The resolution parameter Sigma (A) is currently used for evaluating the degree of similarity between a model and the target structure. Here, quasi-Wilson distributions are used to represent the local statistics of the normalized amplitudes both for the target and for the model structure. The study uses the joint probability distribution approach to provide (i) a description of the statistical properties of the Sigma (A) parameter; (ii) a deeper insight into the role, for the Sigma (A) estimate, of the high-order moments of the target and of the model structure-factor distributions; and (iii) new statistical formulas for estimating Sigma (A) . The theoretical results have been checked using test proteins.

The paper shows how a table top superbright microfocus laboratory X-ray source and an innovative restoring-data algorithm, used in combination, allow to analyze the super molecular structure of soft matter by means of Small Angle X-ray Scattering ex-situ experiments. The proposed theoretical approach is aimed to restore diffraction features from SAXS profiles collected from low scattering biomaterials or soft tissues, and therefore to deal with extremely noisy diffraction SAXS profiles/maps. As biological test cases we inspected: i) residues of exosomes' drops from healthy epithelial colon cell line and colorectal cancer cells; ii) collagen/human elastin artificial scaffolds developed for vascular tissue engineering applications; iii) apoferritin protein in solution. Our results show how this combination can provide morphological/structural nanoscale information to characterize new artificial biomaterials and/or to get insight into the transition between healthy and pathological tissues during the progression of a disease, or to morphologically characterize nanoscale proteins, based on SAXS data collected in a room-sized laboratory.

In this chapter, we will focus on a specific X-ray-based technique among those employed in surface science and which is especially suitable for the study of self-assembled nanocrystals: Grazing Incidence Small Angle X-ray Scattering (GISAXS). We will first introduce the main field of investigation considered herein, with basic notions of X-ray scattering from surfaces, and then address basic concepts about GISAXS. Finally, we will describe a few relevant examples of studies, of nanostructured architectures, through ex situ and in situ experiments of grazing incidence X-ray scattering. This manuscript is focused on the former, showing that they can be performed by using laboratory instruments. In situ investigations still need synchrotron radiation sources in most cases; therefore, only a few examples selected from the literature are reported here, for the sake of completeness. The experiments described are mainly performed in the small angle range, providing information on the size and shape of nanocrystals, together with their spatial arrangement. Both 2D and 3D architectures are considered. In particular, GISAXS measurements of 2D superlattices of nano-octapods, performed both at a third generation synchrotron beamline and with a table-top set-up, are compared; the employed table-top set-up is described in a dedicated paragraph. Further examples of grazing incidence studies as performed by the authors with a table-top set-up are reported: a GISAXS study of 3D iron oxide nanocrystal superlattices, showing the importance of modelling in order to obtain structural information from data; a combined small/wide angle scattering (GISAXS/GIWAXS) study of 3D PbS nanocrystal superlattices; and a GIWAXS study of P3HT nanofibres, showing how the ordering at the molecular and atomic length scales can be obtained by exploring different angular ranges in the same grazing incidence geometry. Finally, selected examples of in situ GISAXS studies, performed with synchrotron radiation sources, are described.

Here we describe a new TEM-based coherent diffraction imaging (CDI) method to achieve sub-ångström resolution in lattice images of nanoparticles. The experiments were performed by using a TEM/STEM JEOL 2010F UHR (objective lens spherical aberration coefficient of (0.47±0.01) mm) with resolution at optimum defocus of 0.19nm and equipped by Schottky cathode. The experiments for CDI require the acquisition of HRTEM image and diffraction from the same region of the sample illuminated by a coherent electron probe. The experimental nano-diffraction must be recorded with the beam coherence length larger than the region to be imaged according to the oversampling requirements [1]. A new efficient iterative phase retrieval algorithm [2] based on pioneering work of Gerchberg and Saxton [3] has been developed and successfully applied. Figure 1 is an example of phase retrieval of a TiO2 particle at a resolution of 70 pm that allows to distinguish the O atoms appreciating subtle alterations in the unit cell structure of the nano-crystals, which would not be otherwise detectable by conventional HRTEM. Such structural deviations could be at the origin of peculiar size-dependent physical-chemical properties of the concerned oxide material in the nanoscale regime [2].

