X-ray linear dichroic ptychography Yuan Hung Loa,b,c,d, Jihan Zhoua,b,c, Arjun Ranaa,b,c, Drew Morrillb,e,f, Christian Gentryb,e,f, Bjoern Endersg, Young-Sang Yuh, Chang-Yu Suni, David A. Shapiroh, Roger W. Falconeb,h,j, Henry C. Kapteynb,e,f, Margaret M. Murnaneb,e,f, Pupa U. P. A. Gilberti,k,l,m,n, and Jianwei Miaoa,b,c,1 aDepartment of Physics and Astronomy, University of California, Los Angeles, CA 90095; bSTROBE NSF Science and Technology Center, University of Colorado Boulder, Boulder, CO 80309; cCalifornia NanoSystems Institute, University of California, Los Angeles, CA 90095; dDepartment of Bioengineering, University of California, Los Angeles, CA 90095; eJILA, University of Colorado Boulder and National Institute of Standards and Technology, Boulder, CO 80309; fDepartment of Physics, University of Colorado Boulder, Boulder, CO 80309; gNational Energy Research Scientific Computing Center, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 hAdvanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; iDepartment of Physics, University of Wisconsin, Madison, WI 53706; jDepartment of Physics, University of California, Berkeley, CA, 94720; kDepartment of Chemistry, University of Wisconsin, Madison, WI 53706; lDepartment of Materials Science and Engineering, University of Wisconsin, Madison, WI 53706; mDepartment of Geoscience, University of Wisconsin, Madison, WI 53706; and nChemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved November 28, 2020 (received for review September 9, 2020) Biominerals such as seashells, coral skeletons, bone, and tooth polarimetry to study crystal orientation uniformity (12, 13). In enamel are optically anisotropic crystalline materials with unique the shorter-wavelength regime, X-ray absorption near-edge nanoscale and microscale organization that translates into excep- structure spectroscopy has been used to study the orientations tional macroscopic mechanical properties, providing inspiration for of various polymorphs of CaCO3 (14, 15), and polarization- engineering new and superior biomimetic structures. Using Seria- dependent imaging contrast (PIC) mapping using X-ray photo- topora aculeata coral skeleton as a model, here, we experimen- emission electron microscopy (X-PEEM) has been demonstrated tally demonstrate X-ray linear dichroic ptychography and map the to quantitatively map crystal orientations in CaCO3 (15–17,). c-axis orientations of the aragonite (CaCO3) crystals. Linear di- Currently, PIC mapping mostly uses X-PEEM in reflection ge- chroic phase imaging at the oxygen K-edge energy shows strong ometry to achieve tens-of-nanometer resolution. However, polarization-dependent contrast and reveals the presence of both PEEM’s limited achievable spatial resolution (∼20 nm) and the narrow (<35°) and wide (>35°) c-axis angular spread in the coral confinement to polished two-dimensional surfaces are insur- samples. These X-ray ptychography results are corroborated by mountable limits. Scanning transmission X-ray microscopy four-dimensional (4D) scanning transmission electron microscopy (STXM) has taken advantage of dichroic contrast to study APPLIED PHYSICAL SCIENCES (STEM) on the same samples. Evidence of co-oriented, but discon- polymer fibers (18) to resolve 30-nm features, but it is limited in nected, corallite subdomains indicates jagged crystal boundaries achievable spatial resolution by the focusing optics, which also consistent with formation by amorphous nanoparticle attachment. has a low efficiency and a short working-distance constraint. We expect that the combination of X-ray linear dichroic ptychog- Although macroscopic morphologies in biominerals have been raphy and 4D STEM could be an important multimodal tool to studied extensively, their nanoscopic structures are still not study nano-crystallites, interfaces, nucleation, and mineral growth studied routinely in a quantitative fashion, mostly due to the lack of optically anisotropic materials at multiple length scales. of a proper transmission microscope that offers bulk-sensitive information with spatial resolution down to the nanometer BIOPHYSICS AND COMPUTATIONAL BIOLOGY coherent diffractive imaging | ptychography | X-ray linear dichroism | scale. With the development of high-brilliance synchrotron biominerals | 4D scanning transmission electron microscopy Significance umans have been using biogenic materials as tools since the Hdawn of humanity. Biominerals such as bone, teeth, sea- Ptychography, a powerful scanning coherent diffractive imag- shells, and coral skeletons exhibit remarkable mechanical prop- ing method, has attracted considerable attention for its gen- erties and complex hierarchical organization (1). Due