Transformation mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule

Abstract

Sea urchin larval spicules transform amorphous calcium carbonate (ACC) into calcite single crystals. The mechanism of transformation is enigmatic: the transforming spicule displays both amorphous and crystalline properties, with no defined crystallization front. Here, we use X-ray photoelectron emission spectromicroscopy with probing size of 40-200 nm. We resolve 3 distinct mineral phases: An initial short-lived, presumably hydrated ACC phase, followed by an intermediate transient form of ACC, and finally the biogenic crystalline calcite phase. The amorphous and crystalline phases are juxtaposed, often appearing in adjacent sites at a scale of tens of nanometers. We propose that the amorphous-crystal transformation propagates in a tortuous path through preexisting 40- to 100-nm amorphous units, via a secondary nucleation mechanism.

Fig. 1.

Ca L-edge XANES spectra and an X-PEEM micrograph of a 48-h spicule. (A and B) Ca L-edge XANES spectra extracted from: near the tip (left yellow line in C) (A) and middle part of the spicule (right yellow line in C) (B). (C) X-PEEM micrograph of part of a fresh 48-h spicule. (D) Ca L-edge XANES spectra of synthetic calcite; the L₂ peak is split into peaks 1 and 2, and the L₃ is split into peaks 3 and 4. The main peaks are 1 and 3, and the crystal field peaks are 2 and 4. (E) Ca L-edge XANES spectra of synthetic ACC. These and all spectra hereafter were extracted from adjacent pixels along a line. The bold spectra at the top of A, B, D, and E are the averages of all spectra below. Blue in A and B highlights a spectrum similar to calcite. Green highlights a spectrum with intense peak 2 and small peak 4. Red, present in A but not in B, highlights a spectrum similar to synthetic hydrated ACC. Each spectrum in A, B, D, and E was extracted from a 200-nm pixel.

Fig. 2.

X-PEEM micrographs and Ca L-edge XANES spectra of a fresh 72-h spicule, and the same spicule after 10 months. (A–C) X-PEEM micrographs of the 72-h spicule. (A) Fresh spicule: the dark region immediately below the spicule is its shadow. (B) The same spicule measured 10 months later. The yellow lines indicate the pixels from which the spectra in E and F were extracted. (C) High magnification of the area in A: colored pixels are those from which the corresponding colored spectra in E and F were extracted. (D) SEM micrograph of the same spicule taken after 10 months. (E and F) Ca L-edge spectra extracted from the lines in A and B, respectively. The blue curve in E corresponds to the type 2 ACC phase, which clearly became calcitic in F. Scale bar in A also applies to B, and scale bar in C also applies to D. Pixel size is 100 × 100 nm². Highlighted in black is the average spectrum.

Fig. 3.

Ca L-edge XANES spectra from a fresh 72-h spicule. The spectra are extracted from individual pixels along a straight line. Each pixel represents 40 × 40 nm². The 2 series of spectra show different patterns of mineral phase distribution in adjacent pixels. (A) We observe large blocks of 5–10 adjacent spectra of type 3 calcite interspersed with smaller series of spectra of type 2 ACC. (B) The transitions between type 2 and 3 spectra are abrupt between the bottom 2 spectra and gradual for the others. Highlighted in black are the average spectra. Blue highlights 1 of the type 3 calcite spectra, green highlights type 2 ACC.

Fig. 4.

Peak splitting anaylsis of Ca L-edge XANES spectra from 48-h and 72-h spicules, as well as those from ACC and calcite. (A) Ca L-edge XANES spectra extracted from single pixels of synthetic ACC (bottom red curve) and calcite (top blue curve) and 3 spectra from a 48-h spicule. The 3 spicule spectra are representative of the 3 mineral phases identified in Fig. 1: red is type 1, green is type 2, and blue is type 3. (B) A plot of SR(L₃) vs. SR(L₂) (see Methods and Fig. S5). The spicule samples indicated by triangles (48-h spicule tip, light blue; middle, purple; 72-h spicule fresh, green; 10 month olds, blue), and the adult sea urchin spine (squares, brown), span 3 quadrants, and synthetic ACC (red circles) is located in the bottom left quadrant, where L₂, L₃ SR < 1. Calcite (blue diamonds) is located in the top right quadrant where L₂, L₃ SR > 1. (C) Spicule ratios are shown separately with the relevant quadrants shaded in gray. Color code is as in B.