Fetal and postnatal mouse bone tissue contains more calcium than is present in hydroxyapatite

It has been shown for developing enamel and zebrafish fin that hydroxyapatite (HA) is preceded by an amorphous precursor, motivating us to examine the mineral development in mammalian bone, particularly femur and tibia of fetal and young mice. Mineral particle thickness and arrangement were characterized by (synchrotron) small-angle X-ray scattering (SAXS) combined with wide-angle X-ray diffraction (WAXD) and X-ray fluorescence (XRF) analysis. Simultaneous measurements of the local calcium content and the HA content via XRF and WAXD, respectively, revealed the total calcium contained in HA crystals. Interestingly, bones of fetal as well as newborn mice contained a certain fraction of calcium which is not part of the HA crystals. Mineral deposition could be first detected in fetal tibia at day 16.5 by environmental scanning electron microscopy (ESEM). SAXS revealed a complete lack of orientation in the mineral particles at this stage, whereas 1 day after birth particles were predominantly aligned parallel to the longitudinal bone axis, with the highest degree of alignment in the midshaft. Moreover, we found that mineral particle length increased with age as well as the thickness, while fetal particles were thicker but much shorter. In summary, this study revealed strong differences in size and orientation of the mineral particles between fetal and postnatal bone, with bulkier, randomly oriented particles at the fetal stage, and highly aligned, much longer particles after birth. Moreover, a part of the calcium seems to be present in other form than HA at all stages of development.     

The utility of bulk wood density for tree-ring research

Bulk wood density measurements are recognized for their utility in ecology, industry, and biomass estimations. In tree-ring research, microdensitometric techniques are widely used, but their ability to determine the correct central tendency has been questioned. Though rarely used, it may be possible to use bulk wood density as a tool to check the accuracy of and even correct microdensitometric measurements. Since measuring bulk wood density in parallel with X-ray densitometry is quickly and easily done, we suspect that its omission is largely due to a lack of awareness of the procedure and/or its importance. In this study, we describe a simple protocol for measuring bulk wood density tailored for tree-ring researchers and demonstrate a few possible applications. To implement real-world examples of the applications, we used a sample of existing X-ray and Blue Intensity (BI) measurements from 127 living and dead Pinus sylvestris trees from northern Sweden to produce new measurements of bulk wood density.We can confirm that the central tendency in this sample material is offset using X-ray densitometry and that the diagnosis and correction of X-ray density is easily done using bulk wood density in linear transfer functions. However, this approach was not suitable for our BI measurements due to heavy discoloration. Nevertheless, we were able to use bulk wood density to diagnose and improve the use of deltaBI (latewood BI – earlywood BI) with regard to its overall trends and multi-centennial variability in a dendroclimatological application. Moreover, we experimented with percent of latewood width, scaled with bulk wood density, as a time- and cost-effective proxy for annual ring density. Although our reconstruction only explains about half of the variation in ring density, it is most likely superior to using fixed literature values of density in allometric equations aimed at biomass estimations.With this study, we hope to raise new awareness regarding the versatility and importance of bulk wood density for dendrochronology by demonstrating its simplicity, relevance, and applicability.     

Application of X-ray absorption and X-ray fluorescence techniques to the study of metallodrug action

X-ray absorption spectroscopy and X-ray fluorescence microscopy are two synchrotron-based techniques frequently deployed either individually or in tandem to investigate the fates of metallodrugs and their biotransformation products in physiologically relevant sample material. These X-ray methods confer advantages over other analytical techniques in that they are nondestructive and require minimal chemical or physical manipulation of the sample before analysis, conserving both chemical and spatial information of the element(s) under investigation. In this review, we present selected examples of the use of X-ray absorption spectroscopy and X-ray fluorescence microscopy in studies of metallodrug speciation and localisation in vivo, in cell spheroids and in intact tissues and organs, and offer recent highlights in the advances of these techniques as they pertain to research on metallodrug action.     

