Current approaches to 3D imaging at subcellular resolution using confocal microscopy and electron tomography, while powerful, are limited to relatively thin and transparent specimens. Here we report on the use of a new generation of dual beam electron microscopes capable of site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions about an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have explored the potential of this sequential "slice-and-view" strategy for site-specific 3D imaging of frozen yeast cells and tumor tissue, and establish that this approach can identify the locations of intracellular features such as the 100 nm-wide yeast nuclear pore complex. We also show that 200 nm thick sections can be generated in situ by "milling" of resin-embedded specimens using the ion beam, providing a valuable alternative to manual sectioning of cells and tissues using an ultramicrotome. Our results demonstrate that dual beam imaging is a powerful new tool for cellular and subcellular imaging in 3D for both basic biomedical and clinical applications.

Antineutrophil cytoplasmic antibodies (ANCA) are frequently associated with chronic inflammatory bowel diseases (IBD) and hepatobiliary disorders. However, their target antigens have not been identified yet. Recently, we observed an atypical perinuclear ANCA fluorescence (p-ANCA) together with an intranuclear staining using ANCA-positive sera from patients with IBD and hepatobiliary disorders. This observation suggests that the target antigens are localized within the nucleus of neutrophilic granulocytes. To further investigate this hypothesis, we examined sera from patients with ulcerative colitis, primary sclerosing cholangitis, autoimmune hepatitis or systemic vasculitis on ethanol or formaldehyde-fixed neutrophils using confocal laser scanning microscopy and immunoelectron microscopy. Counterstaining with propidium iodide, a DNA-specific dye, showed that ANCA-positive sera in IBD and heptobiliary disorders react with intranuclear antigens at the nuclear periphery of the neutrophils. Double immunolabeling techniques revealed that nuclear lamina proteins, lamins A, C and B1, and lamin B receptor were colocalized with the antigen(s) recognized by atypical p-ANCA. No colocalization was observed with classical p-ANCA and antibodies against histones (H1-H4). Our study showed that atypical p-ANCA are antinuclear antibodies reactive with granulocyte-specific antigens present in the nuclear lamina. (HEPATOLOGY 1998;28:332-340.)

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Multimerization of thyroglobulin (TG) takes place extracellularly in the thyroid follicle lumen and is regarded as a mechanism to store TG at high concentrations. Human thyroglobulin (hTG) has been shown to multimerize mainly by intermolecular disulfide cross-links. We recently noted that TG of various mammalian species contains three highly conserved thioredoxin boxes (CXXC). This sequence is known to underlie the enzymatic activity of protein disulfide isomerase (PDI). As hTG formed intermolecular disulfide bonds in the absence of other proteins depending on the redox conditions and hTG concentration, the CXXC-boxes of TG might provide the structural basis for self-assisted intermolecular cross-linking. To test this hypothesis we prepared a recombinant TG fragment containing the three thioredoxin boxes. This fragment exhibited a redox activity amounting to about 10% of the activity of PDI at redox conditions supposed to be present in the extracellular space. This activity might be supplemented by the oxidizing system of the apical cell surfaces of thyrocytes facing the follicle lumen. Indeed, incubation of hTG with peroxidase and H202 resulted in intermolecular disulfide bridge formation. Our results suggest a combined mechanism of self-assisted and peroxidase-mediated disulfide bond formation leading to the intermolecular cross-linking of lumenal hTG.

Experimental approaches in Drosophila melanogaster over the last 20 years have played a fundamental role in elucidating the function, structure and molecular composition of the centrosome. However, quantitative data on the structure and function of the Drosophila centrosome are still lacking. This study uses, for the first time, whole mount electron microscopy in combination with negative staining on isolated centrosomes from the early Drosophila embryos to analyze its dimensions, structure and capacity to nucleate microtubules in vitro. We show that these organelles are on average 0.75 lm in diameter and have abundant pericentriolar material which often appears fibrillar and with bulbous protrusions. Corresponding to the abundant pericentriolar material, extensive microtubule nucleation occurs. Quantification of the number of microtubules nucleated showed that 50-300 active nucleation sites are present. We examined via electron microscopy immunogold labeling the distribution of c-tubulin, CNN, Asp and the MPM-2 epitopes that are phosphorylated through Polo and the Cdk1 kinase. The distribution of these proteins is homogeneous, with the MPM-2 epitopes exhibiting the highest density. In contrast, centrosomal subdomains are identified using a centriole marker to relate centrosome size to the centriole number by electron microscopy. In conclusion, we present a clear-cut technique assaying and quantifying the microtubule nucleation capacity and antigen distribution complementing molecular studies on centrosome protein complexes, cell organelle assembly and protein composition.