to these eral applicability. Here, we demonstrate X-ray linear dichroic unique characteristics, biominerals often outperform their geo- ptychography to study the crystal orientations of the calcium logic or synthetic inorganic counterparts, thus attracting signifi- carbonate in coral samples. In contrast to the absorption con- cant interest in understanding the mechanisms of the biologically trast commonly observed in linear dichroic imaging, dichroic controlled mineralization processes for modern nanotechnology ptychography at pre-oxygen K-edge shows significant and (2). Careful understanding of the three-dimensional (3D) ar- unique dichroic phase contrast, which has not been reported. rangement of biominerals has important engineering implica- The dichroic ptychography images reveal the presence of both tions and has led to bioinspired materials that outperform narrow and wide c-axis angular spread in the submicrometer nonbiomimetic, inorganic synthetic analogs (3). coral fragments, which are confirmed by 4D STEM. We expect One of the most common natural biominerals is calcium car- X-ray linear dichroic ptychography to be a high-resolution, dose-efficient tool to broadly study optically anisotropic ma- bonate (CaCO3), which occurs in bacteria, algae, marine or- terials such as tooth enamel, bone, seashells, brittlestars, ganisms, and humans (4). CaCO3 absorbs light anisotropically, such that the π-bonded p orbitals of O and C atoms parallel to and polymers. the crystal c axis exhibit maximum absorption when aligned Author contributions: J.M. directed the project; P.U.P.A.G. and J.M. designed research; parallel to linearly polarized light. The absorption intensity Y.H.L., J.Z., A.R., D.M., C.G., B.E., Y.-S.Y., C.-Y.S., D.A.S., R.W.F., H.C.K., M.M.M., P.U.P.A.G., 2 changes with a cos law with respect to the azimuthal orientation and J.M. performed research; Y.H.L., J.Z., P.U.P.A.G., and J.M. analyzed data; and Y.H.L., of the carbonate groups in the crystal. This information can re- P.U.P.A.G., and J.M. wrote the paper. veal structural and mechanical properties in CaCO3 biominerals Competing interest statement: H.K. is partially employed by KMLabs, Inc. (5). Coral biomineralization is a subject of intense studies, and This article is a PNAS Direct Submission. the mechanisms of crystal nucleation and growth in coral skel- Published under the PNAS license. etons are only beginning to be revealed (6–9). 1To whom correspondence may be addressed. Email: [email protected]. The optical anisotropy in CaCO3 has been leveraged in po- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ larized visible light microscopy to study macroscopic biomineral doi:10.1073/pnas.2019068118/-/DCSupplemental. structure and formation mechanisms (10, 11) and with imaging Published January 12, 2021. PNAS 2021 Vol. 118 No. 3 e2019068118 https://doi.org/10.1073/pnas.2019068118 | 1of8 Downloaded by guest on September 28, 2021 radiation facilities worldwide, advancements in high-resolution the most efficient way for aragonite fill 3D space (6). Conse- imaging techniques, and the increasing availability of insertion- quently, this space-filling strategy may endow a unique evolu- device X-ray sources providing polarization control, such as el- tionary advantage to host organisms that adhere to the pattern liptically polarizing undulators (EPUs), new synchrotron-based by providing greater resilience to environmental stresses such as tools are now becoming available for probing nanoscale crystal ocean acidification (42). Therefore, the exact nanoscopic mecha- orientation in CaCO3 minerals and biominerals. By taking ad- nisms of biomineral growth along various crystal axes are of sig- vantage of brilliant X-ray sources, coherent diffractive imaging nificant scientific interest in understanding the macroscopic (CDI) can directly achieve high-resolution structural information structural changes in coral species around the world. of noncrystalline samples and nanocrystals from their diffraction We imaged several coral-skeleton samples on and off the O – patterns (19 28). In particular, ptychography, a scanning CDI K-edge π* peak and observed significant contrast differences technique (28), has attracted considerable attention for its gen- between absorption and phase images. Using three linear di- – eral applicability (29 32). Ptychography measures a series of chroic ptychography images, we performed PIC mapping to diffraction patterns from spatially overlapping illumination quantitatively determine crystal c-axis orientations in the coral probes on a sample, where phase-retrieval algorithms are used to with 35-nm spatial resolution. Our dichroic ptychography results