High resolution imaging as a characterization tool for biological crystals.

Biomolecular crystals consist of large unit cells that form a rather flexible medium that is able to accommodate a certain degree of lattice distortion, leading to several interesting issues ranging from structural to physical properties. Several techniques, from X-ray diffraction to microscopy, have been adapted to study the structural and physical properties of biomolecular crystals systematically. The use of synchrotron-based monochromatic X-ray diffraction topography, with triple axis diffractometry and rocking curve measurements, to characterize biomolecular crystals is reviewed. Recent X-ray diffraction images from gel and solution grown lysozyme crystals are presented. Defect structures in these crystals are discussed, together with reciprocal space mapping, and compared with results obtained from crystals grown in a low gravity environment.

Applications of dynamical diffraction under locally plane wave conditions: defects in nearly perfect crystals and X-ray refractometry.

In a previous paper, the concept of a locally plane wave was explained theoretically. In such a configuration, the fringe pattern recorded on the film can be considered as a phase analyser. Here the experimental analysis is presented, showing examples of interesting applications to X-ray refractometry and to the visualization of the strain field around isolated defects.

X-ray dynamical diffraction: the concept of a locally plane wave

The long distance between the source and the experiment and the small source size, now available at third-generation synchrotron sources, leads to new optical characteristics for X-ray diffraction. It is shown that, under certain conditions, the intensity received by a point located on the exit surface of a crystal is described by the diffraction of a locally plane wave. Each point along the surface is influenced by a plane wave with a varying departure from Bragg angle. In such a case, it is possible to visualize the rocking curve of the crystal as a function of the position along the exit surface. This represents a topographic method to obtain the reflectivity curve of the crystal instead of the usual goniometric method. Applications will be described in a forthcoming paper.

Digital image ratio: a new radiographic method for quantifying changes in alveolar bone. Part II: Clinical application.

As reported in a previous paper (1) we have developed a new technique, Digital Image Ratio (DIR), which theoretically avoids some of the drawbacks of quantitative digital substraction radiography. DIR allows the direct computation and visualization of bone-mass-ratio changes. This second paper describes the use of DIR analysis to examine 20 sites in 8 patients undergoing regenerative periodontal therapy. Standardized reproducible radiographs of these 20 sites were taken before and 12 months after surgery. Ten experimental sites were treated with bone graft substitutes (natural coral or natural coral+collagen), and 10 control sites by debridement alone. None of the experimental sites had a density ratio below 1, where 1 indicates no change. The error was +/- 0.07 (0.93-1.07). The experimental sites showed an 18% mean increase in bone density (1.18), which increased to 23% (1.23) for sites filled with natural coral alone. All the control sites had values close to 1.00 (1.00 +/- 0.07) except for 3 sites, which showed a 9-15% loss of bone density. It is thus possible to compare and quantify the changes in experimental and control sites in the same patient using the percentage gain or loss of bone density. This demonstrates that DIR is suitable for clinical applications, and can be used in clinical analysis when bone changes are expected.

New features of dislocation images in third-generation synchrotron radiation topographs.

Some aspects of the dislocation contrast observed at third-generation synchrotron radiation set-ups are presented. They can be explained by taking into account angular deviation effects on the beam propagation, which are visible because of the ;almost plane-wave' character of these sources. In particular, we show how the evolution of the direct image width of a dislocation as a function of the sample-to-film distance can allow a complete determination of the Burgers vector, i.e. in sign and modulus. In addition, experimental results obtained in monochromatic beam topography are compared with simulated images calculated assuming plane-wave illumination and are demonstrated to show a satisfactory agreement. The utility of the weak-beam technique in enhancing the spatial resolution is demonstrated and a criterion for the selection of experimental conditions depending upon the required spatial resolution, signal-to-noise ratio and exposure time is presented.

Digital image ratio: a new radiographic method for quantifying changes in alveolar bone. Part 1: Theory and methodology.

A new world, digital image ratio (DIR), has been developed for directly measuring changes in alveolar bone. The image on the computer monitor represents the relative mass change between two radiographs. Fourier filtering is used to reduce noise artefacts. This method is validated through an experiment with a step wedge. DIR needs only a preliminary calibration of the experimental conditions of operation and avoids tedious calibrations for each measurement as in the case of digital image substraction. Low-voltage X-ray techniques are suggested for long-term quantitative studies of patients to minimize irradiation doses.

Three-dimensional computer reconstructions from serial sections of cell nuclei.

The analysis of ultrathin serial sections as 3-dimensional (3D) information requires interpretation and display of a large amount of data. We suggest a simple way to solve this problem; it permits presentation of a series of sections as a 3D color image of good quality. It involves a picture system with specialized hardware and software written for this purpose. 3D images of cellular organelles have been drawn either by manually defining the contour of the objects or by thresholding of the volumes in the structures. These 2 methods allow rapid drawing of the image on the screen. It is possible to determine the position, shape and size of 3D structures. This interactive system allows the user to choose between several options: colors, removal of parts of the object, and cutout.

Refinement of the C222(1) crystal form of oxidized uteroglobin at 1.34 A resolution.

The structure of uteroglobin, a progesterone binding protein from rabbit uterine fluid, was determined and refined at 1.34 A resolution to a conventional R-factor of 0.229. The accuracy of the co-ordinates is estimated to be 0.15 A. The isotropic temperature factor of individual atoms was refined and its average value is 11.9 A2 for the 548 non-hydrogen atoms of the protein monomer. A total of 83 water molecules was located in difference electron density maps and refined, first using a constant occupancy factor of 1 and then variable occupancy, the final (Q) being 0.63. The mean temperature factor of the water oxygen atoms is 26.4 A2. Uteroglobin is a dimer and its secondary structure consists of four alpha-helices per monomer that align in an anti-parallel fashion. There is one beta-turn between helix 2 and helix 3 (Lys26 to Glu29); 76% of the residues are part of the alpha-helices. In the core of the dimeric protein molecule, between the two monomers that are held together by two disulfide bridges, we have observed a closed cavity. Its length is 15.6 A and its width is 9 A; 14 water molecules could be positioned inside. In the "bottom" part of the protein, near the C terminus, we have observed a smaller cavity, occupied by two water molecules. The calculation of the molecular surface revealed four surface pockets whose possible functional implications are discussed below.