Associating X-Ray Microtomography with Permeability Contrasts in Bioturbated Media

The use of X-ray microtomography (micro-CT) to evaluate the contrasts in permeability associated with bioturbation in two- and three-dimensions (2D and 3D) is investigated in this article. Core analysis, spot-permeametry measurements, and petrographic thin-sections of the six different datasets from Western Canada (Debolt Formation, Wabamun Group, and Medicine Hat Member) and offshore Norway (Ula Formation, Lysing Formation, and Nise Formation) show that the trace fossils commonly modify the permeability and porosity of the reservoir through sediment reworking and diagenesis of the sediment. In this study, micro-CT techniques are used to analyze the X-ray attenuation associated with bioturbation in siliciclastics and carbonates. Because X-ray attenuation represents the transmissivity or absorptivity of a sample to the incident X-rays, each of which is related to sediment composition and sediment densities, micro-CT images are integrated with sedimentological data and petrographic data to identify the trace fossils and matrix in 2D and 3D. Due to the difference in sediment grain size and sorting (i.e., porosity) within the burrows and matrix, analysis of the X-ray attenuations allows for their delineation in 2D cross-sectional images. When processed as 3D volumes, the micro-CT images show the complexity in burrow connectivity and burrow orientations within a sample. More importantly, the 3D volumes help show the distribution of porosity that can be linked to measured permeability values within a sample. Quantification of the heterogeneous burrow fabric on reservoir and resource quality is made from these processed micro-CT images.     

The superstructure of chromatin and its condensation mechanism

Electric dichroism and X-ray scattering measurements on solutions of uncondensed and condensed chicken erythrocyte chromatin were interpreted on the basis of model calculations. Information about the state of uncondensed fibers in the conditions of electric dichroism measurements was obtained from scattering patterns recorded as a function of pH, in the presence of spermine and at very low monovalent cation concentrations. Electric dichroism measurements on a complex of uncondensed chromatin with methylene blue were made to determine the contribution of the linker and of the nucleosomes to the total dichroism.A new approach to calculate the dichroism from realistic structural models, which also yields other structural parameters (radius of gyration, radius of gyration of the cross-section, mass per unit length) was used. Only a restricted range of structures is simultaneously compatible with all experimental results. Further, it is shown that previous interpretations of dichroism measurements on chromatin were in contradiction with X-ray scattering data and failed to take into account the distribution of orientation of the nucleosomes in the fibers. When this is done, it is found that the linker DNA in chicken erythrocyte and sea urchin chromatin must run nearly perpendicularly to the fibre axis. Taken together with the dependence of the fibre diameter on the linker length, these results provede the strongest evidence hitherto available for a model in which the linker crosses the central part of the fibre.     

Cryo-electron microscopy reveals macromolecular organization within biological liquid cyrstals seen in the polarizing microscope

A method is described for examining water dispersible biopolymers in the frozen, hydrated state by electron microscopy using the filamentous bacterial viruses Pf1 and fd as examples. The technique reveals liquid-crystalline textures that correlate well with polarizing microscopy of magnetically oriented specimens. At higher magnification the packing of the virus particle is revealed to a spatial resolution of better than 30 Å, thus linking directly with data from X-ray diffraction and optical microscopy. Electron diffraction confirms that the structure is preserved to high resolution (4 Å). The technique permits a detailed understanding of the processes involved in the orientation of these samples in a strong magnetic field and clarifies the long-range bi-axial properties of some fibres as seen by X-ray diffraction and optical microscopy.     

Nanostructure and elastic modulus of single trabecula in bovine cancellous bone

We aimed to investigate the elastic modulus of trabeculae using tensile tests and assess the effects of nanostructure at the hydroxyapatite (HAp) crystal scale on the elastic modulus. In the experiments, 18 trabeculae that were at least 3 mm in length in the proximal epiphysis of three adult bovine femurs were used. Tensile tests were conducted using a small tensile testing device coupled with microscopy under air-dried condition. The c-axis orientation of HAp crystals and the degree of orientation were measured by X-ray diffraction. To observe the deformation behavior of HAp crystals under tensile loading, the same tensile tests were conducted in X-ray diffraction measurements. The mineral content of specimens was evaluated using energy dispersive X-ray spectrometry. The elastic modulus of a single trabecula varied from 4.5 to 23.6 GPa, and the average was 11.5±5.0 GPa. The c-axis of HAp crystals was aligned with the trabecular axis and the crystals were lineally deformed under tensile loading. The ratio of the HAp crystal strain to the tissue strain (strain ratio) had a significant correlation with the elastic modulus (r=0.79; P<0.001). However, the mineral content and the degree of orientation did not vary widely and did not correlate with the elastic modulus in this study. It suggests that the strain ratio may represent the nanostructure of a single trabecula and would determine the elastic modulus as well as mineral content and orientation.     