The paper focuses on the development of electron coherent diffraction imaging intransmission electron microscopy, made in the, approximately, last ten years in our collaborativeresearch group, to study the properties of materials at atomic resolution, overcoming the limitationsdue to the aberrations of the electron lenses and obtaining atomic resolution images, in whichthe distribution of the maxima is directly related to the specimen atomic potentials projected ontothe microscope image detector. Here, it is shown how augmented coherent diffraction imagingmakes it possible to achieve quantitative atomic resolution maps of the specimen atomic species,even in the presence of low atomic number atoms within a crystal matrix containing heavy atoms.This aim is achieved by: (i) tailoring the experimental set-up, (ii) improving the experimental data byproperly treating parasitic diffused intensities to maximize the measure of the significant information,(iii) developing efficient methods to merge the information acquired in both direct and reciprocalspaces, (iv) treating the dynamical diffused intensities to accurately measure the specimen projectedpotentials, (v) improving the phase retrieval algorithms to better explore the space of solutions.Finally, some of the future perspectives of coherent diffraction imaging in a transmission electronmicroscope are given.

The dynamism of proteins is central to their function, and several proteins have beendescribed as flexible, as consisting of multiple domains joined by flexible linkers, and even asintrinsically disordered. Several techniques exist to study protein structures, but small angle X-rayscattering (SAXS) has proven to be particularly powerful for the quantitative analysis of such flexiblesystems. In the present report, we have used SAXS in combination with X-ray crystallographyto highlight their usefulness at characterizing flexible proteins, using as examples two proteinsinvolved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein,Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightlyelongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets.As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome)protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutatedtarget in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble ofthree main conformations, with the N- and C-terminal ends adopting different orientations withrespect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis.

Scanning small and wide angle X-ray scattering (scanning SWAXS) experiments were performed on healthy and pathologic human bone sections. Via crystallographic tools the data were transformed into quantitative images and as such compared with circularly polarized light (CPL) microscopy images. SWAXS and CPL images allowed extracting information of the mineral nanocrystalline phase embedded, with and without preferred orientation, in the collagen fibrils, mapping local changes at sub-osteon resolution. This favorable combination has been applied for the first time to biopsies of dwarfism syndrome and Paget's disease to shed light onto the cortical structure of natural bone in healthy and pathologic sections.

The metal-binding ability of human ubiquitin (hUb) has been probed towards a selection of biologically relevant metal-ions and complexes. Different techniques have been used to obtain crystals suitable for crystallographic analysis. In a first type of experiments, crystals of hUb have been soaked in solutions containing copper(II) acetate and two metallodrugs: Zeise's salt and cisplatin. Zeise's salt is used in a test for hepatitis while cisplatin is one of the most powerful anticancer drugs in clinical use. Zeise's salt reacts smoothly with hUb crystals affording an adduct with three platinum residues per protein molecule: Pt3-hUb. In contrast, copper(II) acetate and cisplatin were found to be unreactive for contact times up to one hour and to cause degradation of the hUb crystals for longer times. In a second type of experiments, human ubiquitin was co-crystallized with solutions of copper(II) and zinc(II) acetate and with cisplatin. Zinc(II) acetate gives, at low metal-to-protein molar ratio, crystals containing one metal ion per three molecules of protein, Zn-hUb3 (already reported in a previous work), while at high metal-to-protein ratio (70:1) gives crystals containing three Zn(II) ions per protein molecule: Zn3-hUb. In contrast, once again, copper(II) acetate and cisplatin, even at low metal-to-protein ratio, do not give crystalline material. In the soaking experiment, Zeise's anion leads to simultaneous platination of His68, Met1 and Lys6. Present and previous results of co-crystallization experiments performed with zinc(II) and other group-12 metal ions allow to reach a comprehensive understanding of the metal-ion binding properties of hUb with His68 as main anchoring site, followed by Met1 and Glu18. The amount of metal ion, with respect to that of the protein, appears to be a relevant parameter influencing the crystal packing.

The title compound, C24H25NO3·2CH3OH, which crystallized as a methanol disolvate, has applications as a PET radiotracer in the early diagnosis of Alzheimer's disease. The dihedral angle between the biphenyl rings is 8.2 (2)° and the heterocyclic ring adopts a half-chair conformation with the N atom adopting a pyramidal geometry (bond-angle sum = 327.6°). The C atoms of both meth­oxy groups lie close to the plane of their attached ring [deviations = 0.107 (6) and 0.031 (6) Å]. In the crystal, the components are linked by O-H...O and O-H...N hydrogen bonds, generating [010] chains. C-H...O inter­actions are also observed.

We present a method to treat spurious intensities in electron diffraction experiments. Coherent electron diffraction imaging requires proper data reduction before the application of phase retrieval algorithms. The presence of spurious intensities in the electron diffraction patterns makes the data reduction complicated and time consuming and jeopardizes the application of mathematical constraints to maximize the information that can be extracted from the experimental data. Here we show how the experimental diffraction patterns can be treated to remove the unwanted artifacts without corrupting the genuine intensities scattered by the specimen. The resulting diffraction patterns are suitable for the application of further processes and constraints aimed at deriving fundamental structural information by applying phase retrieval algorithms or other approaches capable of deriving quantitative atomic resolution information about the specimen structure.

