Structure Determination of Biogenic Crystals Directly from 3D Electron Diffraction Data.

Highly reflective assemblies of purine, pteridine, and flavin crystals are used in the coloration and visual systems of many different animals. However, structure determination of biogenic crystals by single-crystal XRD is challenging due to the submicrometer size and beam sensitivity of the crystals, and powder XRD is inhibited due to the small volumes of powders, crystalline impurity phases, and significant preferred orientation. Consequently, the crystal structures of many biogenic materials remain unknown. Herein, we demonstrate that the 3D electron diffraction (3D ED) technique provides a powerful alternative approach, reporting the successful structure determination of biogenic guanine crystals (from spider integument, fish scales, and scallop eyes) from 3D ED data confirmed by analysis of powder XRD data. The results show that all biogenic guanine crystals studied are the previously known ß-polymorph. This study highlights the considerable potential of 3D ED for elucidating the structures of biogenic molecular crystals in the nanometer-to-micrometer size range. This opens up an important opportunity in the development of organic biomineralization, for which structural knowledge is critical for understanding the optical functions of biogenic materials and their possible applications as sustainable, biocompatible optical materials.

Brilliant whiteness in shrimp from ultra-thin layers of birefringent nanospheres.

A fundamental question regarding light scattering is how whiteness, generated from multiple scattering, can be obtained from thin layers of materials. This challenge arises from the phenomenon of optical crowding, whereby, for scatterers packed with filling fractions higher than ~30%, reflectance is drastically reduced due to near-field coupling between the scatterers. Here we show that the extreme birefringence of isoxanthopterin nanospheres overcomes optical crowding effects, enabling multiple scattering and brilliant whiteness from ultra-thin chromatophore cells in shrimp. Strikingly, numerical simulations reveal that birefringence, originating from the spherulitic arrangement of isoxanthopterin molecules, enables intense broadband scattering almost up to the maximal packing for random spheres. This reduces the thickness of material required to produce brilliant whiteness, resulting in a photonic system that is more efficient than other biogenic or biomimetic white materials which operate in the lower refractive index medium of air. These results highlight the importance of birefringence as a structural variable to enhance the performance of such materials and could contribute to the design of biologically inspired replacements for artificial scatterers like titanium dioxide.

Lizards exploit the changing optics of developing chromatophore cells to switch defensive colors during ontogeny.

Many animals undergo changes in functional colors during development, requiring the replacement of integument or pigment cells. A classic example of defensive color switching is found in hatchling lizards, which use conspicuous tail colors to deflect predator attacks away from vital organs. These tail colors usually fade to concealing colors during ontogeny. Here, we show that the ontogenetic blue-to-brown tail color change in Acanthodactylus beershebensis lizards results from the changing optical properties of single types of developing chromatophore cells. The blue tail colors of hatchlings are produced by incoherent scattering from premature guanine crystals in underdeveloped iridophore cells. Cryptic tail colors emerge during chromatophore maturation upon reorganization of the guanine crystals into a multilayer reflector concomitantly with pigment deposition in the xanthophores. Ontogenetic changes in adaptive colors can thus arise not via the exchange of different optical systems, but by harnessing the timing of natural chromatophore development. The incoherent scattering blue color here differs from the multilayer interference mechanism used in other blue-tailed lizards, indicating that a similar trait can be generated in at least two ways. This supports a phylogenetic analysis showing that conspicuous tail colors are prevalent in lizards and that they evolved convergently. Our results provide an explanation for why certain lizards lose their defensive colors during ontogeny and yield a hypothesis for the evolution of transiently functional adaptive colors.

A tunable reflector enabling crustaceans to see but not be seen.

Many oceanic prey animals use transparent bodies to avoid detection. However, conspicuous eye pigments, required for vision, compromise the organisms' ability to remain unseen. We report the discovery of a reflector overlying the eye pigments in larval decapod crustaceans and show how it is tuned to render the organisms inconspicuous against the background. The ultracompact reflector is constructed from a photonic glass of crystalline isoxanthopterin nanospheres. The nanospheres' size and ordering are modulated to tune the reflectance from deep blue to yellow, enabling concealment in different habitats. The reflector may also function to enhance the acuity or sensitivity of the minute eyes by acting as an optical screen between photoreceptors. This multifunctional reflector offers inspiration for constructing tunable artificial photonic materials from biocompatible organic molecules.

