Eric F. Eikenberry

Charge-coupled device area x-ray detectors

Charge-coupled device (CCD) area x-ray detector technology is reviewed. CCD detectors consist of a serial chain of signal components, such as phosphors, fiber optics or lenses, image intensifiers and the CCD which serve to convert the x-ray energy to light or electron-hole pairs and to record the spatially resolved image. The various combinations of components that have been used to make CCD detectors are described and the properties of each of the critical components are discussed. Calibration and correction procedures required for accurate data collection are described. The review closes with a brief description of future directions for solid-state area x-ray detectors.

Glycation induces expansion of the molecular packing of collagen

Exposure of rat tail tendon to a reducing sugar results in covalent attachment of the sugar to collagen, a process termed glycation, and leads to the formation of stable intermolecular cross-links. We have used X-ray diffraction to study the changes in the crystalline unit cell of rat tail tendon collagen brought about by glycation. Ribose was selected as a model compound for most of the study because its reaction with proteins is faster than that of glucose, and therefore more convenient for laboratory studies, but glucose and glyceraldehyde were used as well. A kinetic model describing the process of glycation by ribose and subsequent cross-link formation has been developed. Glycation resulted in an expansion by more than 12% of the unit cell that describes the three-dimensional structure of rat tail tendon collagen. The expansion was in a direction perpendicular to the axes of the rod-shaped molecules, indicating that the intermolecular spacing of the collagen increased. Thus, the structure of collagen in rat tail tendon is significantly altered by glycation in vitro. The expansion was not isotropic, but was directed parallel to the (120) planes, one of the three major planes of the quasi-hexagonal structure that is densely populated by collagen molecules. It is hypothesized that this expansion is brought about by the formation of one, or at most a few, specific intermolecular cross-links in the overlap zone that act to push the molecules apart. It is likely that similar structural changes in collagenous tissues are caused by glycation in vivo during the natural course of aging, and that these changes are accelerated in chronic hyperglycemia such as that associated with diabetes. Analysis of the structure of glycated rat tail tendon potentially can give us new insight into the detailed molecular structure of collagen.

Nonbilayer phases of membrane lipids

Numerous liquid crystalline biomembrane lipids are known to exhibit non-lamellar phases characterized by curvature of their component lipid monolayers. An understanding of the phase stability of these systems begins with analysis of the energy of bending the monolayers, the interactions which lead to the bending energy, and the geometrical constraints which lead to competing energy terms which arise when the monolayers are bent and packed onto lattices with different structures. Diffraction and other techniques suitable for probing lipid phase structure are described. A phenomenological model is reviewed which successfully explains many of the qualitative features of lipid mesomorphic phase behavior. A key result of this model is that lipid bilayer compositions which are close to the non-lamellar phase boundaries of their phase diagrams are characterized by a frustrated elastic stress which may modulate the activity of imbedded membrane proteins and which may provide a rationale for the prevalence of non-lamellar-tending lipid species in biomembrane bilayers. Areas in need of future research are discussed.

[5] Characterization of fibrous forms of collagen☆

Publisher Summary Collagen fibers can be studied in tissues or as fibers reprecipitated from solutions of purified collagen. The biochemical data establishing differences in the collagenous and noncollagenous components of various connective tissues has clarified the need to compare structures of fibrils in different tissues and of fibers reprecipitated from different genetic types of collagen. Such investigations have been carried out in this chapter using electron microscopy and X-ray diffraction on both native tissues and reprecipitated fibers. From electron microscopy, one gets an image of the fibril showing the gap: overlap regions (by negative staining) or the distribution of charged residues (by positive staining). In examination it is found that the SLS banding patterns of different genetic types of collagen can be distinguished, but no differences can be seen in the native-type fibrils reprecipitated from purified type I, type II, or type III collagens. In addition, X-Ray diffraction studies provide information on the axial fibril structure and the lateral packing of molecules in the fibril. Examination of various tissues and reprecipitated fibers has indicated that the X-ray patterns vary from the well-studied one of rat tail tendon.

Crystalline fibril structure of type II collagen in lamprey notochord sheath

We report here the existence of a crystalline molecular packing of type II collagen in the fibrils of the lamprey notochord sheath. This is the first finding of a crystalline structure in any collagen other than type I. The lamprey notochord sheath has a composition similar to that of cartilage, with type II collagen, a minor collagen component with 1 alpha, 2 alpha and 3 alpha chains, and cartilage-like proteoglycan. The high degree of orientation of fibrils in the notochord makes it possible to use X-ray diffraction to determine collagen fibril organization in this type II-containing tissue. The low angle equatorial scattering shows the fibrils are all about 17 nm in diameter and have an average center-to-center separation of 31 nm. These results are supported by electron microscope observations. A set of broad equatorial diffraction maxima at higher angles represents the sampling of the collagen molecular transform by a limited crystalline lattice, extending over a lateral dimension close to the diameter of one fibril. This indicates that each 17 nm fibril contains a crystalline array of molecules and, although a unit cell is difficult to determine because of the broad overlapping reflections, it is clear that the quasi-hexagonal triclinic unit cell of type I collagen in rat tail tendon is not consistent with the data. The meridional diffraction pattern showed 26 orders with the characteristic 67 nm periodicity found for tendon. However, the intensities of these reflections differ markedly from those found for tendon and cannot be explained by an unmodified gap/overlap model within each 67 nm period. Both X-ray diffraction and electron microscope data indicate a low degree of contrast along the fibril axis and are consistent with a periodic binding of a non-collagenous component in such a way as to obscure the gap region.

