Prospects for microcomputerized-tomography using synchrotron radiation

A microversion of a computerized tomograph (CT) is described, in which the object is subjected to a successive series of translations with rotation by a small angle in between. The spatial resolution is determined by collimators and translation step lengths and is today, with clinical X-ray tube, of the order of 100 μm. The use of synchrotron radiation instead of X-ray tubes offers the advantages of much higher fluence rates, which can be used to diminish the exposure times from days to minutes or to increase the spatial resolution from 100 μm to about 1 μm. The possibility to receive monoenergetic photons of selectable energy makes it possible to avoid spectral hardening image artifacts, as well as to optimize the information sampling with regard to average absorbed dose or exposure time. Selectable photon energies are valuable also for tomochemistry applications.     

Biological trace-element measurements using synchrotron radiation

The feasibility of performing X-ray fluorescence trace-element determinations at concentrations substantially below the ppm level for biological materials is demonstrated. Conditions for achieving optimum sensitivity were ascertained. Results achieved for five standard reference materials were, in most cases, in excellent agreement with listed values. Minimum detectable limits of 20 ppb were measured for most elements.     

An X-ray microprobe facility using synchrotron radiation

Biological Trace Element Research - An X-ray microprobe for trace elemental analysis at micrometer spatial resolutions, using synchrotron radiation (SR), is under development. The facility consists...     

Perspectives in three-dimensional analysis of bone samples using synchrotron radiation microtomography.

In this paper we present a methodology based on 3D synchrotron radiation microtomography to analyze non-destructively 3D bone samples. After a technical presentation of the imaging system and the image analysis techniques, we report results on three-dimensional analysis of vertebral samples from women of different ages. The new capabilities of this technique for the investigation of bone are discussed. They include a high spatial resolution down to the micron level, a high density resolution allowing a local quantification of bone mineralization, phase contrast imaging and advances in 3D image analysis.     

Structure and growth of ultrasmall protein microcrystals by synchrotron radiation: II. microGISAX and microscopy of lysozyme

The early steps of growth and nucleation of the lysozyme microcrystals by classical and nanotemplate-based hanging vapor diffusion methods are studied using microGISAXS at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Out-of-plane cuts in the Yoneda regions of the 2D scattering profiles point to the detection of ultrasmall lysozyme crystals by microGISAXS quite before than by light microscopy. Furthermore lysozyme crystal formation occurs quite earlier with the nanotemplate than with the classical method. Our data are compatible with two distinct modes of crystal nucleation and growth for P450sc and lysozyme.     

Ultrasonic absorption in aqueous solution of lysozyme.

The titration curve of ultrasonic absorption at 2.82 MHz in aqueous solutions of lysozyme measured by Zana and Lang [J. Phys. Chem., 74 , 2734 (1970)] is theoretically analyzed. The maxima at pH 3 and pH 11 are describable with proton-transfer reactions of dissociable carboxyl and amino groups by assuming that volume changes due to the reactions are 2.3 and 5.2 cm³/mole, respectively, which are appreciably smaller than those of simple amino acids. The remaining, pH-independent excess absorption over solvent is measured at frequencies ranging from 3 to 150 MHz. The absorption is ascribed to the internal loss of protein. The complex compressibility β′_p ? iβ″_p of lysozyme molecule is evaluated as β′_p = 7.2 × 10^?12 cm²/dyne and β″_P = 4.3 × 10^?14 cm²/dyne from the increments over solvent in absorption as well as in sound velocity.     

Imaging of the pulmonary circulation in the closed-chest rat using synchrotron radiation microangiography.

Structural changes of the pulmonary circulation during the pathogenesis of pulmonary arterial hypertension remain to be fully elucidated. Although angiography has been used for visualizing the pulmonary circulation, conventional angiography systems have considerable limitations for visualizing small microvessels (diameters < 200 microm), particularly within a closed-chest animal model. In this study we assess the effectiveness of monochromatic synchrotron radiation (SR) for microangiography of the pulmonary circulation in the intact-chest rat. Male adult Sprague-Dawley rats were anesthetized, and a catheter was positioned within the right ventricle, for administering iodinated contrast agent (Iomeron 350). Subsequently, microangiography of pulmonary arterial branches within the left lung was performed using monochromatic SR. Additionally, we assessed dynamic changes in vessel diameter during acute hypoxic (10% and 8% O2 for 4 min each) pulmonary vasoconstriction (HPV). Using SR we were able to visualize pulmonary microvessels with a diameter of <100 microm (the 4th generation of branching from the left axial artery). Acute hypoxia caused a significant decrease in the diameter of all vessels less than 500 microm. The greatest degree of pulmonary vasoconstriction was observed in vessels with a diameter between 200 and 300 microm. These results demonstrate the effectiveness of SR for visualizing pulmonary vessels in a closed-chest rat model and for assessing dynamic changes associated with HPV. More importantly, these observations implicate SR as an effective tool in future research for assessing gross structural changes associated with the pathogenesis of pulmonary arterial hypertension.     