Macromolecular sheets direct the morphology and orientation of plate-like biogenic guanine crystals.

Animals precisely control the morphology and assembly of guanine crystals to produce diverse optical phenomena in coloration and vision. However, little is known about how organisms regulate crystallization to produce optically useful morphologies which express highly reflective crystal faces. Guanine crystals form inside iridosome vesicles within chromatophore cells called iridophores. By following iridosome formation in developing scallop eyes, we show that pre-assembled, fibrillar sheets provide an interface for nucleation and direct the orientation of the guanine crystals. The macromolecular sheets cap the (100) faces of immature guanine crystals, inhibiting growth along the π-stacking growth direction. Crystal growth then occurs preferentially along the sheets to generate highly reflective plates. Despite their different physical properties, the morphogenesis of iridosomes bears a striking resemblance to melanosome morphogenesis in vertebrates, where amyloid sheets template melanin deposition. The common control mechanisms for melanin and guanine formation inspire new approaches for manipulating the morphologies and properties of molecular materials.

Diabetes Device Downloading: Benefits and Barriers Among Youth With Type 1 Diabetes.

BACKGROUND: The majority of youth with type 1 diabetes (T1D) fail to meet glycemic targets despite increasing continuous glucose monitoring (CGM) use. We therefore aimed to determine the proportion of caregivers who review recent glycemic trends ("retrospective review") and make ensuant insulin adjustments based on this data ("retroactive insulin adjustments"). We additionally considered that fear of hypoglycemia and frequency of severe hypoglycemia would be associated with performing retrospective review. METHODS: We conducted a cross-sectional survey of caregivers of youth with T1D, collecting demographics, diabetes technology usage, patterns of glucose data review/insulin dose self-adjustment, and Hypoglycemia Fear Survey (HFS). RESULTS: Nineteen percent of eligible caregivers (191/1003) responded. Performing retrospective review was associated with younger child age (12.2 versus 15.4, P = .0001) and CGM use (92% versus 73%, P = .004), but was not associated with a significant improvement in child's HbA1c (7.89 versus 8.04, P = .65). Retrospective reviewers had significantly higher HFS-behavior scores (31.9 versus 27.7, P = .0002), which remained significantly higher when adjusted for child's age and CGM use (P = .005). Linear regression identified a significant negative association between HbA1c (%) and number of retroactive insulin adjustments (0.24 percent lower mean HbA1c per additional adjustment made, P = .02). CONCLUSIONS: Retrospective glucose data review is associated with improved HbA1c when coupled with data-driven retroactive insulin adjustments. Barriers to data downloading existed even in this cohort of predominantly CGM-using T1D families.

The Non-Classical Crystallization Mechanism of a Composite Biogenic Guanine Crystal.

Spectacular colors and visual phenomena in animals are produced by light interference from highly reflective guanine crystals. Little is known about how organisms regulate crystal morphology to tune the optics of these systems. By following guanine crystal formation in developing spiders, a crystallization mechanism is elucidated. Guanine crystallization is a "non-classical," multistep process involving a progressive ordering of states. Crystallization begins with nucleation of partially ordered nanogranules from a disordered precursor phase. Growth proceeds by orientated attachment of the nanogranules into platelets which coalesce into single crystals, via progressive relaxation of structural defects. Despite their prismatic morphology, the platelet texture is retained in the final crystals, which are composites of crystal lamellae and interlamellar sheets. Interactions between the macromolecular sheets and the planar face of guanine appear to direct nucleation, favoring platelet formation. These findings provide insights on how organisms control the morphology and optical properties of molecular crystals.

Biogenic Guanine Crystals Are Solid Solutions of Guanine and Other Purine Metabolites.