The aim of this work was to investigate the structural features of type I collagen isoforms and collagen-based films at atomic and molecular scales, in order to evaluate whether and to what extent different protocols of slurry synthesis may change the protein structure and the final properties of the developed scaffolds. Wide Angle X-ray Scattering data on raw materials demonstrated the preferential orientation of collagen molecules in equine tendon-derived collagens, while randomly oriented molecules were found in bovine skin collagens, together with a lower crystalline degree, analyzed by the assessment of FWHM (Full Width at Half Maximum), and a certain degree of salt contamination. WAXS and FT-IR (Fourier Transform Infrared) analyses on bovine collagen-based films, showed that mechanical homogenization of slurry in acidic solution was the treatment ensuring a high content of super-organization of collagen into triple helices and a high crystalline domain into the material. In vitro tests on rat Schwannoma cells showed that Schwann cell differentiation into myelinating cells was dependent on the specific collagen film being used, and was found to be stimulated in case of homogenization-treated samples. Finally DHT/EDC crosslinking treatment was shown to affect mechanical stiffness of films depending on collagen source and processing conditions.

Since its early days electron microscopy has represented a splendid way to study the interactions between charged particles and electromagnetic fields. These efforts produced a flexible and powerful tool to investigate the properties of the matter at the highest spatial resolution. One of the main reason for the high resolution achievable in electron microscopy is related to the small wavelength, l, associated to high-energy electrons, i.e. for 200 keV electrons l = 2.5pm. Unfortunately, the quality of the electron lenses is relatively poor and the diffraction limit is still unreached. The proof of Otto Scherzer in 1936 that skilful lens design could never eliminate the spherical and chromatic aberration of rotationally symmetric lenses [1] promote the efforts of the scientific community to find a gateway. Since that time many attempts were made from one side to find a way to correct spherical and chromatic aberrations via hardware and from the othe r to find different approaches to recovery the information lost in TEM imaging. In the latter approaches it might be mentioned, for example, the invention of Gabor of the holography [2] or the through focal reconstruction of the exit phase wave in high resolution TEM (HRTEM) [3]. Very recently, electron optical devices capable of correcting spherical aberration are available and sub-ångström resolution have been achieved by HRTEM [4] and incoherent imaging in scanning transmission electron microscopy (STEM) by using high angle annular dark field detector [5]. Nevertheless, the diffraction limit in electron microscopy is still not reached. Coherent diffraction imaging (CDI) is an approach combining real and reciprocal space information to achieve images in principle limited only by diffraction limit [6].

Organic capped Au nanoparticles (NPs) and PbS quantum dots (QDs), synthesized with high control on size and size distribution, were used as building blocks for fabricating solid crystals by solvent evaporation. The superlattice formation process for the two types of nano-objects was investigated as a function of concentration by means of electron microscopy and X-ray techniques. The effect of building block composition, size, geometry, and concentration and the role of the organic coordinating molecules was related to the degree of order in the superlattices. A convenient combination of different complementary X-ray techniques, namely in situ and ex situ GISAXS and GIWAXS, allowed elucidating the most reliable signatures of the superlattices at various stages of the self-assembly process, since their early stage of formation and up to few months of aging. Significantly different assembly behaviour was assessed for the two types of NPs, clearly explained on the basis of their chemical composition, ultimately reflecting on the assembling process and on the final structure characteristics.

The Shwachman-Diamond Syndrome (SDS) is a disorder arising from mutations in thegenes encoding for the Shwachman-Bodian-Diamond Syndrome (SBDS) protein and the GTPaseknown as Elongation Factor Like-1 (EFL1). Together, these proteins remove the anti-associationfactor eIF6 from the surface of the pre-60S ribosomal subunit to promote the formation of matureribosomes. SBDS missense mutations can either destabilize the protein fold or affect surface epitopes.The molecular alterations resulting from the latter remain largely unknown, although some evidencesuggest that binding to EFL1 may be affected. We further explored the effect of these SBDS mutationson the interaction with EFL1, and showed that all tested mutations disrupted the binding to EFL1.Binding was either severely weakened or almost abolished, depending on the assessed mutation.In higher eukaryotes, SBDS is essential for development, and lack of the protein results in earlylethality. The existence of patients whose only source of SBDS consists of that with surface missensemutations highlights the importance of the interaction with EFL1 for their function. Additionally,we studied the interaction mechanism of the proteins in solution and demonstrated that bindingconsists of two independent and cooperative events, with domains 2-3 of SBDS directing the initialinteraction with EFL1, followed by docking of domain 1. In solution, both proteins exhibited largeflexibility and consisted of an ensemble of conformations, as demonstrated by Small Angle X-rayScattering (SAXS) experiments.

Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two ?-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first ?-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.

Bovine cornea was studied with scanning small-angle X-ray scattering (SAXS) microscopy, by using both synchrotron radiation and a microfocus laboratory source. A combination of statistical (adaptive binning and canonical correlation analysis) and crystallographic (pair distribution function analysis) approaches allowed inspection of the collagen lateral packing of the supramolecular structure. Results reveal (i) a decrease of the interfibrillar distance and of the shell thickness around the fibrils from the periphery to the center of the cornea, (ii) a uniform fibril diameter across the explored area, and (iii) a distorted quasi-hexagonal arrangement of the collagen fibrils. The results are in agreement with existing literature. The overlap between laboratory and synchrotron-radiation data opens new perspectives for further studies on collagen-based/engineered tissues by the SAXS microscopy technique at laboratory-scale facilities.Bovine cornea was studied with scanning small-angle X-ray scattering microscopy, by using both synchrotron radiation and a microfocus laboratory source. The supramolecular structure of the collagen fibers is explored thanks to the combination of statistical (adaptive binning and canonical correlation analysis) and crystallographic (pair distribution function analysis) approaches.

Electron diffractive imaging (EDI) relies on combining information from the high-resolution transmission electron microscopy image of an isolated kinematically diffracting nano-particle with the corresponding nano-electron diffraction pattern. Phase-retrieval algorithms allow one to derive the phase, lost in the acquisition of the diffraction pattern, to visualize the actual atomic projected potential within the specimen at sub-ångström resolution, overcoming limitations due to the electron lens aberrations. Here the approach is generalized to study extended crystalline specimens. The new technique has been called keyhole electron diffractive imaging (KEDI) because it aims to investigate nano-regions of extended specimens at sub-ångström resolution by properly confining the illuminated area. Some basic issues of retrieving phase information from the EDI/KEDI measured diffracted amplitudes are discussed. By using the generalized Shannon sampling theorem it is shown that whenever suitable oversampling conditions are satisfied, EDI/KEDI diffraction patterns can contain enough information to lead to reliable phase retrieval of the unknown specimen electrostatic potential. Hence, the KEDI method has been demonstrated by simulations and experiments performed on an Si crystal cross section in the [112] zone-axis orientation, achieving a resolution of 71 pm.

For the final step of the maturation of the ribosome, the nascent 40S and 60S subunits are exported from the nucleus to the cell cytoplasm. To prevent premature association of these ribosomal subunits, eukaryotic initiation factor 6 (eIF6) binds the 60S subunit within the nucleus. Its release in the cytoplasm requires the interaction of EFL1 and SDBS proteins. In Shwachman-Diamond syndrome (SDS), a defective SDBS protein prevents eIF6 eviction, inhibiting its recycle to the nucleus and subsequent formation of the active 80S ribosome. Objective This study aims to identify the molecular basis of an SDS-like disease, manifested by pancytopenia, exocrine pancreatic insufficiency and skeletal abnormalities in six patients from three unrelated families. Methods Whole exome analysis was used for mutation identification. Fluorescence microscopy studies assessed the localisation of Tif6-GFP, the yeast eIF6 homologue, in yeast WT and mutant cells. Human and yeast EFL1 proteins, WT and mutants, were expressed in Saccharomyces cerevisiae BCY123 strain, and circular dichroism and small-angle X-ray scattering were used to assess the folding and flexibility of these proteins. Green malachite colorimetric assay was performed to determine the GTPase activity of WT and Efl1 mutants. Results Four patients were homozygous for p.R1095Q variant and two patients were homozygous for p.M882K variant in EFL1. Residue R1095 and M882 are conserved across species. Neither the GTPase activity of the mutant proteins nor its activation by the SDBD protein or the 60S ribosomal subunit were affected. Complementation of efl1? yeast cells with the EFL1 mutants rescued the slow growth phenotype. Nonetheless, Tif6-GFP was relocalised to the cytoplasm in mutant yeast cells in contrast to its nuclear localisation in WT cells. Conclusions Mutations in EFL1 clinically manifest as SDS-like phenotype. Similar to the molecular pathology of SDS, mutant EFL1 proteins do not promote the release of cytoplasmic Tif6 from the 60S subunit, likely preventing the formation of mature ribosomes.

