Dvir Gur

Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: A cryo-electron microscopy study

Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by specialized cells - the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of developing mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular mineral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precursor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the vesicles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates.

Guanine-Based Photonic Crystals in Fish Scales Form from an Amorphous Precursor

Starting from disorder: anhydrous guanine crystals compose the photonic arrays responsible for the skin and scale iridescence found in Japanese Koi fish. These guanine crystals were found to form in intracellular vesicles through an amorphous precursor phase. A combined cryo-SEM and synchrotron radiation X-ray diffraction study showed the evolution of the crystals in great detail.

Structural Basis for the Brilliant Colors of the Sapphirinid Copepods

Males of sapphirinid copepods use regularly alternating layers of hexagonal-shaped guanine crystals and cytoplasm to produce spectacular structural colors. In order to understand the mechanism by which the different colors are produced, we measured the reflectance of live individuals and then characterized the organization of the crystals and the cytoplasm layers in the same individuals using cryo-SEM. On the basis of these measurements, we calculated the expected reflectance spectra and found that they are strikingly similar to the measured ones. We show that variations in the cytoplasm layer thickness are mainly responsible for the different reflected colors and also that the copepod color strongly depends on the angular orientation relative to the incident light, which can account for its appearance and disappearance during spiral swimming in the natural habitat.

The Structural Basis for Enhanced Silver Reflectance in Koi Fish Scale and Skin

Fish have evolved biogenic multilayer reflectors composed of stacks of intracellular anhydrous guanine crystals separated by cytoplasm, to produce the silvery luster of their skin and scales. Here we compare two different variants of the Japanese Koi fish; one of them with enhanced reflectivity. Our aim is to determine how biology modulates reflectivity, and from this to obtain a mechanistic understanding of the structure and properties governing the intensity of silver reflectance. We measured the reflectance of individual scales with a custom-made microscope, and then for each individual scale we characterized the structure of the guanine crystal/cytoplasm layers using high-resolution cryo-SEM. The measured reflectance and the structural-geometrical parameters were used to calculate the reflectance of each scale, and the results were compared to the experimental measurements. We show that enhanced reflectivity is obtained with the same basic guanine crystal/cytoplasm stacks, but the structural arrangement between the stack, inside the stacks, and relative to the scale surface is varied when reflectivity is enhanced. Finally, we propose a model that incorporates the basic building block parameters, the crystal orientation inside the tissue, and the resulting reflectance and explains the mechanistic basis for reflectance enhancement.

The image-forming mirror in the eye of the scallop

Fine-tuned for image formation We typically think of eyes as having one or more lenses for focusing incoming light onto a surface such as our retina. However, light can also be focused using arrays of mirrors, as is commonly done in telescopes. A biological example of this is the scallop, which can have up to 200 reflecting eyes that focus light onto two retinas. Palmer et al. find that spatial vision in the scallop is achieved through precise control of the size, shape, and packing density of the tiles of guanine that together make up an image-forming mirror at the back of each of the eyes. Science, this issue p. 1172 The crystal morphology, organization, and three-dimensional shape of the scallop eye mirror are finely controlled for image formation. 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.

Biologically controlled morphology and twinning in guanine crystals

Guanine crystals are widely used in nature as components of multilayer reflectors. Guanine-based reflective systems found in the copepod cuticle and in the mirror of the scallop eye are unique in that the multilayered reflectors are tiled to form a contiguous packed array. In the copepod cuticle, hexagonal crystals are closely packed to produce brilliant colors. In the scallop eye, square crystals are tiled to obtain an image-forming reflecting mirror. The tiles are about 1 μm in size and 70 nm thick. According to analysis of their electron diffraction patterns, the hexagon and square tiles are not single crystals. Rather, each tile type is a composite of what appears to be three crystalline domains differently oriented and stacked onto one another, achieved through a twice-repeated twinning about their ⟨011⟩ and ⟨021⟩ crystal axes, respectively. By these means, the monoclinic guanine crystal mimics higher symmetry hexagonal and tetragonal structures to achieve unique morphologies.

Hydrogen Analysis by Ultra-High Energy Resolution EELS

1. Department of Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel 2. Department of Physics, ASU, Tempe, AZ 85287, USA 3. Center for Solid State Science, Arizona State University, Tempe, AZ 85287, USA 4. School for Engineering of Matter, Transport and Energy, ASU, Tempe, AZ 85287, USA 5. Nion Co., 11511 NE 118 St., Kirkland, WA 98034, USA 6. Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel 7. Physique des Solides, University of Paris-Sud, Orsay 91405, France

The Dual Functional Reflecting Iris of the Zebrafish

Abstract Many marine organisms have evolved a reflective iris to prevent unfocused light from reaching the retina. The fish iris has a dual function, both to camouflage the eye and serving as a light barrier. Yet, the physical mechanism that enables this dual functionality and the benefits of using a reflective iris have remained unclear. Using synchrotron microfocused diffraction, cryo‐scanning electron microscopy imaging, and optical analyses on zebrafish at different stages of development, it is shown that the complex optical response of the iris is facilitated by the development of high‐order organization of multilayered guanine‐based crystal reflectors and pigments. It is further demonstrated how the efficient light reflector is established during development to allow the optical functionality of the eye, already at early developmental stages.

Development of High-Order Organization of Guanine-Based Reflectors Underlies the Dual Functionality of the Zebrafish Iris

Many marine organisms have evolved a reflective iris to prevent unfocused light from reaching the retina. The fish iris has a dual function, both to camouflage the eye and serving as a light barrier. Yet, the mechanism that enables this dual functionality and the benefits of using a reflective iris have remained unclear. Using synchrotron micro-focused diffraction, cryo-SEM imaging and optical analyses on zebrafish at different stages of development, we show that the complex optical response of the iris is facilitated by the development a high-order organization of multilayered guanine-based crystal reflectors and pigments. We further demonstrates how the efficient light reflector is established during development to allow the optical functionality of the eye, already at early developmental stages. These results shed light on the evolutionary drive for developing a compact reflective iris, which are widely used by many animal species. One Sentence Summary The dual function of the zebrafish iris as a light barrier and camouflage reflector is enabled by the high-order organization of intracellular guanine crystals and pigments.

Erratum: Damage-free vibrational spectroscopy of biological materials in the electron microscope (Nature Communications (2016) 7 (10945) DOI: 10.1038/ncomms10945)

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