Highly reflective crystals of the nucleotide base guanine are widely distributed in animal coloration and visual systems. Organisms precisely control the morphology and organization of the crystals to optimize different optical effects, but little is known about how this is achieved. Here we examine a fundamental question that has remained unanswered after over 100 years of research on guanine: what are the crystals made of? Using solution-state and solid-state chemical techniques coupled with structural analysis by powder XRD and solid-state NMR, we compare the purine compositions and the structures of seven biogenic guanine crystals with different crystal morphologies, testing the hypothesis that intracrystalline dopants influence the crystal shape. We find that biogenic "guanine" crystals are not pure crystals but molecular alloys (aka solid solutions and mixed crystals) of guanine, hypoxanthine, and sometimes xanthine. Guanine host crystals occlude homogeneous mixtures of other purines, sometimes in remarkably large amounts (up to 20% of hypoxanthine), without significantly altering the crystal structure of the guanine host. We find no correlation between the biogenic crystal morphology and dopant content and conclude that dopants do not dictate the crystal morphology of the guanine host. The ability of guanine crystals to host other molecules enables animals to build physiologically "cheaper" crystals from mixtures of metabolically available purines, without impeding optical functionality. The exceptional levels of doping in biogenic guanine offer inspiration for the design of mixed molecular crystals that incorporate multiple functionalities in a single material.

Measuring the optical properties of nanoscale biogenic spherulites.

Recent studies of optical reflectors as part of the vision apparatus in the eyes of decapod crustaceans revealed assemblies of nanoscale spherulites - spherical core-shell nanoparticles with radial birefringence. Simulations performed on the system highlighted the advantages of optical anisotropy in enhancing the functionality of these structures. So far, calculations of the nanoparticle optical properties have relied on refractive indices obtained using ab-initio calculations. Here we describe a direct measurement of the tangential refractive index of the spherulites, which corresponds to the in-plane refractive index of crystalline isoxanthopterin nanoplatelets. We utilize measurements of scattering spectra of individual spherulites and determine the refractive index by analyzing the spectral signatures of scattering resonances. Our measurements yield a median tangential refractive index of 1.88, which is in reasonable agreement with theoretical predictions. Furthermore, our results indicate that the optical properties of small spherulite assemblies are largely determined by the tangential index.

Characterization and possible function of an enigmatic reflector in the eye of the shrimp Litopenaeus vannamei.

Reflective assemblies of high refractive index organic crystals are used to produce striking optical phenomena in organisms based on light reflection and scattering. In aquatic animals, organic crystal-based reflectors are used both for image-formation and to increase photon capture. Here we report the characterization of a poorly-documented reflector in the eye of the shrimp L. vannamei lying 150 µm below the retina, which we term the proximal reflective layer (PR-layer). The PR-layer is made from a dense but disordered array of polycrystalline isoxanthopterin nanoparticles, similar to those recently reported in the tapetum of the same animal. Each spherical nanoparticle is composed of numerous isoxanthopterin single crystal plates arranged in concentric lamellae around an aqueous core. The highly reflective plate faces of the crystals are all aligned tangentially to the particle surface with the optical axes projecting radially outwards, forming a birefringent spherulite which efficiently scatters light. The nanoparticle assemblies form a broadband reflective sheath around the screening pigments of the eye, resulting in pronounced eye-shine when the animal is viewed from a dorsal-posterior direction, rendering the eye pigments inconspicuous. We assess possible functions of the PR-layer and conclude that it likely functions as a camouflage device to conceal the dark eye pigments in an otherwise largely transparent animal.

A highly reflective biogenic photonic material from core-shell birefringent nanoparticles.

Spectacular natural optical phenomena are produced by highly reflective assemblies of organic crystals. Here we show how the tapetum reflector in a shrimp eye is constructed from arrays of spherical isoxanthopterin nanoparticles and relate the particle properties to their optical function. The nanoparticles are composed of single-crystal isoxanthopterin nanoplates arranged in concentric lamellae around a hollow core. The spherulitic birefringence of the nanoparticles, which originates from the radial alignment of the plates, results in a significant enhancement of the back-scattering. This enables the organism to maximize the reflectivity of the ultrathin tapetum, which functions to increase the eye's sensitivity and preserve visual acuity. The particle size, core/shell ratio and packing are also controlled to optimize the intensity and spectral properties of the tapetum back-scattering. This system offers inspiration for the design of photonic crystals constructed from spherically symmetric birefringent particles for use in ultrathin reflectors and as non-iridescent pigments.