Transition-metal alkane-thiolates (i.e., organic salts with formula Me(SR)x,where R is a linear aliphatic hydrocarbon group, -CnH2n+1) undergo a thermolysis reactionat moderately low temperatures (close to 200 °C), which produces metal atoms or metalsulfide species and an organic by-product, disulfide (RSSR) or thioether (RSR) molecules,respectively. Alkane-thiolates are non-polar chemical compounds that dissolve inmost techno-polymers and the resulting solid solutions can be annealed to generatepolymer-embedded metal or metal sulfide clusters. Here, the preparation of silver and goldclusters embedded into amorphous polystyrene by thermolysis of a dodecyl-thiolateprecursor is described in detail. However, this chemical approach is quite universal and alarge variety of polymer-embedded metals or metal sulfides could be similarly prepared.

Imines represent an important class of molecules that are widely used (as intermediates) in the synthesis of a number of Nheterocycliccompounds [1]. These molecules are a product of reversible condensation of an amine and an aldehyde formed through the dynamic bond (a thermodynamically favored product). General procedure for the synthesis of new carbazole substituted imines: 14.26 mmol of 3-amino-9-ethylcarbazole were dissolved in ethanol (30 ml) and added to 14.26 mmol Na2CO3 and 14.26 mmol aldehydes derivates. The reaction mixture wasthen refluxed for 24 hours at 90°C. The solution was extracted with AcOEt with an aqueous phase. The organic phase was washed three times with water, dried with anhydrous magnesium sulphate, and evaporated under reduced pressure. The X-ray diffraction (XRD) data for the imines samples were collected using a Bruker-Nonius KappaCCD single-Crystal diffractometer (Mo Ka radiation, l = 0.71073 Å), installed at IC-CNR, Bari, ItalyThe structures were solved by Direct Methods implemented in SIR2014 [2] and refined by SHELXL2014 [3] using a full-Matrix least-squares method based on F2. The non-hydrogen atoms were refined anisotropically. All the structures were characterized by non-negligible hydrogen bonds.In Figure the molecular structure of one of the imine compounds [N-(5-nitrobenzylidene)-9-ethyl-9H-carbazol-3-amine (C21H17N3O2)] is shown.The steps of imines synthesis and the main crystallographic results of the single crystal XRD study of the imines compounds will be described.

Two new computational methods dedicated to neutron crystallography, called n-FreeLunch and DNDM-NDM, have been developed and successfully tested. The aim in developing these methods is to determine hydrogen and deuterium positions in macromolecular structures by using information from neutron density maps. Of particular interest is resolving cases in which the geometrically predicted hydrogen or deuterium positions are ambiguous. The methods are an evolution of approaches that are already applied in X-ray crystallography: extrapolation beyond the observed resolution (known as the FreeLunch procedure) and a difference electron-density modification (DEDM) technique combined with the electron-density modification (EDM) tool (known as DEDM-EDM). It is shown that the two methods are complementary to each other and are effective in finding the positions of H and D atoms in neutron density maps.

Fine powders composed of tellurium grains of average size <10 nm were produced by dry vibration milling combined with liquid-phase sedimentation techniques, starting from polycrystalline powders with average grain diameter of ca. 30 ?m. Nanocomposite films were obtained by binding the nanosized tellurium grains with poly(methyl methacrylate). Raman spectroscopy revealed that the films were based on the coexistence of tellurium and tellurium oxide crystalline phases due to a partial oxidation in air of the grains. The optical measurements of the fabricated material showed that the absorbance was nearly constant in the 310-2200 nm range and that a typical UV absorption peak of the nanostructured tellurium was centered at around 260 nm. An extensive characterization of the photoconductivity properties was carried out by illuminating the tellurium-poly(methyl methacrylate) films with white light or radiations of different spectral composition selected from the UV-Vis-NIR region. Data analysis has allowed to demonstrate that the photoresponse is closely related to the optical absorption and is independent of the spectral composition of the incident radiation in the wavelength range from 310 to 2200 nm, while the photocurrent increases linearly as a function of the optical power density over about three orders of magnitude.

In the field of inorganic protective substances of porous calcareous materials alternatives to nanolime[1], [2] the alcoholic dispersion of colloidal strontium hydroxide may show some effectiveness[3], [4] due to the peculiar chemical-physical features and compatibility with the crystal structure of calcareous. In this work the development of a rapid (about 6h) "bottom-up" procedure of the dispersion has been realized. The colloidal particles have an average diameter of 200 nm and PdI (Poly dispersion Index) is equal to 0.144 (mono-disperse). The colloidal particles show a good kinetic stability (600 nm) with a zeta potential of +28mV. Tests conducted in the laboratory on a glass slide show that the transformation of strontium hydroxide into the corresponding carbonate, in air and under controlled humidity and temperature conditions (21°C, 51% R.H.), is complete in 8~10 days. The characterization is performed by powder X-Ray Diffraction[5] and Dynamic Light Scattering (D.L.S.)[6].

