold-scanning electron microscopy (SEM) method was employed which is based on liquid substitution of samples with glycerol. Using this method, we were able to show that the AGP epitopes are distributed along callus and root surfaces and they decorate filamentous structures. In callus cells, the JIM4 epitope was specifically enriched in the outer extracellular layers covering compact clusters of embryogenic meristematic callus cells. In roots, the MAC207 epitope was abundant on the root epidermal surface corresponding to the outer root pellicle, but was only occasionally found on the mucilage layer covering the root cap cells. Silver-enhanced gold particles, indicating AGP epitopes, were often linearly arranged suggesting that AGPs associate with filamentous structures both on the surface of embryogenic calli and root epidermal cells. These results indicate that AGPs are components of the outer extracellular layers and networks that cover the surface of roots and cells undergoing somatic embryogenesis.     

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.     

Application of scanning electron microscopy (SEM) and microbead techniques to study the localization of p24 and p18 antigens of HIV-1 on the surface of HIV-1-infected H9-lymphocytes

Immunofluorescence staining techniques at present, when applied to follow the expression of HIV-1-specific antigens on infected cells, only give the information that the antigens detected are localized in the outer region of the membrane of the infected cell. We therefore set up a procedure using magnetic polystyrol particles coated with antibodies specific for the HIV-1 antigens under study, in combination with scanning electron microscopy. We were able to demonstrate that p24 and p18 structural antigens are clearly expressed on the surface of HIV-1-infected H9 lymphocytes. This means that there was no steric hindrance for structures of cell-like size specific for HIV-1 antigens to interact with their target antigens. Other antigens may be hidden in membrane structures and are therefore inaccessible, for example, to the beads used here, which were of a similar size to antigen-specific cells in vivo. The results of this model system must be seen with respect to the interaction of antigen-specific cell-mediated immunity with full antibody-dependent cellular cytotoxicity, or without cytotoxic T lymphocytes, the mediator function of antibodies.