Guanine and 7,8-Dihydroxanthopterin Reflecting Crystals in the Zander Fish Eye: Crystal Locations, Compositions, and Structures.

The eyes of many fish contain a reflecting layer of organic crystals partially surrounding the photoreceptors of the retina, which are commonly believed to be composed of guanine. Here we study an unusual fish eye from Stizostedion lucioperca that contains two layers of organic crystals. The crystals in the outer layer are thin plates, whereas the crystals in the inner tapetum layer are block-shaped. We show that the outer layer indeed contains guanine crystals. Analyses of solutions of crystals from the inner layer indicated that the block-shaped crystals are composed of xanthopterin. A model of the structure of the block-shaped crystals was produced using symmetry arguments based on electron diffraction data followed by dispersion-augmented DFT calculations. The resulting crystal structure of xanthopterin included, however, a problematic repulsive interaction between C═O and N of two adjacent molecules. Knowing that dissolved 7,8-dihydroxanthopterin can oxidize to xanthopterin, we replaced xanthopterin with 7,8-dihydroxanthopterin in the model. An excellent fit was obtained with the powder X-ray diffraction pattern of the biogenic crystals. We then analyzed the biogenic block-shaped crystals in their solid state, using MALDI-TOF and Raman spectroscopy. All three methods unequivocally prove that the block-shaped crystals in the eye of S. lucioperca are crystals of 7,8-dihydroxanthopterin. On the basis of the eye anatomy, we deduce that the guanine crystals form a reflective layer producing the silvery color present on part of the eye surface, whereas the block-shaped crystals backscatter light into the retina in order to increase the light sensitivity of the eye.

Animal Eyes: Filtering Out the Background.

Animals use photonic structures in their eyes to form images, enhance sensitivity and provide camouflage. A recent exciting discovery shows that the eyes of some larval mantis shrimp possess photonic crystals that function as color filters to detect bioluminescence.

Reply to comment on Couzi et al. (2018): a phenomenological model for structural phase transitions in incommensurate alkane/urea inclusion compounds.

In a recent paper (Couzi et al. 2018 R. Soc. open sci. 5, 180058. (doi:10.1098/rsos.180058)), we proposed a new phenomenological model to account for the I↔II↔"III" phase sequence in incommensurate n-alkane/urea inclusion compounds, which represents an alternative interpretation to that proposed in work of Toudic et al. In a Comment (Toudic et al. 2019 R. Soc. open sci. 6, 182073. (doi:10.1098/rsos.182073)), Toudic et al. have questioned our assignment of the superspace group of phase II of n-nonadecane/urea, which they have previously assigned, based on a (3 + 2)-dimensional superspace, as C2221(00γ)(10δ). In this Reply, we present new results from a comprehensive synchrotron single-crystal X-ray diffraction study of n-nonadecane/urea, involving measurements as a detailed function of temperature across the I↔II↔"III" phase transition sequence. Our results demonstrate conclusively that "main reflections" (h, k, l, 0) with h+k odd are observed in phase II of n-nonadecane/urea (including temperatures in phase II that are just below the transition from phase I to phase II), in full support of our assignment of the (3+1)-dimensional superspace group P212121(00γ) to phase II. As our phenomenological model is based on phase II and phase "III" of this incommensurate material having the same (3+1)-dimensional superspace group P212121(00γ), it follows that the new X-ray diffraction results are in full support of our phenomenological model.

Spatially resolved mapping of phase transitions in liquid-crystalline materials by X-ray birefringence imaging.

The X-ray Birefringence Imaging (XBI) technique, first reported in 2014, is a sensitive method for spatially resolved mapping of the local orientational properties of anisotropic materials. We report the first application of the XBI technique to characterize molecular orientational ordering in a liquid crystalline material, demonstrating significant potential for exploiting XBI measurements to advance structural understanding of liquid crystal phases.