Research on composite materials is facing, among others, the challenging task of incorporating nanocrystals, and their superstructures, in polymer matrices. Electron microscopy can typically image nanometre-scale structures embedded in thin polymer films, but not in films that are micron size thick. Here, X-ray Ptychography was used to visualize, with a resolution of a few tens of nanometers, how CdSe/CdS octapod-shaped nanocrystals self-assemble in polystyrene films of 24 ± 4 ?m, providing a unique means for non-destructive investigation of nanoparticles distribution and organization in thick polymer films.

The nanoscale structural order of air-dried rat-tail tendon is investigated using small-angle X-ray scattering (SAXS). SAXS fiber diffraction patterns were collected with a superbright laboratory microsource at XMI-LAB [Altamura, Lassandro, Vittoria, De Caro, Siliqi, Ladisa & Giannini (2012). J. Appl. Cryst. 45, 869-873] for increasing integration times (up to 10 h) and a novel algorithm was used to estimate and subtract background, and to deconvolve the beam-divergence effects. Once the algorithm is applied, the peak visibility improves considerably and reciprocal space information up to the 22nd diffraction order is retrieved (q = 0.21 angstrom(-1), d = 29 angstrom) for an 8-10 h integration time. The gain in the visibility is already significant for patterns collected for 0.5 h, at least on the more intense peaks. This demonstrates the viability of detecting structural changes on a molecular/nanoscale level in tissues with state-of-the-art laboratory sources and also the technical feasibility to adopt SAXS fiber diffraction as a future potential clinical indicator for disease.

Films obtained by casting, starting from conventional emulsions (CE), nanoemulsions (NE) or their gels, whichled to different structures, with the aim of explore the relationship between structure and physical properties,were prepared. Sodium caseinate was used as the matrix, glycerol as plasticizer, glucono-delta-lactone asacidulant to form the gels, and TiO2 nanoparticles as reinforcement to improve physical behavior. Structuralcharacterization was performed by SAXS and WAXS (Small and Wide Angle X-ray Scattering, respectively),combined with confocal and scanning electron microscopy. The results demonstrate that the incorporation of thelipid phase does not notably modify the mechanical properties of the films compared to solution films. Filmsfrom NE were more stable against oil release than those from CE. Incorporation of TiO2 improved mechanicalproperties as measured by dynamical mechanical analysis (DMA) and uniaxial tensile tests. TiO2 macroscopicspatial distribution homogeneity and the nanostructure character of NE films were confirmed by mapping the qdependentscattering intensity in scanning SAXS experiments. SAXS microscopies indicated a higher intrinsichomogeneity of NE films compared to CE films, independently of the TiO2 load. NE-films containing structureswith smaller and more homogeneously distributed building blocks showed greater potential for food applicationsthan the films prepared from sodium caseinate solutions, which are the best known films.

Osteoarthritis (OA), among other bone pathologies, is expected to determine supramolecular changes at the level of the mineralized collagen fiber. In a proof-of-principle study, bone biopsies were collected from six coxarthritis-affected patients, aged 62-87 years, during hip prosthesis implant surgery, sliced down to 100 mm-thick tissues, and investigated using scanning small-angle and wide-angle X-ray scattering (SAXS and WAXS) transmission microscopy. A multimodal imaging evaluation of the SAXS and WAXS data, combined with principal component and canonical correlation analyses, allowed the transformation of the raw data into microscopy images and inspection of the nanoscale structure of the mineralized collagen fibers across mm 2 tissue areas. The combined scanning SAXS and WAXS microscopy is shown to be a suitable choice for characterizing and quantifying the nanostructural properties of collagen over extended areas. The results suggest the existence of a correlation between age and cross-linking-induced rigidity of collagen fibers.

This study is aimed at investigating the structure of a scaffold made of bovine gelatin and hydroxyapatite for bone tissue engineering purposes. In particular, the detailed characterization of such a material has a great relevance because of its application in the healing process of the osteochondral defect that consists of a damage of cartilage and injury of the adjacent subchondral bone, significantly compromising millions of patient's quality of life. Two different techniques exploiting X-ray radiation, with table-top setups, are used: microtomography (micro-CT) and microdiffraction. Micro-CT characterizes the microstructure in the three dimensions at the micrometer scale spatial resolution, whereas microdiffraction provides combined structural/morphological information at the atomic and nanoscale, in two dimensional microscopy images with a hundred micrometer spatial resolution. The combination of these two techniques allowed an appropriate structural characterization for the purpose of validating the engineering approach used for the realization of the hydroxyapatite gradient across the scaffold, with properties close to the natural model.

