Guy C. Cox

Electron tomography and computer visualisation of a three-dimensional ‘photonic’ crystal in a butterfly wing-scale

A combination of transmission electron tomography and computer modelling has been used to determine the three-dimensional structure of the photonic crystals found in the wing-scales of the Kaiser-I-Hind butterfly (Teinopalpus imperialis). These scales presented challenges for electron microscopy because the periodicity of the structure was comparable to the thickness of a section and because of the complex connectivity of the object. The structure obtained has been confirmed by taking slices of the three-dimensional computer model constructed from the tomography and comparing these with transmission electron microscope (TEM) images of microtomed sections of the actual scale. The crystal was found to have chiral tetrahedral repeating units packed in a triclinic lattice.

Second-harmonic imaging of plant polysaccharides

The application of second-harmonic generation (SHG) microscopy to plant materials has been neglected hitherto even though it would seem to have promise for identification and characterization of biologically and commercially important plant polysaccharides. We find that imaging of cellulose requires rather high laser powers, which are above optimal values for live cell imaging. Starch, however, is easily imaged by the technique at laser fluences compatible with extended cell viability. This also has useful applications in imaging plant-derived starchy food products. Lignin in plant cell walls shows a strong three-photon excited fluorescence, which may be enhanced by resonance effects.

Super-resolution imaging of the bacterial cytokinetic protein FtsZ.

The idea of a bacterial cytoskeleton arose just 10 years ago with the identification of the cell division protein, FtsZ, as a tubulin homolog. FtsZ plays a pivotal role in bacterial division, and is present in virtually all prokaryotes and in some eukaryotic organelles. The earliest stage of bacterial cell division is the assembly of FtsZ into a Z ring at the division site, which subsequently constricts during cytokinesis. FtsZ also assembles into dynamic helical structures along the bacterial cell, which are thought to act as precursors to the Z ring via a cell cycle-mediated FtsZ polymer remodelling. The fine structures of the FtsZ helix and ring are unknown but crucial for identifying the molecular details of Z ring assembly and its regulation. We now reveal using STED microscopy that the FtsZ helical structure in cells of the gram positive bacterium, Bacillus subtilis, is a highly irregular and discontinuous helix of FtsZ; very different to the smooth cable-like appearance observed by conventional fluorescence optics. STED also identifies a novel FtsZ helical structure of smaller pitch that is invisible to standard optical methods, identifying a possible third intermediate in the pathway to Z ring assembly, which commits bacterial cells to divide.

Visualisation of mitochondria in living neurons with single- and two-photon fluorescence laser microscopy

In this work we investigated the relative merits of conventional single-photon confocal laser scanning fluorescence microscopy (CLSM) and two-photon laser scanning fluorescence microscopy (2p-LSM) for the study of mitochondria in living neurons. Dorsal root ganglion neurons were loaded with the mitochondrion-specific fluorescent dye JC-1, the ratio between red (J-aggregates) and green (monomer) fluorescence of which reflects mitochondrial membrane potential. Cells were illuminated at 488 nm for single-photon excitation or at 870 nm for two-photon excitation. In both modalities we found that mitochondria showed: (i) similar appearance; (ii) similar fluorescence ratio values over both whole cell bodies and individual mitochondria; and (iii) similar responses to mitochondrial uncoupler, which dropped the ratio values by 50%. However, 2p-LSM exhibited several advantages over CLSM: (i) better signal/noise ratio in the green emission channel; (ii) less phototoxicity upon repetitive scanning in the focal plane; and (iii) no significant loss of image quality upon repetitive scans in the z direction. We conclude that, while both techniques enable visualisation of individual mitochondria in living cells, 2p-LSM has significant advantages for physiological work requiring time-lapse experiments or four-dimensional reconstructions of mitochondria.

Filamentous cyanophytes containing phycourobilin and in symbiosis with sponges and an ascidian of coral reefs

A study was made of the ultrastructure and pigment composition of filamentous cyanophytes living in symbiosis with several sponges and a colonial didemnid ascidian collected from the southern end of the Great Barrier Reef, Australia, between 1983 and 1986. The sponges were Dysidea herbacea Keller and several other encrusting sponges which have not been identified; the ascidian was Trididemnum miniatum Kott (1977). The cyanophyte Oscillatoria spongeliae (Shultz) Hauck was identified as the symbiont of several of the sponges, including D. herbacea. Two other unidentified Oscillatoria species were found in a bristly papillate sponge and in T. miniatum. Chlorophyll a, alone, was present in all the symbionts with the exception of T. miniatum, which contained the cosymbiont Prochloron and where chlorophyll b was also present. Two phycoerythrins were isolated by chromatography and chromatofocusing. Both resembled C-phycoerythrin, but one of the two carried the chromophore phycourobilin as well as phycoerythrobilin possibly on both the α and β subunits, which had apparent molecular masses of 18 and 20 kdaltons. No γ subunit was present. Ultrastructurally, the three Oscillatoria species were distinguished by an unusual type of parallel, longitudinal, thylakoid organisation; the arrangement was different in detail in each species.

