Three-dimensional Replica Research Articles

Immunoelectron Microscopy of <italic>Proteus vulgaris</italic> by the Plasma Polymerization Metal-Extraction Replica Method: Differential Staining of Flagellar (H) and Somatic (O) Antigens by Colloidal Golds

Flagellar (H) and somatic (O) antigens of Proteus vulgaris were differentially stained with antibodies coupled to different sizes of colloidal gold particles, and the distribution of these antigens was visualized by the plasma polymerization metal-extraction replica (PMR) method. The H antigen, labeled with 5 nm colloidal gold, was almost exclusively located on the flagella, whereas the O antigen, labeled with 10 nm colloidal gold, was almost exclusively located on the bacterial body. The marker gold particles were clearly observed as electron-dense particles on the relatively low contrast background of three-dimensional replica image of the flagellated bacteria. Thus, the PMR method may prove to be a useful tool for studying the localization of multiple substances on the cell surface, at a high resolution and in three dimensions. The diameter of the flagella measured by the replica method was about 15 nm, close to the value obtained by negative staining (16 nm). When treated with anti-flagellar (H) factor serum and protein A-gold, the diameter of flagella was significantly increased to about 35 nm. This increase in diameter was presumably caused by binding of immunoglobulins to H antigens of flagella.

Selective contrast in images of crystalline cytochrome oxidase dimers

Vesicle crystals of cytochrome oxidase dimers embedded in the phospholipid bilayer of a collapsed vesicle are produced when beef heart mitochondria are treated with Triton detergents. The crystals have the symmetry of plane group p22121 with unit cell dimensions of a=95 A, b=125 A, and c=210 A. The molecules protrude 60 A beyond the lipid bilayer on the inside of the vesicle, but very little beyond the bilayer on the outside. Decoration of crys- stals with subunit-specific antibodies identified the outside surface of the vesicle crystals as corresponding to the M-side (matrix) of the inner mitochondrial membrane and the inside surface as the C-side (cytoplasmic).We have used various methods of specimen preparation to selectively image different regions of the dimer crystals in order to correlate the structure of dimers with that of 'Y'-shaped monomers. Electron micrographs of negatively stained specimens (Fig. 1a, 2a) give a two-dimensional projection of the three-dimensional stain replica of the entire structure.

Porcine Tracheal Goblet Cell Ultrastructure: A Three-Dimensional Reconstruction

In order to study specific anatomical relationships among organelles involved in the movement and secretion of mucin granules by airway epithelium, a three-dimensional replica of a single goblet cell from porcine trachea was constructed by means of transmission electron microscopy of serially prepared ultrathin sections. Many features of the goblet cell that could not be deduced from single planes of section were revealed. The cell is columnar in configuration. The cytoplasm contains not one, but several discrete clusters of mucin granules, each associated with a separate Golgi apparatus. Microtubules and microfilaments often are observed in close association with both mucin granules and coiled filamentous mitochondria, suggesting a role in the intracellular movement of these organelles. These morphological observations are in agreement with previous functional studies of airway mucin secretion, and elucidate further the cellular biology of airway mucosal goblet cells and the secretory process.