We crystallised SdsA, a sodium dodecyl sulphate hydrolase, from Pseudomonas aeruginosa in three different crystal polymorphs and determined their three-dimensional structure. The different polymorphs present different crystal packing habits. One of the polymorphs suggests the existence of a tetramer, an oligomeric state not observed previously, while the crystal packing of the remaining two polymorphs obstructs the active site entrance but stabilizes flexible regions of the protein. Non-conventional crystallisation methods that minimise convection such as counter-diffusion in polyvinyl alcohol gel coupled with the influence of a 500 MHz (10.2 Tesla) magnetic field were necessary to isolate the poorest diffracting polymorph and increase its internal order to determine its structure by X-ray diffraction. Our results show the effectiveness of non-conventional crystallographic methods to isolate different crystal polymorphs.

Nanocomposites based on colloidal CdSe nanocrystals (NCs) and a poly(styrene-co-4-vinylpyridine), able to specifically coordinate the NC surface, have been designed and prepared. For first time, the polymer synthesis has been performed by using 2,2,5-tri-methyl-4-phenyl-3-azahexane-3-nitroxide as a mediator, increasing the percentage of 4-vinylpyridine monomeric unit, thus obtaining a random copolymer. The nanocomposite properties have been investigated as a function of NC surface chemistry and copolymer composition, by means of spectroscopic, morphological and structural characterization techniques. An improved uniformity of NC dispersion in the nanocomposite has been found at increased percentage of 4-vinylpyridine in the copolymer. The improved NC dispersion in the nanocomposite films has been discussed in terms of the ability of the copolymer to act as a multivalent ligand. The reported results offer a valuable contribution toward the design and the fabrication of innovative nanocomposite material, formed of copolymers and colloidal NCs, specifically suited for energy conversion applications.

Ribosome biogenesis is closely linked to the cell growth and proliferation. Dysregulation of this process causes several diseases collectively known as ribosomopathies. One of them is the Shwachman-Diamond Syndrome, and the SBDS protein mutated in this disease participates with EFL1 in the cytoplasmic maturation of the 60S subunit. Recently, we have shown that the interaction of EFL1 with SBDS resulted in a decrease of the Michaelis-Menten constant (KM) for GTP and thus SBDS acts as a GEF for EFL1 (1). Subsequent studies demonstrated that SBDS greatly debilitates the interaction of EFL1 with GDP without altering that for GTP. The interaction of EFL1 alone or in complex with SBDS to guanine nucleotides is followed by a conformational rearrangement. Understanding the molecular strategy used by SBDS to disrupt the binding of EFL1 for GDP and the associated conformational changes will be key to understand their mode of action and alterations occurring in the disease. The structure of the GTPase EFL1 is not known and its crystallization has been unsuccessful at least in our hands. In this study, we aim to show the conformational changes resulting from the interactions between EFL1 and its binding partners, the SBDS protein and the guanine nucleotides using SAXS technique (2, 3). SAXS will provide structural information of the proteins and their conformational changes (4). For the SAXS data analysis we have built models of EFL1 using by EF-2 as homology template and of SBDS using the crystal structures of the archaea orthologues.

SUNBIM (Supramolecular & SUbmolecular Nano & Bio Materials X-ray IMaging Project) is a suite of integrated programs developed, in collaboration with Rigaku Innovative Technologies, to treat Small and Wide Angle X-ray Scattering data, collected either in transmission geometry (SAXS/WAXS) or in reflection geometry (GISAXS/GIWAXS). In addition, a specific routine to collect and analyze data in SAXS scanning transmission microscopy has been developed as additional tool to investigate tissues or material science samples through a focused X-ray beam which is used to raster scan a specimen while acquiring SAXS scattering patterns with a 2D detector. Indeed, a first-generation-synchrotron-class FrE+ SuperBright Rigaku microsource, coupled to a three pinhole S-MAX3000 camera, was recently installed at the X-ray MicroImaging Laboratory (XMI-L@b) and used with success in SAXS/WAXS/GISAXS/GIWAXS experiments (De Caro et al, 2012; 2013)and for SAXS scanning microscopy (Altamura et al, 2012; Giannini et al, 2013).

SUNBIM (supramolecular and submolecular nano- and biomaterials X-rayimaging) is a suite of integrated programs which, through a user-friendlygraphical user interface, are optimized to perform the following: (i) q-scalecalibration and two-dimensional ! one-dimensional folding on small- andwide-angle X-ray scattering (SAXS/WAXS) and grazing-incidence SAXS/WAXS (GISAXS/GIWAXS) data, also including possible eccentricity correctionsfor WAXS/GIWAXS data; (ii) background evaluation and subtraction,denoising, and deconvolution of the primary beam angular divergence onSAXS/GISAXS profiles; (iii) indexing of two-dimensional GISAXS frames andextraction of one-dimensional GISAXS profiles along specific cuts; (iv) scanningmicroscopy in absorption and SAXS contrast. The latter includes collection oftransmission and SAXS data, respectively, in a mesh across a mm2 area,organization of the as-collected data into a single composite image oftransmission values or two-dimensional SAXS frames, analysis of the composeddata to derive the absorption map and/or the spatial distribution, andorientation of nanoscale structures over the scanned area.

