The Biomineral-Cell Interface in the Sea Urchin Embryo

As man-made materials become more similar to the biological structures that inspire them, they
increasingly combine nano-sized hard and soft, synthetic and biological components. This creates new
challenges for characterization, especially in those materials where water is an integral part of the
structure. Cryogenic sample preparation and imaging is often necessary for such specimens. Imaging of
cryo-fixed, freeze-fractured samples by cryo-SEM is particularly efficient. However, the propagation of
the fracture plane is unpredictable at best, and frequently the fracture surface fails to reveal the interface
of interest [1]. This can be a major complicating factor, for example in the analysis of the interaction of
cells and the endoskeleton in the sea urchin embryo [2]. Herein, we describe a cryogenic sample
preparation workflow for cryo-planing and imaging large areas of frozen-hydrated samples, using whole
sea urchin embryos as an example for a hybrid material with large hardness contrast between the organic
and biomineralized tissues.

The central innovation of the cryo triple ion gun milling (CryoTIGM) method is a custom-built tool
based on ion mill slope cutter. Specifically, a Leica TIC3X unit was fitted with a vacuum load lock that
allows cryo-transfer of a vitrified sample. Sea urchin embryo suspensions were high pressure-frozen
between aluminum planchettes and trimmed using a custom-built cryo-saw. The cryo-saw consists of a
liquid nitrogen reservoir, a sample compartment, a diamond blade, and a VCT-docking port (Fig. 1A).
Trimming was performed under liquid nitrogen, and samples were then positioned in a sample holder
next to a milling mask (Fig. 1B). The sample was transferred to the CryoTIGM tool (Fig. 1C), where
three broad Ar+ beams converge at the mask shielding the trimmed sample edge (Fig. 1D). Material
above the mask was removed, creating a cross-section in the sample at the level of the mask (Fig. 1E).
The ion-milled sample was subsequently freeze-etched and coated with Pt to increase contrast.

For whole, frozen-hydrated sea urchin embryos, we find that ion milling with Ar+ at an acceleration
voltage of 3.0 kV, a current of 1.0 mA/gun, a base temperature of -120o C, and for 2 h results in very
smooth cryo-planed area of ~700,000 µm 2 (Fig. 1E). Sections clearly revealed cell-endoskeleton
interfaces (Fig. 2A). The membranes of the syncytium that envelopes the endoskeleton appear welldefined
(Fig. 2B). Numerous organelles are observed within the syncytial compartment, indicating
excellent preservation of cellular ultrastructure. These results suggest that CryoTIGM is a promising new
tool for interfacial studies of hybrid hard/soft materials. Given the large and smooth cryo-planed surface,
microanalysis by cryo-SEM-EDS appears particularly promising. In this context, I will discuss recent
attempts to identify vesicles that store and/or transport biomineral precursors in the sea urchin embryo.