In this work we have studied in situ the formation and growth of calcium phosphate (CaP) nanoparticles (NPs) in the presence of three calcium-binding carboxylate molecules having different affinities for Ca2+ ions: citrate (Cit), hydroxycitrate (CitOH), and glutarate (Glr). The formation of CaP NPs at several reaction temperatures ranging from 25 °C to 80 °C was monitored in situ through simultaneous Small and Wide X-ray Scattering (SAXS/WAXS) using synchrotron light. SAXS was used to investigate the first stages of NP formation where a crystalline order is not yet formed. In this regard we have developed a new bivariate mesh data analysis method for identifying the SAXS curves associated with the most relevant timeframes for performing curve modeling. WAXS was used to study the formation of crystalline phases and their evolution over time. The combined SAXS/WAXS data allowed us to track NP nucleation, their size and morphology, and their evolution up to mature hydroxyapatite (HA) nanocrystals. We have assessed that in the first stages of reaction (80 seconds) amorphous, elongated primary NPs nucleate whose size and morphology depend on the temperature and type of carboxylate molecule. The temperature controls the release of Ca2+ ions from carboxylate molecules, and thus induces the formation of a higher amount of amorphous particles and increases their size and aspect ratio. As the reaction time progresses, amorphous particles evolve into crystalline ones, whose kinetics of crystal growth are controlled by temperature and carboxylate ions. Stronger Ca-binding carboxylates (CitOH > Cit > Glr) have a more pronounced inhibiting effect on HA crystallization, retarding the formation and growth of crystalline domains, while a rise of temperature promotes crystallization. This work allowed us to shed more light on the formation of HA in the presence of growth-controlling molecules, as well as present the potential of combined SAXS/WAXS for studying the formation of highly relevant NPs for different applications.

A mm thick free-standing gel containing lipid vesicles made of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) was studied by scanning Small Angle X-ray Scattering (SAXS) and X-ray Transmission (XT) microscopies. Raster scanning relatively large volumes, besides reducing the risk of radiation damage, allows signal integration, improving the signal-to-noise ratio (SNR), as well as high statistical significance of the dataset. The persistence of lipid vesicles in gel was demonstrated, while mapping their spatial distribution and concentration gradients. Information about lipid aggregation and packing, as well as about gel density gradients, was obtained.A posterioriconfirmation of lipid presence in well-defined sample areas was obtained by studying the dried sample, featuring clear Bragg peaks from stacked bilayers. The comparison between wet and dry samples allowed it to be proved that lipids do not significantly migrate within the gel even upon drying, whereas bilayer curvature is lost by removing water, resulting in lipids packed in ordered lamellae. Suitable algorithms were successfully employed for enhancing transmission microscopy sensitivity to low absorbing objects, and allowing full SAXS intensity normalization as a general approach. In particular, data reduction includes normalization of the SAXS intensity against the local sample thickness derived from absorption contrast maps. The proposed study was demonstrated by a room-sized instrumentation, although equipped with a high brilliance X-ray micro-source, and is expected to be applicable to a wide variety of organic, inorganic, and multicomponent systems, including biomaterials. The employed routines for data reduction and microscopy, including Gaussian filter for contrast enhancement of low absorbing objects and a region growing segmentation algorithm to exclude no-sample regions, have been implemented and made freely available through the updated in-house developed software SUNBIM.

X-ray Small and Wide Scattering scanning microscopies have been adopted to inspect morphological and structural properties of collagen-based tissues at the atomic and nano scale 1 . Examples will be discussed on specific pathologies: o osteoarthritis of the hip, also named osteoarthrosis of the hip or coxarthrosis, which is a chronic degenerative disorder of the hip joint, causing growing articular pain that can bring the patient to lifestyle limitations until surgical intervention is needed 2 o keratoconus, a pathology affecting cornea, which causes progressive thinning of the stroma and consequently abnormal curvature, inducing irregular astigmatism and myopia, corneal fibrosis, and distortion of vision, due to the modification in the organization of the corneal collagen 3 o abdominal aortic aneurysm, that occurs in the major artery from the hearth that supplies blood to the abdomen, and popliteal aneurysm, that takes place in the legs, behind the knees, characterized by alteration of collagen structure into vessel's wall of the aneurysm tissues, heterogeneous grade of inflammation related to infiltrating cells and extracellular matrix changes, in particular disruption of elastic fibers, fibrosis and calcifications 4 o diabetes mellitus, a metabolic disorder characterized by high blood sugar levels over a prolonged period due to defects in insulin action or secretion, which causes collagen to have a fixed orientation, stiffen the tissue and is likely to disrupt the normal cell interactions.