Characteristics of mineral particles in the human bone/cartilage interface

Bone and cartilage consist of different organic matrices, which can both be mineralized by the deposition of nano-sized calcium phosphate particles. We have studied these mineral particles in the mineralized cartilage layer between bone and different types of cartilage (bone/articular cartilage, bone/intervertebral disk, and bone/growth cartilage) of individuals aged 54 years, 12 years, and 6 months. Quantitative backscattered electron imaging and scanning small-angle X-ray scattering at a synchrotron radiation source were combined with light microscopy to determine calcium content, mineral particle size and alignment, and collagen orientation, respectively. Mineralized cartilage revealed a higher calcium content than the adjacent bone (p<0.05 for all samples), whereas the highest values were found in growth cartilage. Surprisingly, we found the mineral platelet width similar for bone and mineralized cartilage, with the exception of the growth cartilage sample. The most striking result, however, was the abrupt change of mineral particle orientation at the interface between the two tissues. While the particles were aligned perpendicular to the interface in cartilage, they were oriented parallel to it in bone, reflecting the morphology of the underlying organic matrices. The tight bonding of mineralized cartilage to bone suggests a mechanical role for the interface of the two elastically different tissues, bone and cartilage.     

Biomedical applications of photon-induced X-ray fluorescence

An X-ray fluorescence method for in vitro analysis of trace elements is presented. The method is characterized by the use of an X-ray tube with secondary targets as a quasimonoenergetic radiation source, and by “infinitely thin” specimens. Different aspects have been examined in order to optimize the sensitivity of the method. It is extremely important to use secondary targets as pure as possible and collimators internally covered by the same element as the secondary target. It is also important to reduce the contribution at the XRF spectrum of photons scattered by the sample, by the sample support, and by the air. Preconcentration techniques can conveniently also be used to this purpose. In this work, biological samples are preconcentrated by reducing them to ash. Typical sensitivities obtained are in the order of 1–5 ng/cm² in a counting time of 10³ s for elements with atomic number ranging from 24 (chromium) to 40 (zirconium).     

Determination of protein orientation on surfaces with X-ray photoelectron spectroscopy

The use of X-ray photoelectron spectroscopy (XPS) for determination of the preferred orientation of proteins on metal surfaces is demonstrated. A myoglobin derivative in which a pentaamineruthenium (III) group is attached to a specific histidine (residue 81) is used for this purpose, with the Ru and Fe acting as a double marker. The relative intensities of the XPS photoelectron spectra for these two markers are shown to be a sensitive measure of orientation of the protein film. On both aluminum and indium-tin-oxide surfaces, XPS indicates that the Ru-Fe axis of the myoglobin derivative is preferentially oriented with the Ru furthest from the substrate.     

High-resolution structure of the photosynthetic Mn4Ca catalyst from X-ray spectroscopy

The application of high-resolution X-ray spectroscopy methods to study the photosynthetic water oxidizing complex, which contains a unique hetero-nuclear catalytic Mn4Ca cluster, is described. Issues of X-ray damage, especially at the metal sites in the Mn4Ca cluster, are discussed. The structure of the Mn4Ca catalyst at high resolution, which has so far eluded attempts of determination by X-ray diffraction, X-ray absorption fine structure (EXAFS) and other spectroscopic techniques, has been addressed using polarized EXAFS techniques applied to oriented photosystem II (PSII) membrane preparations and PSII single crystals. A review of how the resolution of traditional EXAFS techniques can be improved, using methods such as range-extended EXAFS, is presented, and the changes that occur in the structure of the cluster as it advances through the catalytic cycle are described. X-ray absorption and emission techniques (XANES and Kbeta emission) have been used earlier to determine the oxidation states of the Mn4Ca cluster, and in this report we review the use of X-ray resonant Raman spectroscopy to understand the electronic structure of the Mn4Ca cluster as it cycles through the intermediate S-states.     

