Introduction

Biominerals are crystalline1,2,3,4,5,6,7,8,9,10,46,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127 but it is limited to detecting the orientation of the crystalline c-axis, it cannot provide the a- and b-axis orientation, and it is only available at selected synchrotrons already equipped with a photoemission electron microscope (PEEM) and an elliptically polarizing undulator (EPU). Fortunately, nearly all synchrotrons in every country have a PEEM and an EPU, as these are quite useful for research on magnetic materials. One advantage of PIC map** is the spatial resolution, which is 20 nm,128,129 as opposed to EBSD’s 200 nm. Another advantage is that PIC map** can easily analyze enamel, which is inaccessible to EBSD. The third advantage of PIC map**, compared to x-ray diffraction methods, is the surface sensitivity: 3 nm at the Ca L-edge (where enamel is measured) and 5 nm at the O K-edge (where calcite, aragonite, and vaterite are measured). Thus, when a 20 nm × 20 nm × 3 nm pixel contains the full polarization spectrum, the spectrum is reliably from one crystallite, or two at grain boundaries, not thousands of crystallites as in micro- or nano-diffraction of a bulk biomineral. Thus, PIC map** results are mathematically tractable.

Mesostructure revealed formation mechanisms

One of the first discoveries based on the mesostructure of biominerals was that amorphous nanoparticles first attach to one another and then crystallize,46 as opposed to oriented attachment of particles that crystallize first, and then attach.64,130,131,132,133 Polycrystalline systems formed by oriented attachment, called mesocrystals, were observed extensively in synthetic materials, but not conclusively in biominerals.67,134,135,136,137,138

In the calcite prisms of mollusk shells, the mesostructure demonstrated that the crystal lattice gradually tilts and suddenly splits within prisms.115,139,140

In nacre, the mesostructure revealed that tablets grow near-epitaxially,114,141 that nacre tablet thickness correlates with the temperature at which the shell formed,142 and that plastic deformation of stacks of co-oriented aragonite tablets cooperatively toughen the material.123 Figure 2 shows the mesostructure of nacre through time.

In enamel, the mesostructure showed that, unexpectedly, in whisker-shaped nanocrystals the elongation direction and the crystalline c-axis are not aligned, as they always are in synthetic calcium phosphate whisker crystals. The mesostructure of adjacent whisker crystals, parallel in morphology but forming small-angle boundaries with one another, toughens enamel.121 Figure 3 shows the mesostructure of human enamel and the small misorientation of adjacent crystals.

Figure 3
figure 3

Human enamel has a complex 3D structure, including 50-nm-wide whisker crystals bundled into 5-µm rods that vary in orientation according to a decussation pattern. Here, the rods are in the plane of the image on the left and top right of the image, and perpendicular to the image plane at the center of the image. Pixel size in this polarization-dependent imaging contrast map is matched to the size of the whisker crystals, ~60 nm. Notice the gradients in color, indicating that at grain boundaries between crystals there is only small misorientation within each rod, which toughens enamel. Note: Hs, Homo sapiens. Data from Reference 121.

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In coral skeletons, the mesostructure enabled the discovery that sprinkles, that is, equant, randomly oriented nanocrystals are the first to nucleate in spherulites.126 Figure 4 shows the spherulitic mesostructure of coral skeletons.119,126 The spherulitic fibers start from sprinkles, which can be either at the growth fronts of spherulites or in the centers of calcification, as observed in different coral species presented in Figure 5. The same mechanism is expected to lead to spherulite formation in other systems besides coral skeletons known to grow spherulitically. These include polymers, organic molecules in food or drugs, or geologic minerals.

Figure 4
figure 4

Coral skeleton mesostructure shows spherulitic growth, with crystalline acicular fibers radiating from so-called centers of calcification. Here too, the two letters on top right of each panel indicate the genus and species, (a) Stylophora pistillata and (b) Porites lutea. In these polarization-dependent imaging contrast maps, there are few or no sprinkles. Data from Reference 126.

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Figure 5
figure 5

Polarization-dependent imaging contrast maps of coral skeleton from Balanophyllia europea (Be) and Acropora pharaonis (Ap), two very different coral skeletons that retain sprinkles even after they are mature and fully crystallized. In Be, sprinkles are at growth fronts of spherulites (a), in Ap, they are localized at the centers of calcification (b). Data from Reference 126.

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Important questions thus far unanswered

  1. 1.

    What controls the curvature of shells, and shell morphogenesis in general? It is possible that mesostructure at different locations in a shell, corresponding to different developmental stages, will provide the answer.

  2. 2.

    In forming columnar nacre, how do ions or particles reach the growing tablets sites? The pores in the organic sheets are too small143,144,145,146,147 for particles66 to squeeze through them, and there are tens of organic sheets that particles would have to go through148,149 from the depositing cell to the tablet growth site.148,149 It must be ions, or a dense liquid precursor62 that pass through the pores. But: can ions, easily going through pores of many organic sheets, really diffuse thermodynamically uphill from a less to a more concentrated solution?

  3. 3.

    Why are mollusk shells and sea urchin spines less sensitive than coral skeletons65 to anthropogenic-CO2-induced ocean acidification? The material is precisely the same aragonite or calcite (CaCO3); thus, their solubility is identical. Does the mesostructure contribute to the solubility of marine biominerals?

  4. 4.

    Can single whisker nanocrystals of human enamel be separated and analyzed by TEM to determine if the crystalline c-axis is completely uncorrelated with the whisker elongation direction? PIC map** suggests so.121 EBSD of enamel does not work at all, presumably because the whisker crystals are too small, and TEM thus far did not shed light on this key question to understand the single-crystal behavior in tooth enamel. It is simple to obtain samples from dentists who collect third molars from young adults every day and are usually happy to donate them with minimal paperwork, thus providing an excellent source of healthy human enamel. We use enamel every day when smiling or eating, thus, revealing the crystallography of its fundamental units will provide us with a knowing smile.