Crystallization of Organic Molecules: Nonclassical Mechanism Revealed by Direct Imaging.

Organic crystals are of primary importance in pharmaceuticals, functional materials, and biological systems; however, organic crystallization mechanisms are not well-understood. It has been recognized that "nonclassical" organic crystallization from solution involving transient amorphous precursors is ubiquitous. Understanding how these precursors evolve into crystals is a key challenge. Here, we uncover the crystallization mechanisms of two simple aromatic compounds (perylene diimides), employing direct structural imaging by cryogenic electron microscopy. We reveal the continuous evolution of density, morphology, and order during the crystallization of very different amorphous precursors (well-defined aggregates and diffuse dense liquid phase). Crystallization starts from initial densification of the precursors. Subsequent evolution of crystalline order is gradual, involving further densification concurrent with optimization of molecular ordering and morphology. These findings may have implications for the rational design of organic crystals.

The Organic Crystalline Materials of Vision: Structure-Function Considerations from the Nanometer to the Millimeter Scale.

Vision mechanisms in animals, especially those living in water, are diverse. Many eyes have reflective elements that consist of multilayers of nanometer-sized crystalline plates, composed of organic molecules. The crystal multilayer assemblies owe their enhanced reflectivity to the high refractive indices of the crystals in preferred crystallographic directions. The high refractive indices are due to the molecular arrangements in their crystal structures. Herein, data regarding these difficult-to-characterize crystals are reviewed. This is followed by a discussion on the function of these crystalline assemblies, especially in visual systems whose anatomy has been well characterized under close to in vivo conditions. Three test cases are presented, and then the relations between the reflecting crystalline components and their functions, including the relations between molecular structure, crystal structure, and reflecting properties are discussed. Some of the underlying mechanisms are also discussed, and finally open questions in the field are identified.

Optically functional isoxanthopterin crystals in the mirrored eyes of decapod crustaceans.

The eyes of some aquatic animals form images through reflective optics. Shrimp, lobsters, crayfish, and prawns possess reflecting superposition compound eyes, composed of thousands of square-faceted eye units (ommatidia). Mirrors in the upper part of the eye (the distal mirror) reflect light collected from many ommatidia onto the photosensitive elements of the retina, the rhabdoms. A second reflector, the tapetum, underlying the retina, back-scatters dispersed light onto the rhabdoms. Using microCT and cryo-SEM imaging accompanied by in situ micro-X-ray diffraction and micro-Raman spectroscopy, we investigated the hierarchical organization and materials properties of the reflective systems at high resolution and under close-to-physiological conditions. We show that the distal mirror consists of three or four layers of plate-like nanocrystals. The tapetum is a diffuse reflector composed of hollow nanoparticles constructed from concentric lamellae of crystals. Isoxanthopterin, a pteridine analog of guanine, forms both the reflectors in the distal mirror and in the tapetum. The crystal structure of isoxanthopterin was determined from crystal-structure prediction calculations and verified by comparison with experimental X-ray diffraction. The extended hydrogen-bonded layers of the molecules result in an extremely high calculated refractive index in the H-bonded plane, n = 1.96, which makes isoxanthopterin crystals an ideal reflecting material. The crystal structure of isoxanthopterin, together with a detailed knowledge of the reflector superstructures, provide a rationalization of the reflective optics of the crustacean eye.

The image-forming mirror in the eye of the scallop.

Scallops possess a visual system comprising up to 200 eyes, each containing a concave mirror rather than a lens to focus light. The hierarchical organization of the multilayered mirror is controlled for image formation, from the component guanine crystals at the nanoscale to the complex three-dimensional morphology at the millimeter level. The layered structure of the mirror is tuned to reflect the wavelengths of light penetrating the scallop's habitat and is tiled with a mosaic of square guanine crystals, which reduces optical aberrations. The mirror forms images on a double-layered retina used for separately imaging the peripheral and central fields of view. The tiled, off-axis mirror of the scallop eye bears a striking resemblance to the segmented mirrors of reflecting telescopes.