Sleep-enhancing effects of far-infrared radiation in rats

Unrestrained male rats continuously exposed to far-infrared radiation exhibited a significant increase in slow wave sleep (SWS) during the light period but not in the dark period. The change was largely due to the elevated occurrence of SWS episodes but not to the prolongation of their duration. Paradoxical sleep was not affected throughout the observation period except for a significant decrease at the end of the dark period. Thus the far-infrared radiation exerted a sleep modulatory effect closely related to the circadian activity-rest cycle.     

X-ray fluorescence analysis using synchrotron radiation at the photon factory

X-ray fluorescence experiments at the Photon Factory in Japan are described. An energy-dispersive X-ray fluorescence system was combined with various excitation modes, i.e., a continuum and a monochromatic excitation, which consist of a crystal monochromator or a wide band pass monochromator. These excitation modes provide a wide range of band width and photon flux of excitation beams. Minimum detection limits obtained for a thin sample were less than 0.1 ppm and 0.1 pg when there was no line interference. Advantages of using monochromatic excitation are discussed, with emphasis on the possibility of chemical state analysis. Grazing incidence X-ray fluorescence is a technique very appropriate to synchrotron radiation characteristics. Near-surface analysis and trace analysis of solution samples placed on a total reflection support were made. Future plans are discussed, including microbeam analysis, tomography, X-ray excited optical fluorescence, and applications of insertion devices (undulator and wiggler).     

Survey of chemical speciation of trace elements using synchrotron radiation

Information concerning the chemical state of trace elements in biological systems generally has not been available. Such information for toxic elements and metals in metalloproteins could prove extremely valuable in the elucidation of their metabolism and other biological processes. The shielding of core electrons by binding electrons affect the energy required for creating inner-shell holes. Furthermore, the molecular binding and symmetry of the local environment of an atom affect the absorption spectrum in the neighborhood of the absorption edge. X-ray absorption near-edge structure (XANES) using synchrotron radiation excitation can be used to provide chemical speciation information for trace elements at concentrations as low as 10 ppm. The structure and position of the absorption curve in the region of an edge can yield vital data about the local structure and oxidation state of the trace element in question. Data are most easily interpreted by comparing the observed edge structure and position with those of model compounds of the element covering the entire range of possible oxidation states. Examples of such analyses will be reviewed.     

In vivo K-edge imaging with synchrotron radiation.

We present in this paper two imaging techniques using contrast agents assessed with in vivo experiments. Both methods are based on the same physical principle, and were implemented at the European Synchrotron Radiation Facility medical beamline. The first one is intravenous coronary angiography using synchrotron radiation X-rays. This imaging technique has been planned for human studies in the near future. We describe the first experiments that were carried out with pigs at the ESRF. The second imaging mode is computed tomography using synchrotron radiation on rats bearing brain tumors. Owing to synchrotron radiation physical properties, these new imaging methods provide additional information compared to conventional techniques. After infusion of the contrast agent, it is possible to derive from the images the concentration of the contrast agent in the tumor area for the computed tomography and in any visible vessel for the angiography method.     

Imaging of the closed-chest mouse pulmonary circulation using synchrotron radiation microangiography

Structural and functional changes of pulmonary circulation related to pathophysiology of pulmonary arterial hypertension (PAH) remain to be fully elucidated. Angiographic visualization in in vivo animals provided a powerful tool for assessing the major indexes associated with the pathogenesis of PAH. In this study, we have exploited the full potential of synchrotron radiation (SR) microangiography to show the ability to visualize pulmonary hemodynamics in a closed-chest mouse. Male adult mice were anesthetized and cannulated with a customized 24-gauge catheter into the right ventricle via the jugular vein for administering iodine contrast agent. The microangiography was performed on the left lung. We measured dynamic changes in vessel diameter in response to acetylcholine (ACh) and acute exposure to hypoxic gas (10% O(2)). Moreover, the pulmonary transit time was estimated by the time of contrast agent circulating. We were able to visualize the pulmonary arteries from the left pulmonary artery (LPA) to the third generation of branching (inner diameter <100 μm). ACh and acute hypoxia induced vascular responses chiefly in the second and third branching vessels rather than the LPA and the first branching vessels. The transit time was only 0.83 s. These results demonstrate the effectiveness of SR for visualizing the pulmonary circulation in a closed-chest mouse. Future studies using SR microangiography on specific gene-targeted knockout and transgenic mice will provide new insights into the pathophysiology of pulmonary dysfunction and functional adaptation to survive in hypoxic condition.     