Mesoscopic analysis of motion and conformation of cross-bridges

The orientation of a cross-bridge is widely used as a parameter in determining the state of muscle. The conventional measurements of orientation, such as that made by wide-field fluorescence microscopy, electron paramagnetic resonance (EPR) or X-ray diffraction or scattering, report the average orientation of 10¹²–10⁹ myosin cross-bridges. Under conditions where all the cross-bridges are immobile and assume the same orientation, for example in normal skeletal muscle in rigor, it is possible to determine the average orientation from such global measurements. But in actively contracting muscle, where a parameter indicating orientation fluctuates in time, the measurements of the average value provide no information about cross-bridge kinetics. To avoid problems associated with averaging information from trillions of cross-bridges, it is necessary to decrease the number of observed cross-bridges to a mesoscopic value (i.e. the value affected by fluctuations around the average). In such mesoscopic regimes, the averaging of the signal is minimal and dynamic behavior can be examined in great detail. Examples of mesoscopic analysis on skeletal and cardiac muscle are provided.     

A means for orienting flat cells in flow systems.

Flattened cells, such as red blood cells, epithelial cells, and sperm of many species, cause problems for fluorescence-activated cell analysis and sorting machines because the flow systems of such devices are unable to control the orientation of these cells as they flow past the detectors. For this reason, the fluorescence or scattered light measurements for identical cells may vary greatly. A flow geometry is here described that orients flat cells in a coaxial flow system so that each cell presents the same aspect to the observation device. A wedge-shaped exit on the sample injection tube in a coaxial flow system is sufficient to produce the desired orientation effect when used with low sample flow rates. Data is presented showing the effect of orientation of fixed chicken erythrocytes on histograms of small forward-angle light-scattering measurements.     

Liquid sample delivery techniques for serial femtosecond crystallography

X-ray free-electron lasers overcome the problem of radiation damage in protein crystallography and allow structure determination from micro- and nanocrystals at room temperature. To ensure that consecutive X-ray pulses do not probe previously exposed crystals, the sample needs to be replaced with the X-ray repetition rate, which ranges from 120 Hz at warm linac-based free-electron lasers to 1 MHz at superconducting linacs. Liquid injectors are therefore an essential part of a serial femtosecond crystallography experiment at an X-ray free-electron laser. Here, we compare different techniques of injecting microcrystals in solution into the pulsed X-ray beam in vacuum. Sample waste due to mismatch of the liquid flow rate to the X-ray repetition rate can be addressed through various techniques.     

Cell wall dimensions reign supreme: cell wall composition is irrelevant for the temperature signal of latewood density/blue intensity in Scots pine

Many microdensitometric techniques are available for deriving maximum latewood density (MXD), which is the state-of-the-art proxy parameter for local to hemispheric-scale temperature reconstructions of the last millennium. Techniques based on X-ray radiation and visible light reflection, such as “blue intensity” (BI), integrate both the density/composition and the dimensions of the cell walls to derive microdensitometric data. In contrast, the dendroanatomical technique relies only on the dimensions of the cell walls. It is therefore possible to isolate cell wall variables by subtracting data derived using the dendroanatomical technique from data derived using X-ray and BI-based techniques.In this study, we explore differences in well-replicated data from parallel X-ray, BI, and dendroanatomical measurements of temperature-sensitive Pinus sylvestris trees from northern Finland. We aim to determine whether cell wall density is critical to the success of X-ray-based MXD, and whether the BI-based parameter counterpart, here termed MXBI, contains useful information about the composition of the cell wall (specifically the lignin).Our results indicate that cell wall density and cell wall BI have no relevant influence on MXD and MXBI measurements. Even in years with severely reduced lignification, identified as so-called “blue rings”, dendroanatomical MXD (aMXD) measurements do not deviate significantly from their MXD or MXBI counterparts. Moreover, derived chronologies of cell wall density and cell wall BI contain no significant climate signals when correlated with local climate. Maximum latewood density of conifers can thus be obtained without bias using the dendroanatomical technique. Because lignin content appears to play a negligible role for cell wall BI, the cell wall BI likely presents the biggest challenge when producing unbiased MXBI data. This is because BI data is notorious for cell wall color distortion across the heartwood and sapwood, and between living wood and dead wood, and may therefore distort the otherwise strong link with wood density on multidecadal scales.     