SUNBIM (Supramolecular and sUbmolecular Nano- and Biomaterials X-ray IMaging) is a computer suite of integrated programs which, through a user-friendly graphical interface, is able to perform a number of functions for (GI)SAXS-(GI)WAXS data analysis [1] such as: centering, q-scale calibration, two-dimensional to one-dimensional folding of small- and wide-angle X-ray scattering (SAXS/WAXS) data, also in grazing-incidence (GISAXS/GIWAXS); indexing of two-dimensional GISAXS frames and extraction of one-dimensional GISAXS profiles along specific cuts; quantitative scanning microscopy in absorption and SAXS contrast. SUNBIM consists of five main programs: (1) Calibration package, a set of functions allow one to find all of the geometrical parameters needed to extract a one-dimensional profile out of a two-dimensional image; (2) Batch Script & 2D Mesh Composite, to prepare batch script files (ASCII files) to run a sequential acquisition of two-dimensional frames (in scanning mode) and to perform a composite of the as-collected two-dimensional SAXS frames into a single image; (3) Single-scan (GI)SAXS and (GI)WAXS data analysis, to calibrate and fold the two-dimensional data, in order to extract relevant information from the experimental data and to fold 2D data into 1D profiles; (4) Multi-scan SAXS and WAXS data analysis, to fold each two-dimensional frame of the mesh into a one-dimensional profile and extract scattering features of the sample with a multi-modal imaging approach; (5) One-D Data Analysis Manager, a package that in addition to basic operations on one dimensional profiles (such as change of the plot representation from pixels to q, change from linear scale to logarithmic scale of the axes, choice of colors and plot thickness, inserting the legend, etc. as well as import, trigger, save and export plots) gives the possibility to denoise the folded profile and/or to deconvolute the primary beam angular divergence from the SAXS/WAXS profiles, particularly useful for a complete data analysis. SUNBIM combines in the same package both originally developed algorithms (i.e denoising, beam centering etc.) and reliable methods documented in the literature (multi-modal imaging [2], GIXAXS three-dimensional frame indexing [3]). New tools have been developed to enrich SUNBIM suite. The main novelty is the possibility to perform a deeper data reduction including dark current subtraction, background evaluation and subtraction, normalization of the SAXS intensity against the local sample thickness derived from absorption contrast maps. The advances of the new release with respect to previous one include also an automatic background subtraction from the 1D profile of the azimuthal integration to enhance peak visibility at large scattering angles (WAXS), to correct geometric aberration for small sample-to-detector distance. The previous release of the software has already been used successfully to analyse several nano-structured samples [4][5][6]. We are confident that the new features will allow a more correct and extensive analysis of the (GI)SAXS/(GI)WAXS data. SUNBIM is developed in the MATLAB language and it is distributed free of charge to the academic user (downloadable after a valid registration from http://www.ba.ic.cnr.it/softwareic/sunbimweb/)

A novel method is described that combines high-resolution scanning microdiffraction techniques, Rietveld quantitative phase analysis and a statistical method known as canonical correlation analysis (CCA). The method has been applied to a sample taken from a bone-tissue-engineered bioceramic porous scaffold implanted in a mouse for six months. The CCA technique allows the detection of those pixels throughout the investigated sample that best correlate with signal models. Besides the standard usage of this approach, which requires theoretical profiles as signal models, a novel application is presented here, which consists of picking the model spectra out of the experimental data set. Patterns representative of a reasonable range of phase compositions were selected among the huge number of two-dimensional patterns ( folded in onedimensional profiles) to extract quantitative phase fractions. At this stage, the CCA approach was also used to overcome the low Poisson statistic of signal models, so making Rietveld quantitative analysis more reliable. These patterns have been used as profile models for CCA. The final classification map, obtained by assigning the considered pixel to the model spectrum with the highest canonical coefficient, provides the spatial variation of phase concentration.