Dynamic In Vivo Chest X-ray Dark-Field Imaging in Mice.

X-ray grating interferometry is a powerful emerging tool in biomedical imaging, providing access to three complementary image modalities. In addition to the conventional attenuation modality, interferometry provides a phase modality, which visualizes soft tissue structures, and a dark-field modality, which relates to the number and size of sub-resolution scattering objects. A particularly strong dark-field signal originates from the alveoli or air sacs in the lung. Dark-field lung radiographs in animal models have already shown increased sensitivity in diagnosing lung diseases, such as lung cancer or emphysema, compared to conventional X-ray chest radiography. However, to date, X-ray dark-field lung imaging has either averaged information over several breaths or has been captured during a breath hold. In this paper, we demonstrate the first time-resolved dark-field imaging of a breath cycle in a mechanically ventilated mouse, in vivo, which was obtained using a grating interferometer. We achieved a time resolution of 0.1 s, visualizing the changes in the dark-field, phase, and attenuation images during inhalation and exhalation. These measurements show that the dark-field signal depends on the air volume and, hence, the alveolar dimensions of the lung. Conducting this type of scan with animal disease models would help to locate the optimum breath point for single-image diagnostic dark-field imaging and could indicate if the changes in the dark-field signal during breath provide a diagnostically useful complementary measure.

Application of sensitive, high-resolution imaging at a commercial lab-based X-ray micro-CT system using propagation-based phase retrieval.

Several dedicated commercial lab-based micro-computed tomography (µCT) systems exist, which provide high-resolution images of samples, with the capability to also deliver in-line phase contrast. X-ray phase contrast is particularly beneficial when visualizing very small features and weakly absorbing samples. The raw measured projections will include both phase and absorption effects. Extending our previous work that addressed the optimization of experimental conditions at the commercial ZEISS Xradia 500 Versa system, single-distance phase-contrast imaging is demonstrated on complex biological and material samples. From data captured at this system, we demonstrate extraction of the phase signal or the correction of the mixed image for the phase shift, and show how this procedure increases the contrast and removes artefacts. These high-quality images, measured without the use of a synchrotron X-ray source, demonstrate that highly sensitive, micrometre-resolution imaging of 3D volumes is widely accessible using commercially advanced laboratory devices.

Quantitative breast tissue characterization using grating-based x-ray phase-contrast imaging.

X-ray phase-contrast imaging has received growing interest in recent years due to its high capability in visualizing soft tissue. Breast imaging became the focus of particular attention as it is considered the most promising candidate for a first clinical application of this contrast modality. In this study, we investigate quantitative breast tissue characterization using grating-based phase-contrast computed tomography (CT) at conventional polychromatic x-ray sources. Different breast specimens have been scanned at a laboratory phase-contrast imaging setup and were correlated to histopathology. Ascertained tumor types include phylloides tumor, fibroadenoma and infiltrating lobular carcinoma. Identified tissue types comprising adipose, fibroglandular and tumor tissue have been analyzed in terms of phase-contrast Hounsfield units and are compared to high-quality, high-resolution data obtained with monochromatic synchrotron radiation, as well as calculated values based on tabulated tissue properties. The results give a good impression of the method's prospects and limitations for potential tumor detection and the associated demands on such a phase-contrast breast CT system. Furthermore, the evaluated quantitative tissue values serve as a reference for simulations and the design of dedicated phantoms for phase-contrast mammography.

Energy-resolved visibility analysis of grating interferometers operated at polychromatic X-ray sources.

Grating interferometry has been successfully adapted at standard X-ray tubes and is a promising candidate for a broad use of phase-contrast imaging in medical diagnostics or industrial testing. The achievable image quality using this technique is mainly dependent on the interferometer performance with the interferometric visibility as crucial parameter. The presented study deals with experimental investigations of the spectral dependence of the visibility in order to understand the interaction between the single contributing energies. Especially for the choice which type of setup has to be preferred using a polychromatic source, this knowledge is highly relevant. Our results affirm previous findings from theoretical investigations but also show that measurements of the spectral contributions to the visibility are necessary to fully characterize and optimize a grating interferometer and cannot be replaced by only relying on simulated data up to now.