Structural organization of collagen in Metridium senile

The structure and distribution of collagen fibres in Metridium senile mesoglea has been investigated using high and small angle X-ray diffraction techniques on conventional and synchrotron sources. The mesoglea collagen axial spacing appears very close to that of rat tail tendon, which is at variance with the values previously obtained from electron microscopic observations. The different intensity distribution of the small angle X-ray diffraction maxima recorded for mesoglea and rat tail tendon indicates a different distribution of electron density inside the repeating period. Furthermore the absence of the first order, the weak second order and the strong third and sixth orders in the patterns of wet and dry mesogleal collagen could explain that only a periodicity of 20–22 nm corresponding to one-third of the true axial period observed in the electron micrographs. The analysis of the reflections at 0.29 and 1.1–1.4 nm characteristics of the collagen molecular structure have been used to determine the distribution and orientation of the collagen fibres in unstretched and stretched samples     

X-ray interference studies of crossbridge action in muscle contraction: evidence from quick releases

We have used a high-resolution small angle X-ray scattering system, together with a high-performance CCD camera, on the BioCAT beamline at the APS synchrotron radiation facility at the Argonne National Laboratory, to study X-ray interference effects in the meridional reflections generated by the arrays of myosin crossbridges in contracting muscle. These give information about axial movements of the myosin heads during contraction with sub-nanometer resolution. Using whole intact muscle preparations (frog sartorius) we have been able to record the detailed behavior of M3 (the first order meridional reflection from the myosin crossbridges, at 14.56 nm) at each of a number of quick releases of increasing magnitude, on the same specimen, and at the same time make similar measurements on higher order myosin meridional reflections, particularly M6. The latter provides information about the dispersion of lever arm angles of the actin-attached myosin heads. The observations show that in isometric contraction the lever arm angles are dispersed through +/- 20-25 degrees on either side of a mean orientation that is about 60 degrees away from their orientation at the end of the working stroke: and that they move towards that orientation in synchronized fashion, with constant dispersion, during quick releases. The relationship between the shift in the interference fringes (which measures the shift of the myosin heads scattering mass towards the center of the sarcomere, and the changes in the total intensity of the reflections, which measures the changes in the axial profile of the heads, is consistent with the tilting lever arm mechanism of muscle contraction. Significant fixed contributions to the meridional reflections come from unattached myosin heads and from backbone components of the myosin filaments, and the interaction of these with the contributions from actin-attached myosin heads determines the behavior of these reflections.     

Observation of correlated X-ray scattering at atomic resolution

Tools to study disordered systems with local structural order, such as proteins in solution, remain limited. Such understanding is essential for e.g. rational drug design. Correlated X-ray scattering (CXS) has recently attracted new interest as a way to leverage next-generation light sources to study such disordered matter. The CXS experiment measures angular correlations of the intensity caused by the scattering of X-rays from an ensemble of identical particles, with disordered orientation and position. Averaging over 15 496 snapshot images obtained by exposing a sample of silver nanoparticles in solution to a micro-focused synchrotron radiation beam, we report on experimental efforts to obtain CXS signal from an ensemble in three dimensions. A correlation function was measured at wide angles corresponding to atomic resolution that matches theoretical predictions. These preliminary results suggest that other CXS experiments on disordered ensembles—such as proteins in solution—may be feasible in the future.     

Opportunities for biomineralization research using multiscale computed X-ray tomography as exemplified by bone imaging

Biominerals typically have complex hierarchical structures traversing many length scales. This makes their structural characterization complicated, since it requires 3D techniques that can probe full specimens at down to nanometer-resolution, a combination that is difficult – if not impossible – to achieve simultaneously. One challenging example is bone, a mineralized tissue with a highly complex architecture that is replete with a network of cells. X-ray computed tomography techniques enable multiscale structural characterization through the combination of various equipment and emerge as promising tools for characterizing biominerals. Using bone as an example, we discuss how combining different X-ray imaging instruments allow characterizing bone structures from the nano- to the organ-scale. In particular, we compare and contrast human and rodent bone, emphasize the importance of the osteocyte lacuno-canalicular network in bone, and finally illustrate how combining synchrotron X-ray imaging with laboratory instrumentation for computed tomography is especially helpful for multiscale characterization of biominerals.