Time-resolved X-ray diffraction from tendon collagen during creep using synchrotron radiation

In order to understand the molecular mechanism of relaxation phenomena in collagenous tissue, time-resolved, small-angle X-ray diffraction measurements were performed on bovine Achilles tendon collagen under creep. A tension-induced increase in the 67 nm period (D-period) was observed, and the strain in the D-period, epsilon D, was found to be almost proportional to the external force per unit cross-sectional area (average stress) of the specimen. With an increase in epsilon D, a change in the ratio of intensities of the third-order reflection peak of the D-period to that of the second-order peak was also observed. The increase in epsilon D was decomposed into three elementary processes of D-period deformation, which are presented on the basis of the Hodge-Petruska model: (1) molecular elongation, (2) increase in gap region, and (3) relative slippage of lateral adjoining molecules. Up to 8 MPa of average stress, the contribution to epsilon D originated mostly from only mode (1). At more than 10 MPa of average stress, modes (2) and (3) also contributed to fibril elongation. For epsilon D by molecular elongation (mode (1)), the time dependence of the D-period change in the immediate response region is a sharply shaped step function, while the contribution to epsilon D by molecular rearranging modes gives a slight creep nature at the immediate response region in the time dependence of epsilon D. Because this creep nature is observed at the immediate response, it is related qualitatively to the KWW function in a stress-relaxation modulus of collagenous tissue observed in an immediate response region (Sasaki et al. (1993). Journal of Biomechanics 26, 1369-1376). The elementary process of KWW-type relaxation is concluded to be related to the tension-induced molecular rearrangement within a D-period.     

Contact microscopy with synchrotron radiation

Soft X-ray contact microscopy with synchrotron radiation offers the biologist, and especially the microscopist, a way to morphologically study specimens that could not be imaged by conventional TEM, STEM, or SEM methods (i.e., hydrated samples, samples easily damaged by an electron beam, electron-dense samples, thick specimens, unstained, low-contrast specimens) at spatial resolutions approaching those of the TEM, with the additional possibility to obtain compositional (elemental) information about the sample as well. Although flash X-ray sources offer faster exposure times, synchrotron radiation provides a highly collimated, intense radiation that can be tuned to select specific discrete ranges of X-ray wavelengths or specific individual wavelengths that optimize imaging or microanalysis of a specific sample. This paper presents an overview of the applications of X-ray contact microscopy to biological research and some current research results using monochromatic synchrotron radiation to image biological samples.     

Anomalous X-ray diffraction with soft X-ray synchrotron radiation.

Anomalous diffraction with soft X-ray synchrotron radiation opens new possibilities in protein crystallography and materials science. Low-Z elements like silicon, phosphorus, sulfur and chlorine become accessible as new labels in structural studies. Some of the heavy elements like uranium exhibit an unusually strong dispersion at their M(V) absorption edge (lambdaMV = 3.497 A, E(MV) = 3545 eV) and so does thorium. Two different test experiments are reported here showing the feasibility of anomalous X-ray diffraction at long wavelengths with a protein containing uranium and with a salt containing chlorine atoms. With 110 electrons the anomalous scattering amplitude of uranium exceeds by a factor of 4 the resonance scattering of other strong anomalous scatterers like that of the lanthanides at their L(III) edge. The resulting exceptional phasing power of uranium is most attractive in protein crystallography using the multi-wavelength anomalous diffraction (MAD) method. The anomalous dispersion of an uranium derivative of asparaginyl-tRNA synthetase (hexagonal unit cell; a = 123.4 A, c = 124.4 A) has been measured for the first time at 4 wavelengths near the M(V) edge using the beamline ID1 of ESRF (Grenoble, France). The present set up allowed to measure only 30% of the possible reflections at a resolution of 4 A, mainly because of the low sensitivity of the CCD detector. In the second experiment, the dispersion of the intensity of 5 X-ray diffraction peaks from pentakismethylammonium undecachlorodibismuthate (PMACB, orthorhombic unit cell; a = 13.003 A, b = 14.038 A, c = 15.450 A) has been measured at 30 wavelengths near the K absorption edge of chlorine (lambdaK = 4.397 A, EK= 2819.6 eV). All reflections within the resolution range from 6.4 A to 3.4 A expected in the 20 degree scan were observed. The chemical state varies between different chlorine atoms of PMACB, and so does the dispersion of different Bragg peaks near the K-edge of chlorine. The results reflect the performance of the beamline ID1 of ESRF at wavelengths beyond 3 A at the end of 1998. A gain by a factor 100 for diffraction experiments with 4.4 A photons was achieved in Autumn 1999 when two focusing mirrors had been added to the X-ray optics. Further progress is expected from area detectors more sensitive to soft X-rays. Both CCD detectors and image plates would provide a gain of two orders of measured intensity. Image plates would have the additional advantage that they can be bent cylindrically and thus cover a larger solid angle in reciprocal space. In many cases, samples need to be cooled: closed and open systems are presented. A comparison with the state of art of soft X-ray diffraction, as it had been reached at HASYLAB (Hamburg, Germany), and as it is developing at the Brookhaven National Laboratory (USA), is given.     

