Coccolithophores are unicellular marine phytoplankton that produce complex and intricately shaped mineralised scales called coccoliths. Coccoliths are produced in an intracellular vesicle where crystal nucleation occurs, from which several individual calcite units develop with anisotropic crystallographic facets, prompting studies into the cellular mechanisms which control crystal growth within the cell. Here, we characterise those morphological developments in 3D that occur during the formation of coccoliths by the species Gephyrocapsa huxleyi using cryo-ptychographic X-ray computed tomography. This technique is ideally suited to study coccolith mineral development, as intracellular structures can be imaged intact in their native state without needing to disrupt cells. Combined with additional imaging of developing coccoliths using cryo-transmission electron microscopy and scanning electron microscopy, we report the developmental stages involved in coccolith growth across the complete mineralisation period, while also showing that the constrained space created by individual crystal units growing in close confinement affects the final crystal morphology and overall mineral structure. These findings provide clarification on the mineralisation pathways that coccolithophores and other biomineralising organisms use to control the formation of highly functionalised crystalline structures, particularly relevant in the design of materials with tunable properties.

Bragg coherent diffraction imaging (BCDI) fails to reliably retrieve phases in micro-crystals exhibiting strong strain inhomogeneities, which restricts its applicability. Here we show that three-dimensional Bragg ptychography (3DBP) overcomes this limitation by enabling stable inversion for large lattice distortions. Using a combination of experimental measurements and numerical tests, we compare the performance limits of the two approaches and demonstrate that 3DBP tolerates lattice distortions more than six times larger than BCDI. We also establish the sensitivity of both methods on a weakly distorted crystal, for which 3DBP yields smoother amplitude and phase fields with reduced short-length-scale artifacts. 3DBP thus provides a reliable route for imaging micro-crystals with large lattice distortions, expanding the scope of coherent X-ray Bragg microscopy to strongly deformed systems.

We present a highly sensitive coherent Raman microscopy approach, which allows for the tridimensional (3D) imaging of a series of carbonate polymorphs in marine organisms. CaCO3 biomineralization occurs from the transformation of metastable amorphous precursors and other crystalline phases into a final crystalline phase. Understanding biomineralization pathways requires identifying this physico-chemical temporal sequence. Our approach exploits the different vibrational signatures of amorphous calcium carbonate, aragonite, calcite, Mg-calcite or hemi-hydrated calcium carbonate. This optical method enables the production of spatially and spectrally resolved images of the different compounds. When applied on the growing edge of post-mortem samples of both Pinctada margaritifera pearl oyster shell and Stylophora pistillata coral, it allows for inferring a temporal crystallisation sequence. We thus highlight the existence of intermediate crystalline phases, involving magnesian calcite or hemi-hydrated calcium carbonate, respectively.

Biomineralisation integrates complex biologically assisted physico-chemical processes leading to an extraordinary diversity of calcareous biomineral crystalline architectures, in intriguing contrast with the consistent presence of a submicrometric granular structure. While the repeated observation of amorphous calcium carbonate is interpreted as a precursor to the crystalline phase, the crystalline transition mechanisms are poorly understood. Access to the crystalline architecture at the mesoscale, i.e., over a few granules, is key to building realistic crystallisation models. Here we exploit three-dimensional X-ray Bragg ptychography microscopy to provide a series of nanoscale maps of the crystalline structure within the "single-crystalline" prism of the prismatic layer of a Pinctada margaritifera shell. The mesocrystalline organisation exhibits several micrometre-sized iso-oriented/iso-strained crystalline domains, the detailed studies of which reveal the presence of crystalline coherence domains ranging from 130 to 550 nm in size. The further increase in the lattice parameter with the size of the coherence domain likely results from the crystallisation mechanism, pointing towards a maturation process occurring after the initial amorphous-to-crystalline transition.

Coccolithophores are abundant marine phytoplankton that produce biomineralized calcite scales, called coccoliths, which sequester substantial amounts of carbon and play a substantial role in biogeochemical cycles. However, mechanisms underlying the storage and transport of ions essential for calcification remain unresolved. We used ptychographic x-ray computed tomography under cryogenic conditions to visualize intracellular calcium-rich structures involved in the storage of calcium ions in the coccolithophore species Chrysotila carterae. During calcification, we observed a range of structures, from small electron-dense bodies within larger compartments to denser and distributed globular compartments, before returning to small bodies once scale formation is complete. Nanobeam-scanning x-ray fluorescence measurements further revealed that these electron-dense bodies are rich in phosphorus and calcium (molar ratio of ~4:1). The dynamic nature of structures suggests that these bodies are part of the required cellular calcium ion transport pathways, a fundamental process critical for understanding the response of coccolithophores to climate change.

