Guy Cox

Fluorescent pigments in corals are photoprotective.

All reef-forming corals depend on the photosynthesis performed by their algal symbiont, and such corals are therefore restricted to the photic zone. The intensity of light in this zone declines over several orders of magnitude—from high and damaging levels at the surface to extreme shade conditions at the lower limit. The ability of corals to tolerate this range implies effective mechanisms for light acclimation and adaptation. Here we show that the fluorescent pigments (FPs) of corals provide a photobiological system for regulating the light environment of coral host tissue. Previous studies have suggested that under low light, FPs may enhance light availability. We now report that in excessive sunlight FPs are photoprotective; they achieve this by dissipating excess energy at wavelengths of low photosynthetic activity, as well as by reflecting of visible and infrared light by FP-containing chromatophores. We also show that FPs enhance the resistance to mass bleaching of corals during periods of heat stress, which has implications for the effect of environmental stress on the diversity of reef-building corals, such as enhanced survival of a broad range of corals allowing maintenance of habitat diversity.

3-Dimensional imaging of collagen using second harmonic generation

Collagen is the most important structural protein of the animal body. Its unique triple-helix structure and extremely high level of crystallinity make it exceptionally efficient in generating the second harmonic of incident light, and we show here how this leads to a novel mode of microscopy of immediate practical significance in medicine and biology. In particular, it provides sensitive and high-resolution information on collagen distribution, discriminates between type I and type III collagen, and allows both a greater understanding of and a sensitive test for cirrhosis of the liver. Future research applications could include wound healing and hereditary collagen diseases such as osteogenesis imperfecta.

Cyanobacterially deposited speleothems: Subaerial stromatolites

Abstract Stalagmites, which appear to be deposited by cyanobacterial action, have been identified in two caves in New South Wales (Australia). These have a characteristic morphology, which has given rise to local names likening them to crustaceans. We have studied these speleothems, and the cyanobacteria covering them, by microscopic and chemical techniques. Both calcite precipitation and aeolian sediment trapping are involved in their deposition, which is strongly controlled by environmental conditions. They can be regarded as stromatolites within currently accepted definitions of the term.

Visualization of second-order nonlinear layer in thermally poled fused silica glass

Second-harmonic microscopy has been applied to characterize the second-order nonlinear layer in fused silica plates thermally poled at 280 °C and 3.5 kV for different time intervals. The nonlinear layer is found only under the anode surface and to be ∼5μm deep under the anode for a poling time of 30 min. Progression of this layer into the bulk glass with poling time is also characterized.

Second-Harmonic Imaging of Collagen

Molecules that have no center of symmetry are able to convert light to its second harmonic, at twice the frequency and half the wavelength. This only happens with any efficiency at very high light intensities such as are given by a pulsed laser, and because the efficiency of the process depends on the square of the intensity, it will be focal plane selective in exactly the same way as two-photon excitation of fluorescence. Because of its unusual molecular structure and its high degree of crystallinity, collagen is, by far, the strongest source of second harmonics in animal tissue. Because collagen is also the most important structural protein in the mammalian body, this provides a very useful imaging tool for studying its distribution. No energy is lost in second-harmonic imaging, so the image will not fade, and because it is at a shorter wavelength than can be excited by two-photon fluorescence, it can be separated easily from multiple fluorescent probes. It is already proving useful in imaging collagen with high sensitivity in various tissues, including cirrhotic liver, normal and carious teeth, and surgical repair of tendons.

Electron microscopy of cellulose in entire tissue

Microfibrillar structures can be seen in ultrathin sections of cell walls which have been post‐stained with uranyl acetate and lead citrate. This stain is removable by washing, suggesting that a physical mechanism is involved. It is suggested that these structures are the cellulose microfibrils of the wall; they appear to be 3.5 nm in diameter and not fasciated into larger units. Freeze‐etching of untreated tissue supports this conclusion. These two techniques seem to have many advantages over methods previously used for the study of microfibril arrangement and structure.

High-voltage electron microscopy of whole, critical-point dried plant cells

SummaryThe lower epidermis ofSelaginella Helvetica leaves has numerous chloroplasts. In the diffuse light of the plants normal habitat these are distributed over the inner wall of the cell, while in bright sunlight they move to the lateral walls. High voltage electron microscopy of whole critical-point dried cells shows that in the diffuse-light position the chloroplasts are connected by bundles of tightly-packed parallel filaments; these are distinct from, but seem to interconnect with, the filaments of the cytomatrix. In thin sections these appear as conventional microfilament bundles, while staining with rhodamineconjugated phalloidin implies that they are composed of actin. In bright light, when the chloroplasts have moved to the lateral walls, these microfilament bundles completely disappear, while filaments of the cytomatrix system remain attached to the chloroplasts. These results suggest that the function of the microfilament bundles may be to anchor the chloroplasts as much as to move them, and that the cytomatrix system may play a part in the movement; it is possible that actin microfilament bundles may actually dissociate into separate filaments within the cytomatrix. Staining of cryo-sections with FITC-labelled antitubulin reveals a typical “cortical” pattern of microtubules which appears to play no part in chloroplast motility.

Intrahepatic expression of collagen and fibroblast activation protein (FAP) in hepatitis C virus infection.

Mark D. Gorrell, Xin M. Wang, Miriam T. Levy, Eleanor Kable, George Marinos , Guy Cox and Geoffrey W. McCaughan 1. A. W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Centenary Institute of Cancer Medicine and Cell Biology and the University of Sydney, NSW Australia. 2. Electron Microscope Unit, University of Sydney. 3. Gastroenterology Department, Prince of Wales Hospital, Sydney

Ultrastructure of a cave-wall cyanophyte-Gloeocapsa NS4

Gloeocapsa strain NS4, a cyanophyte (cyanobacterium) which grows in low light levels inside cave entrances, was studied in the electron microscope by thin sectioning and freeze-etching. The cells are surrounded by a microfibrillar sheath divided by ‘dense lamellae’, which are probably an acidic mucopolysaccharide. Inside this is a typical Gramnegative cell wall. Double-replica freeze-fracture showed that the outer envelope of the wall fractures to give two faces each consisting of densely-packed particles; the particles of the outer leaflet seem to consist of subunits arranged in a hollow cylinder. A structural model of the outer envelope is proposed. The plasma membrane fractures to give a PF face with 3000 ∼ 9 nm particles μm-1 and an EF face with 150–700 11–12 nm particles μm-1. The thylakoids are arranged in a pattern not previously found in a unicellular cyanophyte, parallel arrays which intersect, and may fuse with, the plasma membrane. The thylakoid membranes have 2,850 particles μm-1, mean size 10.9 nm, on the PF face and 560 particles μm-1, mean size 12.3 nm, on the EF face. Phycobilisomes are difficult to see, but may be unusually large. These ultrastructural features may be adaptations to a very low light habitat.

Computer-generated, three-dimensional reconstruction of histological parallel serial sections displaying microvascular and glandular structures in human endometrium

This paper describes a technique to develop high-resolution three-dimensional (3D) images of microvasculature structures in curettage, hysterectomy or endometrial resection biopsies using parallel histological serial sections. Employing a labelled streptavidin-biotin-alkaline phosphatase (LSAB(+)) method and visualising by using DAB(+) with the primary antibody, mouse anti human Q-Bend-10, the images were directly digitised from a light microscope into the KS400 Universal Image Processing and Analysis software via a CCD colour camera; binary images of the structures were created and the binary images were exported into VoxBlast 3D rendering software to view still and rotating 3D images on a computer monitor. This in turn enabled hard copies of the full sequence to be printed.