Changes in macrovessel pulmonary blood flow distribution following chronic hypoxia: assessed using synchrotron radiation microangiography.

Structural and functional changes of the pulmonary circulation, particularly during the pathogenesis of pulmonary arterial hypertension (PAH), remain to be fully elucidated. In this study, we utilized monochromatic synchrotron radiation (SR) microangiography to assess changes in pulmonary arteriole blood flow in the intact-chest rat after 4 wk of chronic hypoxia. Sprague-Dawley rats were exposed to normoxia (N-rats) or chronic hypoxia (10% O(2); CH-rats) for 28 days. Rats were anesthetized, and microangiography was performed on the left lung to assess 1) the branching distribution of pulmonary arteriole blood flow (internal diameter >80 microm) and 2) dynamic changes in vessel lumen diameter during acute hypoxic (8% O(2) for 4 min) pulmonary vasoconstriction (HPV) before and after beta-adrenoceptor blockade (2 mg/kg i.v. propranolol). Using SR angiography, we observed that the number of opaque third- and fourth-generation vessels (100-300 microm) for CH-rats was significantly fewer than the number for N-rats. The magnitude of HPV was not different between CH-rats and N-rats. Beta-adrenoceptor blockade accentuated the HPV in 200- to 300-microm vessels for CH-rats, but even more so in N-rats. However, in CH-rats, beta-adrenoceptor blockade also accentuated the HPV in 100- to 200-microm vessels. In summary, we utilized SR to assess gross blood flow changes and functional changes (i.e., HPV) of the pulmonary circulation in PAH. These results highlight the benefits of SR for assessing pulmonary circulatory pathology. Of particular importance, future use of SR will provide an effective method for assessing potential therapeutic treatments for PAH.     

Energy dispersive X-ray diffraction by collagen fibrils in costal cartilage using synchrotron radiation

Low-angle X-ray diffraction patterns from the isotropic distribution of collagen fibrils, which occur in low concentrations in costal cartilage, were recorded using synchrotron radiation. An energy dispersive technique was used to exploit the properties of synchrotron radiation to the full. The third, fourth, fifth and sixth diffraction orders from the axial periodicity of the fibrils were recorded and used to calculate a value for this periodicity of 67 ± 1 nm. This result is in good agreement with measurements made on amianthoid areas as well as from fibrils in tendon, which consist of a chemically distinct form of collagen.     

Non-destructive three-dimensional evaluation of a polymer sponge by micro-tomography using synchrotron radiation

X-ray micro-tomography, a non-destructive technique is used to uncover the complex 3-D micro-architecture of a degradable polymer sponge designed for bone augmentation. The measurements performed at HASYLAB at DESY are based on a synchrotron radiation source resulting in a spatial resolution of about 5.4 microm. In the present communication we report the quantitative analysis of the porosity and of the pore architecture. First, we elucidate that synchrotron radiation at the photon energy of 9 keV has an appropriate cross section for this low-weight material. Modifications in sponge micro-architecture during measurement are not detected. Second, the treatment of the data, an amount of 2.5 Gbyte to generate binary data is described. We compare the 3-D with the 2-D analysis in a quantitative manner. The obtained values for the mean distance to material within the sponge calculated from 2-D and 3-D data of the whole tomogram differ significantly: 12.5 microm for 3-D and 17.6 microm for 2-D analysis. If the pores exhibit a spherical shape as frequently found, the derived mean pore diameter, however, is overestimated only by 6% in the 2-D image analysis with respect to the 3-D evaluation. This approach can be applied to different porous biomaterials and composites even in a hydrated state close to physiological conditions, where any surface preparation artifact is avoided.     

Evaluating protein:protein complex formation using synchrotron radiation circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy beamlines at synchrotrons produce dramatically higher light flux than conventional CD instruments. This property of synchrotron radiation circular dichroism (SRCD) results in improved signal-to-noise ratios and allows data collection to lower wavelengths, characteristics that have led to the development of novel SRCD applications. Here we describe the use of SRCD to study protein complex formation, specifically evaluating the complex formed between carboxypeptidase A and its protein inhibitor latexin. Crystal structure analyses of this complex and the individual proteins reveal only minor changes in secondary structure of either protein upon complex formation (i.e., it involves only rigid body interactions). Conventional CD spectroscopy reports on changes in secondary structure and would therefore not be expected to be sensitive to such interactions. However, in this study we have shown that SRCD can identify differences in the vacuum ultraviolet CD spectra that are significant and attributable to complex formation.