Monochromatic computed tomography with a compact laser-driven X-ray source.

A laser-driven electron-storage ring can produce nearly monochromatic, tunable X-rays in the keV energy regime by inverse Compton scattering. The small footprint, relative low cost and excellent beam quality provide the prospect for valuable preclinical use in radiography and tomography. The monochromaticity of the beam prevents beam hardening effects that are a serious problem in quantitative determination of absorption coefficients. These values are important e.g. for osteoporosis risk assessment. Here, we report quantitative computed tomography (CT) measurements using a laser-driven compact electron-storage ring X-ray source. The experimental results obtained for quantitative CT measurements on mass absorption coefficients in a phantom sample are compared to results from a rotating anode X-ray tube generator at various peak voltages. The findings confirm that a laser-driven electron-storage ring X-ray source can indeed yield much higher CT image quality, particularly if quantitative aspects of computed tomographic imaging are considered.

Vibrational dynamics of myoglobin determined by the phonon-assisted Mössbauer effect.

The phonon-assisted Mössbauer effect is used to determine the partial phonon density of states of the iron within the active center of deoxymyoglobin, carboxymyoglobin, and dry and wet metmyoglobin between 40 and 300 K. Between 0 and 1 meV the iron density of states increases quadratically with the energy, as in a Debye solid. Mean sound velocities are extracted from this slope. Between 1 and 3 meV a nearly quadratic "Debye-like" increase follows due to the similar strength of intermolecular and intramolecular forces. Above 3 meV, optical vibrations are characteristic for the iron-ligand conformation. The overall mean square displacements of the heme iron atom obtained from the density of states agree well with the values of Mössbauer absorption experiments below 180 K. In the physiological temperature regime the data confirm the existence of harmonic vibrations in addition to the protein specific dynamics measured by Mössbauer absorption. In the Debye energy regime the mean square displacement of the iron is in agreement with that of the hydrogens measured by incoherent neutron scattering demonstrating the global character of these modes. At higher energies the vibration of the heavy iron atom at 33 meV in metmyoglobin is as large as that of the lightweight hydrogens at that energy. A freeze dried, rehydrated (h=0.38 g H2O/g protein) metmyoglobin sample shows an excess of states above the Debye law between 1 and 3 meV, similar to neutron scattering experiments. The room temperature density of states below 3 meV exhibit an increase of the density compared to the low temperature data, which can be interpreted as mode softening.

The X-ray absorption spectroscopy Debye-Waller factors of an iron compound and of met-myoglobin as a function of temperature.

Protein dynamics can be characterized by the mean square displacements of the individual atoms of a molecule. This concept is extended to X-ray absorption spectroscopy (XAS) of proteins where the physical information in the Debye-Waller factor is in general neglected. In a first step, a procedure for the investigation of the temperature dependence of XAS spectra has been developed for a small iron compound. Subsequently, experiments have been performed on met-myoglobin. It is shown that the mean square displacements of XAS are smaller than those obtained by Mössbauer spectroscopy and far smaller than crystallographic mean square displacements. This behavior is explained by the different sensitivity of the methods. XAS measures a relative mean square displacement between the absorbing and backscattering atoms only. A comparison with mean square displacements calculated from normal modes shows that static displacements contribute significantly. It becomes obvious that the atoms of the active center show a high correlation of their motions.

Nuclear forward scattering of synchrotron radiation by deoxymyoglobin.

Nuclear forward scattering of synchrotron radiation is used to determine the quadrupole splitting and the mean square displacement of the iron atom in deoxymyoglobin in the temperature range between 50 K and 243 K. Above 200 K an abnormally fast decay of the forward scattered intensity at short times after the synchrotron flash is observed, which is caused by protein-specific motions. The results strongly support the picture that protein dynamics seen at the position of the iron can be understood by harmonic motions in the low temperature regime while in the physiological regime diffusive motions in limited space are present. The shape of the resonance broadening function is investigated. An inhomogeneous broadening with a Lorentzian distribution indicating dipole interactions results in a better agreement with the experimental data than the common Gaussian distribution.

