Spatial arrangement of physeal cartilage chondrocytes and the structure of the primary spongiosa.

Using laser confocal microscopy and 5-chloromethyl-fluoresceindiacetate (CMFDA) loading of chondrocytes we have investigated the structure of the ovine physis during late fetal development and its relationship to the structure observed in the primary spongiosa. Chondrocytes within the ovine growth plate form nests that together span the growth plate. We propose that all growth plates may be composed of nests of cells, but that the length of the individual nests changes between growth plates and with gestational age. The continuous column of cells seen within some growth plates is a nest of cells that is in the process of being absorbed by the invading metaphyseal front. Scanning electron microscopy of the mineralized portion of the primary spongiosa revealed structures that were consistent with the hypothesis that the cartilage surrounding the nest structure gives rise to the structure in the primary spongiosa. Although mineralization does not occur between cells within a nest, bands of mineral form between nests in the lower hypertrophic region and around the end of the nest as it reaches the hypertrophic region. This pattern of mineralization around and between nest termini yields the complex three-dimensional network of mineralized trabeculae observed in the primary spongosia.

Comparison of microcolony formation between Vibrio sp. strain S141 and a flagellum-negative mutant developing on agar and glass substrata.

A flagellum-negative mutant, M8.2, of the marine bacterium Vibrio sp. S141 was produced by transposon mutagenesis. Time-lapse video imaging of surface colonisation behaviour and microcolony formation of S141 compared to M8.2 cells was carried out to investigate the role of the flagellum of Vibrio sp. S141 in microcolony formation on agar and glass substrata. On an agar surface, S141 cells formed a tetrad pattern after the first two cell divisions, during initial surface colonisation. Developed microcolonies consisted of tight circular arrangements of cells with infrequent branching of cells from the main body. In contrast, M8.2 cells did not form tetrad patterns and micro-colonies generally showed enhanced branching and did not develop circular arrangements of cells. On a glass surface under flow conditions, S141 cells displayed several types of movement behaviours at the surface which may have assisted microcolony formation. M8.2 cells appeared unable to develop micro-colonies, but rather displayed a behaviour which enabled them to spread out across the substratum. Laser scanning confocal microscopy revealed S141 mature biofilms consisted of characteristic towers of bacterial growth with scattered troughs. The flagellum-negative M8.2 biofilm did not form such architecture, displaying a homogeneous distribution of cells throughout the biofilm and across the entire substratum. Although not required for attachment to the glass substratum, the flagellum was required for alignment as well as specific movement behaviours by S141 cells.

Technical Advance: Confocal measurement of the three-dimensional size and shape of plant parenchyma cells in a developing fruit tissue.

Parenchyma cells from the inner mesocarp of a grape berry (Vitis vinifera L. cv. Chardonnay) were visualised in three-dimensions within a whole mount of cleared, stained tissue using confocal laser scanning microscopy and digital image reconstruction. The whole berry was fixed, bisected longitudinally, cleared in methyl salicylate, stained with safranin O and mounted in methyl salicylate. Optical slices were collected at 1.0 µm intervals to a depth of 150 µm. Neighbouring z-series were joined post-collection to double the field-of-view. Attenuation at depth of the fluorescent signal from cell walls was quantified and corrected. Axial distortion due to refractive index mismatch between the immersion and mounting media was calibrated using yellow-green fluorescent microspheres and corrected. Transmission electron microscopy was used to correct fluorescent measurements of cell wall thickness. Digital image reconstructions of wall-enclosed spaces enabled cells to be rendered as geometric solids of measurable surface area and volume. Cell volumes within the inner mesocarp tissue of a single grape berry exhibited a 14-fold range, with polysigmoidal distribution and groupings around specific size classes. Cell shape was irregular and the planes of contact were rarely flat or simple. Variability in cell shape was indicated by the range in surface area to volume ratios, from 0.080 to 0.198 µm-1. Structural detail at the internal surface of the cell wall was apparent. The technique is applicable to a wide range of morphometric analyses in plant cell biology, particularly developmental studies, and reveals details of cell size and shape that were previously unattainable.

Three-dimensional confocal images of microdamage in cancellous bone.

The accumulation of microdamage in bone may contribute to loss of bone quality in osteoporosis, and the role of microdamage in the etiology of fatigue fractures is unknown. Microdamage created during testing, ex vivo, can increase the fragility of bone by decreasing the load necessary to cause fracture. Microdamage can also accumulate in vivo, but its influence on bone fragility is unknown. To date, stained microcracks are the only criteria to have been correlated with bone mechanics, leaving the influence of ultrastructural damage on bone fragility open for scrutiny. Staining en bloc has identified three morphological features in the tissue, discrete microcracks, cross-hatch staining, and diffuse staining. The relationship between these features and their identification as microdamage remains equivocal. The purpose of this study was to investigate the three-dimensional nature of microdamage in cancellous bone and also to describe stained microcracks, cross-hatch staining, and diffuse staining and to determine whether they all relate to microdamage in bone. Laser scanning confocal microscopy that provides improved spatial resolution over bright-field microscopy was used to visualize bone damage. It was found that crack surface density was highly correlated with crack density (r = 0.95, p < 0.0001), suggesting that the crack surface of preexisting cracks increases as new cracks are formed or submicroscopic cracks become visible under bright-field microscopy. Cross-hatch staining and diffuse staining included ultra-microcracks about 10 microm in length. The ultra-microcracks in cross-hatch staining were organized in bands and surrounded by diffuse staining. This study demonstrates that damage in bone occurs over a wide range and that discrete microcracks, cross-hatch staining, and diffuse staining are all indicative of bone damage. The diffuse staining still evident in association with the ultra-microcracks seen in cross-hatch staining and diffuse staining is probably due to damage at a still smaller scale than we have been able to investigate.