XTOP 2018 - the 14th Biennial Conference on High-Resolution X-Ray Diffraction and ImagingBari, 3rd -7th September 2018TABLE-TOP SCANNING SAXS/WAXS MICROSCOPY OF ENGINEERED NANO/BIO-MATERIALSD. Altamura1,*, S.G. Pastore1, D. Siliqi1, M. Ladisa1, F. Scattarella1, A. Terzi1, and C. Giannini11 CNR-Istituto di Cristallografia, Via Amendola 122/O, 70126 Bari, Italy*e-mail: (davide.altamura@ic.cnr.it)INTRODUCTION: Simultaneous SAXS/WAXS data collection allows to directly relate atomic and nano- scale structures at each point on the sample. Synchrotron beamlines typically exploit an off-axis detector for WAXS, although this prevents the collection of diffraction rings over the full azimuthal range, or scanning is performed twice to collect the full scattering signal at small (SAXS) and wide (WAXS) angles. Despite the excellent data quality, a possible drawback can be some information loss and/or mismatch between SAXS and WAXS maps due to sample structural evolution (spontaneous, due to drying, radiation damage, drifts). A different approach, also available in table-top facilities, exploits an image plate detector with central hole, to collect the average 2D WAXS signal along with a spatially resolved SAXS map, and/or single SAXS/WAXS patterns from selected sample points.EXPERIMENTAL: A Rigaku synchrotron-class micro-source, coupled to a SAXS/WAXS camera, allows for double length scale diffraction microscopy with 70?200 ?m spatial resolution1.RESULTS AND DISCUSSION: Even though a scattering component from sample local structure can be partially smeared out in the integrated average, the spatially resolved SAXS/Absorption map can help in discriminating and localizing micro- and nano-scale components (e.g. CaS and HA, respectively, in the Figure), through the following simultaneous SAXS/WAXS signal collection from selected regions of interest. Once the correspondence of SAXS/WAXS features is assessed, the 2D SAXS/Absorption map can be used for qualitative structural mapping, quantitative estimation of concentration gradients (e.g. HA nanocrystals across the scaffold in the Figure)1, or statistical quantitative information can be extracted by averaging the normalized SAXS intensity in selected q-ranges, for comparative studies on nanoparticle abundance (e.g. oil nanodroplets in films made from emulsions)2, among others.CONCLUSIONS:The simultaneous SAXS/WAXS-integrated full pattern collection allows for double length scale description of the real-time status of the sample, with 2D spatial resolution, without loss of intensity or possibly information in case of preferred orientations, and is feasible with laboratory equipment.REFERENCES:1. D. Altamura, S. G. Pastore, M. G. Raucci, D. Siliqi, F. De Pascalis, M. Nacucchi, L. Ambrosio, and C. Giannini. ACS Appl. Mater. Interfaces (2016), 8, 8728-8736.2. Juan M. Montes de Oca-Ávalosa, D. Altamura, Roberto J. Candal, F. Scattarella, D. Siliqi, C. Giannini, M. Lidia Herrera. Relationship between nano/mi

During recent decades innovative nanomaterials have been extensively studied, aiming at both investigating the structure-property relationship and discovering new properties, in order to achieve relevant improvements in current state-of-the art materials. Lately, controlled growth and/or assembly of nanostructures into hierarchical and complex architectures have played a key role in engineering novel functionalized materials. Since the structural characterization of such materials is a fundamental step, here we discuss X-ray scattering/diffraction techniques to analyze inorganic nanomaterials under different conditions: dispersed in solutions, dried in powders, embedded in matrix, and deposited onto surfaces or underneath them.

A first-generation-synchrotron-class X-ray laboratory microsource, coupled to a three-pinhole camera, is presented. It allows (i) small- and wide-angle X-ray scattering images to be acquired simultaneously, and (ii) scanning small- and wide-angle X-ray scattering microscopy to be carried out. As representative applications, the structural complexity of a biological natural material (human bone biopsy) and of a metamaterial (colloidal nanocrystal assembly) are inspected at different length scales, studying the atomic/molecular ordering by (grazing-incidence) wide-angle X-ray scattering and the morphological/structural conformation by (grazing-incidence) small-angle X-ray scattering. In particular, the grazing-incidence measurement geometries are needed for inspecting materials lying on top of surfaces or buried underneath surfaces.