Identification of key residues in rabbit liver microsomal cytochrome P450 2B4: importance in interactions with NADPH-cytochrome P450 reductase.

A cytochrome P450 2B4 (CYP2B4) model was used to select key residues supposed to serve in interactions with NADPH-cytochrome P450 reductase (P450R). Eight amino acid residues located on the surface of the hemoprotein were chosen for mutagenesis experiments with CYP2B4(Delta2-27) lacking the NH(2)-terminal signal anchor sequence. The mutated proteins were expressed in Escherichia coli, purified, and characterized by EPR- and CD-spectral analysis. Replacement of histidine 226 with alanine caused a 3.8-fold fall in the affinity for P450R with undisturbed reductive capacity of the system. Similarly, the K225A, R232A, and R253A variants exhibited P450R-directed activity that was depressed to about half that of the control enzyme, suggesting that the deletion of positive charges on the surface of CYP2B4(Delta2-27) resulted in impaired electrostatic contacts with complementary amino acids on the P450R protein. While the Y235A mutant did not show appreciably perturbed reduction activity, the conservative substitution with alanine of the phenylalanine residues at positions 223 and 227 gave a 2.1- to 6. 1-fold increase in the K(m) values with unchanged V(max); this was attributed to the disruption of hydrophobic forces rather than to global structural rearrangement(s) of the engineered pigments. Measurement of the stoichiometry of aerobic NADPH consumption and H(2)O(2) formation revealed the oxyferrous forms of the F223A, H226A, and F227A mutants to autoxidize more readily owing to less efficient coupling of the systems. Noteworthy, the F244A enzyme did not exhibit significant reduction activity, suggesting a pivotal role of Phe-244 in the functional coupling of P450R. The residue was predicted to constitute part of an obligatory electron transfer conduit through pi-stacking with Phe-296 located close to the heme unit. All of the residues examined reside in the putative G helix of CYP2B4, so that this domain obviously defines part of the binding site for P450R.

Simultaneous interpretation of Mössbauer, EPR and 57Fe ENDOR spectra of the [Fe4S4] cluster in the high-potential iron protein I from Ectothiorhodospira halophila.

Mössbauer spectra of the oxidized [Fe4S4]3+ and the reduced [Fe4S4]2+ clusters in the high-potential iron protein I from Ectothiorhodospira halophila were measured in a temperature range from 5 K to 240 K. EPR measurements and 57Fe electron-nuclear double resonance (ENDOR) experiments were carried out with the oxidized protein. In the oxidized state the cluster has a net spin S = 1/2 and is paramagnetic. As common in [Fe4S4]3+ clusters, the Mössbauer spectrum was simulated with two species contributing equally to the absorption area: two Fe3+ atoms couple to the "ferric-ferric" pair, and one Fe2+ and one Fe3+ atom give the "ferric-ferrous pair". For the simulation of the Mössbauer spectrum, g-values were taken from EPR measurements. A-tensor components were determined by 57Fe ENDOR experiments that turned out to be a necessary source of estimating parameters independently. In order to obtain a detailed agreement of Mössbauer and ENDOR data, electronic relaxation has to be taken into account. Relaxing the symmetry condition in a way that the electric field gradient tensor does not coincide with g- and A-tensors yielded an even better agreement of experimental and theoretical Mössbauer spectra. Spin-spin and spinlattice relaxation times were estimated by pulsed EPR; the former turned out to be the dominating mechanism at T = 5 K. Relaxation times measured by pulsed EPR and obtained from the Mössbauer fit were compared and yield nearly identical values. The reduced cluster has one additional electron and has a diamagnetic (S = 0) ground state. All the four irons are indistinguishable in the Mössbauer spectrum, indicating a mixed-valence state of Fe2.5+ for each.