This paper proposes an innovative approach to improve Bragg coherent diffraction imaging (BCDI) microscopy applied to time evolving crystals and/or nonhomogeneous crystalline strain fields, identified as two major limitations of BCDI microscopy. Speckle BCDI (spBCDI), introduced here, rests on the ability of a strongly nonuniform illumination to induce a convolution of the three-dimensional (3D) frequency content associated with the finite-size crystal and a kernel acting perpendicularly to the illumination beam. In the framework of Bragg diffraction geometry, this convolution is beneficial as it encodes some 3D information about the sample in a single two-dimensional (2D) measurement, i.e., in the detector plane. With this approach, we demonstrate that we can drastically reduce the sampling frequency along the rocking curve direction and still obtain datasets with enough information to be inverted by a traditional phase retrieval algorithm. Numerical simulations, performed for a highly distorted crystal, show that spBCDI allows a gain in the sampling ratio ranging between 4 and 20 along the rocking curve scan, for a speckle illumination with individual speckle size of 50 nm at the sample position. Furthermore, spBCDI allows working at low intensity levels, leading to an additional gain for the total scanning time. Reductions of a factor of about 40 were numerically observed. Thus, full 3D datasets measured in the 0.2 s time scale at fourth-generation synchrotrons become feasible, with a remarkable potential for the imaging of strongly distorted crystals. Practical details on the implementation of the method are also discussed.

This work proposes an innovative approach to improve Bragg coherent diffraction imaging (BCDI) microscopy applied to time evolving crystals and/or non-homogeneous crystalline strain fields, identified as two major limitations of BCDI microscopy. Speckle BCDI (spBCDI), introduced here, rests on the ability of a strongly non-uniform illumination to induce a convolution of the three-dimensional (3D) frequency content associated with the finite-size crystal and a kernel acting perpendicularly to the illumination beam. In the framework of Bragg diffraction geometry, this convolution is beneficial as it encodes some 3D information about the sample in a single two-dimensional (2D) measurement, i.e., in the detector plane. With this approach, we demonstrate that we can drastically reduce the sampling frequency along the rocking curve direction and still obtain data sets with enough information to be inverted by a traditional phase retrieval algorithm. Numerical simulations, performed for a highly distorted crystal, show that spBCDI allows a gain in the sampling ratio ranging between 4 and 20 along the rocking curve scan, for a speckle illumination with individual speckle size of 50 nm. Furthermore, spBCDI allows working at low intensity levels, leading to an additional gain for the total scanning time. Reductions of a factor of about 32 were numerically observed. Thus, measurements in the 0.3 s time scale at 4th generation synchrotrons become feasible, with a remarkable performance for the imaging of strongly distorted crystals. Practical details on the implementation of the method are also discussed.

1] is a 3D microscopy technique providing sensitivity to crystal strain with nano-scale resolution that has already demonstrated to be a powerful tool in many fields of science [2][3][4].This technique has a high potential for the investigation of functional crystalline nanoscale materials.Here, the understanding of how crystal structure and strain are intertwined with the relevant physical properties (e.g.electrical, magnetic, optical, mechanical ...) is essential for the design of future nanotechnologies.

Biomineralization integrates complex physical and chemical processes bio-controlled by the living organ-isms through ionic concentration regulation and organic molecules production. It allows tuning the struc-tural, optical and mechanical properties of hard tissues during ambient-condition crystallisation, motivat-ing a deeper understanding of the underlying processes. By combining state-of-the-art optical and X-ray microscopy methods, we investigated early-mineralized calcareous units from two bivalve species, Pinc-tada margaritifera and Pinna nobilis, revealing chemical and crystallographic structural insights. In these calcite units, we observed ring-like structural features correlated with a lack of calcite and an increase of amorphous calcium carbonate and proteins contents. The rings also correspond to a larger crystalline disorder and a larger strain level. Based on these observations, we propose a temporal biomineralization cycle, initiated by the production of an amorphous precursor layer, which further crystallizes with a tran-sition front progressing radially from the unit centre, while the organics are expelled towards the prism edge. Simultaneously, along the shell thickness, the growth occurs following a layer-by-layer mode. These findings open biomimetic perspectives for the design of refined crystalline materials. Statement of significance Calcareous biominerals are amongst the most present forms of biominerals. They exhibit astonishing structural, optical and mechanical properties while being formed at ambient synthesis conditions from ubiquitous ions, motivating the deep understanding of biomineralization. Here, we unveil the first forma-tion steps involved in the biomineralization cycle of prismatic units of two bivalve species by applying a new multi-modal non-destructive characterization approach, sensitive to chemical and crystalline proper-ties. The observations of structural features in mineralized units of different ages allowed the derivation of a temporal sequence for prism biomineralization, involving an amorphous precursor, a radial crys-tallisation front and a layer-by-layer sequence. Beyond these chemical and physical findings, the herein introduced multi-modal approach is highly relevant to other biominerals and bio-inspired studies.(c) 2022 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )