X-ray linear dichroic ptychography

Biominerals such as seashells, coral skeletons, bone, and tooth enamel are optically anisotropic crystalline materials with unique nanoscale and microscale organization that translates into exceptional macroscopic mechanical properties, providing inspiration for engineering new and superior biomimetic structures. Using Seriatopora aculeata coral skeleton as a model, here, we experimentally demonstrate X-ray linear dichroic ptychography and map the c-axis orientations of the aragonite (CaCO₃) crystals. Linear dichroic phase imaging at the oxygen K-edge energy shows strong polarization-dependent contrast and reveals the presence of both narrow (<35°) and wide (>35°) c-axis angular spread in the coral samples. These X-ray ptychography results are corroborated by four-dimensional (4D) scanning transmission electron microscopy (STEM) on the same samples. Evidence of co-oriented, but disconnected, corallite subdomains indicates jagged crystal boundaries consistent with formation by amorphous nanoparticle attachment. We expect that the combination of X-ray linear dichroic ptychography and 4D STEM could be an important multimodal tool to study nano-crystallites, interfaces, nucleation, and mineral growth of optically anisotropic materials at multiple length scales.

Humans have been using biogenic materials as tools since the dawn of humanity. Biominerals such as bone, teeth, seashells, and coral skeletons exhibit remarkable mechanical properties and complex hierarchical organization (1). Due to these unique characteristics, biominerals often outperform their geologic or synthetic inorganic counterparts, thus attracting significant interest in understanding the mechanisms of the biologically controlled mineralization processes for modern nanotechnology (2). Careful understanding of the three-dimensional (3D) arrangement of biominerals has important engineering implications and has led to bioinspired materials that outperform nonbiomimetic, inorganic synthetic analogs (3).One of the most common natural biominerals is calcium carbonate (CaCO₃), which occurs in bacteria, algae, marine organisms, and humans (4). CaCO₃ absorbs light anisotropically, such that the π-bonded p orbitals of O and C atoms parallel to the crystal c axis exhibit maximum absorption when aligned parallel to linearly polarized light. The absorption intensity changes with a cos² law with respect to the azimuthal orientation of the carbonate groups in the crystal. This information can reveal structural and mechanical properties in CaCO₃ biominerals (5). Coral biomineralization is a subject of intense studies, and the mechanisms of crystal nucleation and growth in coral skeletons are only beginning to be revealed (6–9).The optical anisotropy in CaCO₃ has been leveraged in polarized visible light microscopy to study macroscopic biomineral structure and formation mechanisms (10, 11) and with imaging polarimetry to study crystal orientation uniformity (12, 13). In the shorter-wavelength regime, X-ray absorption near-edge structure spectroscopy has been used to study the orientations of various polymorphs of CaCO₃ (14, 15), and polarization-dependent imaging contrast (PIC) mapping using X-ray photoemission electron microscopy (X-PEEM) has been demonstrated to quantitatively map crystal orientations in CaCO₃ (15–17,). Currently, PIC mapping mostly uses X-PEEM in reflection geometry to achieve tens-of-nanometer resolution. However, PEEM’s limited achievable spatial resolution (∼20 nm) and the confinement to polished two-dimensional surfaces are insurmountable limits. Scanning transmission X-ray microscopy (STXM) has taken advantage of dichroic contrast to study polymer fibers (18) to resolve 30-nm features, but it is limited in achievable spatial resolution by the focusing optics, which also has a low efficiency and a short working-distance constraint.Although macroscopic morphologies in biominerals have been studied extensively, their nanoscopic structures are still not studied routinely in a quantitative fashion, mostly due to the lack of a proper transmission microscope that offers bulk-sensitive information with spatial resolution down to the nanometer scale. With the development of high-brilliance synchrotron radiation facilities worldwide, advancements in high-resolution imaging techniques, and the increasing availability of insertion-device X-ray sources providing polarization control, such as elliptically polarizing undulators (EPUs), new synchrotron-based tools are now becoming available for probing nanoscale crystal orientation in CaCO₃ minerals and biominerals. By taking advantage of brilliant X-ray sources, coherent diffractive imaging (CDI) can directly achieve high-resolution structural information of noncrystalline samples and nanocrystals from their diffraction patterns (19–28). In particular, ptychography, a scanning CDI technique (28), has attracted considerable attention for its general applicability (29–32). Ptychography measures a series of diffraction patterns from spatially overlapping illumination probes on a sample, where phase-retrieval algorithms are used to iteratively recover the incident wave and complex exit wave of the sample. This versatile diffractive imaging technique has been applied to study various biological materials in two and three dimensions with high resolution (33–40).In this work, we present X-ray linear dichroic ptychography of biominerals using the aragonite (CaCO₃) coral skeleton of Seriatopora aculeata as a model. Aragonite is an orthorhombic CaCO₃ polymorph, with all three crystal axes being unequal in length and perpendicular to one another (1). Carbonate crystals grow acicularly with a needle-like habit and with 10 times greater growth rate along the c axis than along the a axis (41), resulting in densely packed bundles of thin crystals in coral skeletons. It has been hypothesized that this elongated growth pattern with crystals growing mostly along the fast c axis but in all directions is the most efficient way for aragonite fill 3D space (6). Consequently, this space-filling strategy may endow a unique evolutionary advantage to host organisms that adhere to the pattern by providing greater resilience to environmental stresses such as ocean acidification (42). Therefore, the exact nanoscopic mechanisms of biomineral growth along various crystal axes are of significant scientific interest in understanding the macroscopic structural changes in coral species around the world.We imaged several coral-skeleton samples on and off the O K-edge π* peak and observed significant contrast differences between absorption and phase images. Using three linear dichroic ptychography images, we performed PIC mapping to quantitatively determine crystal c-axis orientations in the coral with 35-nm spatial resolution. Our dichroic ptychography results were qualitatively validated by correlating the ptychography PIC maps with four-dimensional (4D) scanning transmission electron microscopy (STEM), a scanning nano-electron diffraction technique for probing crystal orientations in crystalline materials (43). We observed that, at the nanoscale, crystallite orientations can be narrowly distributed, as is characteristic of spherulitic crystals, but also randomly distributed in submicrometer particles. Moreover, we verified linear dichroic phase contrast at a pre-edge energy below the absorption resonance. The use of such phase contrast may lead to new dose-efficient dichroic imaging techniques for studying anisotropic biominerals and has important implications for understanding the nanoscale organization of crystallites in biominerals.