An unusual cyanophyte, containing phycourobilin and symbiotic with ascidians and sponges

A study was made of the pigment composition and ultrastructure of a unicellular cyanophyte living in symbiosis with colonial didemnid ascidians and encrusting sponges collected from the southern end of the Great Barrier Reef, Australia, in 1981–1984. The ascidians were Trididemnum tegulum Kott and T. clinides. Kott; the sponges were Prianos aff. melanos de Laubenfels, Spirastrella aff. decumbens Ridley and an unidentified brown fleshy sponge (BFS). This cyanophyte seems to be identical with Synechocystis trididemni Lafargue et Duclaux. A phycoerythrin containing both phycourobilin and phycoerythrobilin chromophores was shown to be present; the urobilin was carried on α and β subunits, no γ subunit was found. A second phycoerythrin possessing only erythrobilin chromophores was also present. In thin-sections the cells showed no central DNA-containing nucleoid, and an unusual thylakoid arrangement with some thylakoids having greatly expanded lumens forming pseudo-vacuoles in the centre of the cell. Freeze-fracture showed 11 to 12 nm particles on both PF (protoplasmic face) and EF (exoplasmic face) faces of thylakoids. In many ways, the ultrastructure resembled that of the chlorophyll-b containing prokaryote Prochloron spp.

Comparative Cell Shape and Diffusional Water Permeability of Red Blood Cells from Indian Elephant (Elephas maximus) and Man (Homo sapiens)

Abstract: Red blood cells (RBC) from an Indian elephant (Elephas maximus) were studied by light microscopy (LM), scanning electron microscopy (SEM) and a new nuclear magnetic resonance (NMR) ‘imaging’ method based on the translational diffusion of water, NMR q-space analysis. Also, the transmembrane diffusional permeability, Pd of water in RBC was measured by using a Mn2+-doping NMR technique, taking human RBC as a reference. The main diameter of the elephant RBC was measured as 9.3 ± 0.7 μm by LM, 9.3 ± 0.7 μm by ‘shrinkage-corrected’ SEM, and 9.3 ± 0.4 μm by q-space anlaysis. The value is ∼1.4 μm larger than that for the human RBC. The values of Pd were, in the case of elephant RBC, 3.2 × 10−3 cm/s at 25 °C, 3.9 × 10−3 cm/s at 30 °C, 5.2 × 10−3 cm/s at 37 °C and 6.5 × 10−3 cm/s at 42 °C; all values were significantly lower than the corresponding values of Pd for human RBC, namely 4.3 × 10−3 cm/s at 25 °C, 5.2 × 10−3 cm/s at 30 °C, 6.1 × 10−3 cm/s at 37 °C, 7.8 × 10−3 cm/s at 42°C. The maximal inhibition of Pd (56%) was reached in 30 min at 37 °C with 2 mmp-chloromercuribenzene sulphonate (PCMBS) for both species of RBC. The basal permeability to water at 37 °C was estimated to be 2.3 × 10−3 cm/s for elephant and 2.6 × 10−3 cm/s for human RBC. The values of the activation energy for water permeability (Ea,d) was significantly higher for elephant RBC (31.9 kJ/mol) than for human RBC (25.9 kJ/mol). This indicated that features other than the number of transporters per cell are likely to be important in defining the differences in water permeability in the RBC from the two species.

Characterization of the Second Harmonic Signal from Collagen

Collagen is known to be a very effective generator of the second harmonic of incident light from 700 to 1100nm, and second harmonic generation (SHG) microscopy is coming into use as a tool for studying the distribution of collagen in tissue. It also shows promise as a technique for characterizing collagen - both in distinguishing different collagen types and their packing and in identifying degradation of collagen in pathologic conditions. However many aspects of image formation in SHG microscopy of collagen remain imperfectly understood, and we have commenced a rigorous study of these factors. The present paper presents the first results from this program.

Second harmonic imaging of collagen in mammalian tissue

It has recently been demonstrated that collagen is a very effective upconverter of light by second harmonic generation (SHG) but hitherto the potential this offers for biomedical imaging has not been realized. We show that bright SHG images van be obtained over a wide excitation range at illumination levels comparable to or lower than those required for two-photon excitation of fluorescent labels, with no damage to the collagen structure. Both paraffin and cryostat sections have been used, and medically significant results have been obtained in several fields. We show that the signal is easily distinguished from single and two-photon excited fluorescence by its forward propagation and narrow spectral width; in principle it could also be distinguished by lifetime. Key microscope requisites are: immersion objectives and condensers, high-efficiency PMT detectors for transmitted light, suitable filters, and effective blocking of stray light, especially from the mercury lamp.

Biological properties of coral GFP-type proteins provide clues for engineering novel optical probes and biosensors

In recent years, a variety of Green Fluorescent Protein (GFP)-like pigments have been discovered from corals and other marine organisms. They are widely used to expand the range of available GFP-type proteins in imaging applications, such as in vivo markers for gene expression and protein localization studies, FRET-based (Förster resonance energy transfer) multicolor imaging and biosensors. They have known diverse optical and biochemical properties but their in vivo spectral properties and biological function in marine organisms is only beginning to be understood. We have investigated their spectral diversity, optical properties and cellular microstructure in corals of the Great Barrier Reef with the aim of elucidating their photo-biological function/s as well as to identify novel proteins suitable for GFP-based technologies. We found numerous spectral variants, with emissions covering almost the full range of the visible spectrum. Many of these GFP-like proteins, especially in corals from the more extreme habitats, such as sun-exposed shallows or in deep water, showed a range of light-related spectral characteristics: high photostability, spectral tuning for energy transfer and dynamic photo-induced transformation properties. Intra-cellularly they were organized into spectral donor-acceptor pairs or even arrays, tuned for FRET. Coral color proteins thus offer an exciting potential to expand the use of the available GFPs in bio-imaging applications and as a basis for improved protein engineering.