Foam cells in the atherosclerotic lesion have substantial cholesterol stores within large, swollen lysosomes. This feature is mimicked by incubating THP-1 macrophages with mildly oxidized low density lipoprotein (LDL). Incubation of THP-1 cells with acetylated LDL produces cytoplasmic cholesteryl ester accumulation rather than lysosomal storage. The differences could be due to differences in uptake and delivery of lipoprotein to lysosomes or to lysosomal and post-lysosomal processing events. We compared uptake and lysosomal trafficking of acetylated and oxidized LDL using colloidal gold-labeled lipoproteins. Labeling did not alter cellular cholesterol accumulation. We found that uptake and delivery to lysosomes are not different for acetylated and oxidized LDL. In fact, both oxidized and acetylated LDL can be delivered to the same lysosomes. Sequential incubation with oxidized LDL followed by acetylated LDL showed that the lipid-engorged lysosomes are long-lived structures, continuously accepting newly ingested lipoprotein. Comparison of acetylated and oxidized LDL in mouse peritoneal macrophages, a cell which does not accumulate substantial lysosomal lipid, also revealed no differences in uptake. This indicates that in THP-1 cells, the differences in metabolism of oxidized and acetylated LDL are due to cell-specific lysosomal or post-lysosomal events not present in B6C3F1 mouse macrophages.

Apicomplexan parasites employ complex and unconventional mechanisms for cell locomotion, host cell invasion, and cell division that are only poorly understood. While immunofluorescence and conventional transmission electron microscopy have been used to answer questions about the localization of some cytoskeletal proteins and cell organelles, many questions remain unanswered, partly because new methods are needed to study the complex interactions of cytoskeletal proteins and organelles that play a role in cell locomotion, host cell invasion, and cell division. The choice of fixation and preparation methods has proven critical for the analysis of cytoskeletal proteins because of the rapid turnover of actin filaments and the dense spatial organization of the cytoskeleton and its association with the complex membrane system. Here we introduce new methods to study structural aspects of cytoskeletal motility, host cell invasion, and cell division of Toxoplasma gondii, a most suitable laboratory model that is representative of apicomplexan parasites. The novel approach in our experiments is the use of high resolution low voltage field emission scanning electron microscopy (LVFESEM) combined with two new specimen preparation techniques. The first method uses LVFESEM after membrane extraction and stabilization of the cytoskeleton. This method allows viewing of actin filaments which had not been possible with any other method available so far. The second approach of imaging the parasite's ultrastructure and interactions with host cells uses semithick sections (200 nm) that are resin de-embedded (Ris and Malecki, 1993) and imaged with LVFESEM. This method allows analysis of structural detail in the parasite before and after host cell invasion and interactions with the membrane of the parasitophorous vacuole as well as parasite cell division.

Osteocytes communicate through a canalicular system that maintains the vitality and mineral metabolism of bone. Casting the vascular canals and canaliculi of compact bone with methacrylate and viewing them with scanning electron microscopy shows their extent and relationships. Confocal laser scanning microscopy of the same specimen before corrosion establishes the degree of calcification of the different tissue components. These methods were used to compare basal with alveolar compact bone in the rat mandible at different ages. Sections of the mandibular molar region were placed in a methacrylate resin. After polymerization and study with confocal microscopy, the organic matrix was removed. Juvenile rats had large irregular central vascular canals and lacunae that were more concentric in the basal than the alveolar bone. Cast lacunae were round, and the canaliculi from these lacunae were short and thick in both bones. Adult rats had regular concentrically arranged lacunae in the basal bone. Cast lacunae were ellipsoid and flatter in the basal bone than in the alveolar bone. The intercommunicating canaliculi were increased and canaliculi had more branching than the juvenile rats. The aged rats had fewer vascular canals, lacunae, and canaliculi and had osteoporotic changes. The cast lacunae were slender and flat especially in the basal bone. The porosity of the mandible became more pronounced in the alveolar than in the basal bone with aging. The canaliculi of mandibular compact bone thinned and developed extensive branching with adulthood but decreased in size and number with advanced age. Lacunae proceed from the large circular structures of youth to the flat forms of the aged. These studies show that the internal structure of compact bone changes with age and mirrors its functional state.

Modulation of lymphocyte flow in the lymphatic compartment of the lymph node may serve, in part, to promote lymphocyte sensitization during an antigenic challenge. This study was undertaken to show where this might occur by examining the structural relationships of the intranodal lymphatic pathways, blood vessels, and connective tissue support with respect to lymphocyte and lymph flow. Differently stained plastic resins were injected into the blood vessels and lymphatics of the submandibular lymph node and visualized with a confocal laser scanning microscope. The specimens were corroded to study the three-dimensional cast structures by scanning electron microscopy. Alkali digestion was also used to prepare the reticular fiber network in the lymph node for scanning electron microscopic examination. At the hilus of the node, two to three arteries gave off arterioles running in medullary cords towards the cortex. The medullary cords, the periphery of the deep cortex, and the perifollicular zones had dense capillary networks. In contrast, the center of the follicle and the center of the deep cortex were less highly vascularized. High-endothelial venules were restricted to the perifollicular zone and the periphery of the deep cortex. At the cortico-medullary boundary, they abruptly transformed into medullary venules with a normal endothelium. The marginal sinus of the lymph node was crossed by thick reticular fibers that arose from the inner sheets of the capsule. The lymph pathway went through the marginal sinus, into the trabecular sinus, to the cortical perifollicular sinus, the dense lymphatic sinus around the deep cortex, and finally into the medullary sinus. At present, the exact functional significance of the complex lymph node lymphatic architecture is not clear. However, the highly organized structural organization may play a significant role in regulating and directing lymphocyte flow to facilitate antigen presentation.

Electron diffraction has been recently used in the pharmaceutical industry to study the polymorphism in crystalline drug substances. While conventional X-ray diffraction patterns could not be used to determine the cell parameters of two forms of the microcrystalline GP IIb/IIIa receptor antagonist roxifiban, a combination of electron single-crystal and synchrotron powder diffraction techniques were able to clearly distinguish the two polymorphs. The unit-cell parameters of the two polymorphs were ultimately determined using new software routines designed to take advantage of each technique's unique capabilities. The combined use of transmission electron microscopy (TEM) and synchrotron patterns appears to be a good general approach for characterizing complex (low-symmetry, large-unit-cell, micron-sized) polymorphic pharmaceutical compounds.

Multielemental X-ray maps collected by a remote scanning system of the electron beam are processed by a dedicated software program performing accurate modal determination of geological samples. The classification of different mineral phases is based on elemental concentrations. The software program Petromod loads the maps into a database and computes a matrix consisting of numerical values proportional to the elemental concentrations. After an initial calibration, the program can perform the chemical composition calculated on the basis of a fixed number of oxygens for a selected area. In this way, it is possible to identify all the mineral phases occurring in the sample. Up to three elements can be selected to calculate the modal percentage of the identified mineral. An automated routine scans the whole set of maps and assigns each pixel that satisfies the imposed requirements to the selected phase. Repeating this procedure for every mineral phase occurring in the mapped area, a modal distribution of the rock-forming minerals can be performed. The final output consists of a digitized image, which can be further analyzed by common image analysis software, and a table containing the calculated modal percentages. The method is here applied to a volcanic and a metamorphic rock sample.