An unusual collagen periodicity in skin.

The axial periodicity of collagen fibrils was observed by X-ray diffraction to be significantly shorter in wet skin (65.2 nm) than in wet tendon (67.0 nm). This difference appears to be due to some feature in the native skin environment, since purified skin Type I and Type III collagens will both form fibrils with a normal 67.0 nm d-period.

Collagen fibril morphology in developing chick metatarsal tendon: 2. Electron microscope studies

Abstract Transverse section of embryonic chick metatarsal tendons ranging in age from 11 days to 18 days fetal were examined by electron microscopy to determine both the diameters and the lateral arrangements of the cylindrical collagen fibrils. In early developmental stages, from 11 days to 14 days fetal, sharp unimodal distributions of diameters centred near 32 or 40 nm were observed, but increasingly heterogeneous diameters were seen with increasing age. The heterogeneous diameter distributions were not uniform, but showed discrete populations of preferred diameters. The centre-to-centre distance separating the fibrils in the early developmental stages was about twice the fibril diameter and constant with age. Comparison of X-ray diffraction results with these observations indicated that the saptial relationships of the structures are preserved during the preparative procedures for electron microscopy, but that a transverse shrinkage of 25–30% had occurred relative to the wet dimension.

Calibration procedures for charge-coupled device x-ray detectors

Calibration procedures are described for use with electronic x-ray detectors, with an emphasis on detectors based on fiber-optically coupled charge-coupled devices. Methods are detailed for removing spurious events, pixel pedestals, dark-current, spatial distortion, and intensity response variations for both small-angle and wide-angle applications. The accuracy of the calibration procedures is discussed.

Tests of a prototype pixel array detector for microsecond time-resolved X-ray diffraction

X-ray test results from a prototype 92 × 100 pixel array detector (PAD) for use in rapid time-resolved X-ray diffraction studies are described. This integrating detector is capable of taking up to eight full-frame images at microsecond frame times. It consists of a silicon layer, which absorbs the X-rays, bump-bonded to a layer of CMOS electronics in which each pixel has its own processing, storage and readout electronics. Tests indicate signal performance characteristics are comparable with phosphor-based CCD X-ray detectors, with greatly improved time resolution, comparable linearity and enhanced point spread. This prototype is a test module en route to a larger detector suitable for dedicated operation. Areas of needed improvement are discussed.

Correlation Between Lipid Plane Curvature and Lipid Chain Order

The 1-palmitoyl-2-oleoyl-phosphatidylethanolamine: 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE:POPC) system has been investigated by measuring, in the inverted hexagonal (HII) phase, the intercylinder spacings (using x-ray diffraction) and orientational order of the acyl chains (using 2H nuclear magnetic resonance). The presence of 20 wt% dodecane leads to the formation of a HII phase for the composition range from 0 to 39 mol% of POPC in POPE, as ascertained by x-ray diffraction and 2H nuclear magnetic resonance. The addition of the alkane induces a small decrease in chain order, consistent with less stretched chains. An increase in temperature or in POPE proportion leads to a reduction in the intercylinder spacing, primarily due to a decrease in the water core radius. A temperature increase also leads to a reduction in the orientational order of the lipid acyl chains, whereas the POPE proportion has little effect on chain order. A correlation is proposed to relate the radius of curvature of the cylinders in the inverted hexagonal phase to the chain order of the lipids adopting the HII phase. A simple geometrical model is proposed, taking into account the area occupied by the polar headgroup at the interface and the orientational order of the acyl chains reflecting the contribution of the apolar core. From these parameters, intercylinder spacings are calculated that agree well with the values determined experimentally by x-ray diffraction, for the variations of both temperature and POPE:POPC proportion. This model suggests that temperature increases the curvature of lipid layers, mainly by increasing the area subtended by the hydrophobic core through chain conformation disorder, whereas POPC content affects primarily the headgroup interface contribution. The frustration of lipid layer curvature is also shown to be reflected in the acyl chain order measured in the L alpha phase, in the absence of dodecane; for a given temperature, increased order is observed when the curling tendencies of the lipid plane are more pronounced.