In Situ TEM Shows Ion Binding Is Key to Directing CaCO 3 Nucleation in a Biomimetic Matrix P.J.M. Smeets a,b,c, *, K.R.Cho b , R.G.E. Kempen a , N.A.J.M Sommerdijk a , J.J. De Yoreo c a Eindhoven University of Technology, Eindhoven, Netherlands b Lawrence Berkeley National Laboratory, Berkeley, CA c Pacific Northwest National Laboratory, Richland, WA *Corresponding author: e-mail address: P.J.M.Smeets@tue.nl Biominerals possess shapes, structures, and properties not found in synthetic minerals. These defining characteristics arise from the interplay of the min- eral with a macromolecular matrix, which directs crystal nucleation and growth (Lowenstam & Weiner, 1989; Mann, 2001). Within this three- dimensional biomolecular assembly, the developing mineral interacts with acidic macromolecules, either dissolved in the crystallization medium or associated with insoluble framework polymers such as chitin or collagen (Palmer et al., 2008). Although acidic macromolecules are known to affect growth habits and phase selection, or even to completely inhibit precipita- tion in solution (Sommerdijk & With, 2008; Meldrum & C€ olfen, 2008; Gower, 2008), little is known about the role of matrix-immobilized acidic macromolecules in directing mineralization. This lack of understanding is, in part, due to the difficulty of studying biomimetic mineralization systems with sufficient spatial and temporal res- olution (Dey et al., 2010). However, liquid phase transmission electron microscopy (LP-TEM) can visualize events in situ in thin liquid volumes (about 500 nm in height) confined within two electron transparent silicon nitride (SiN) membranes. Here, we use LP-TEM to visualize the nucleation and growth of CaCO 3 in a biomimetic matrix of polystyrene sulfonate (PSS). In particular, we utilized a dual inlet flow stage where we started out with a CaCl 2 solution in the confined cell, after which carbonate was introduced through in-diffusion of vapor released from the decomposition of solid (NH 4 ) 2 CO 3 (mainly CO 2 (g) and NH 3 (g) ) via the second inlet port. Within minutes, we directly observed the nucleation of randomly distrib- uted vaterite nanoparticles over the surface, which we attribute to hetero- geneous nucleation on the SiN. When we introduced a PSS solution together with our CaCl 2 solution, in situ imaging of this model system showed that the PSS by binding of Ca 2+ 35 CISCEM 2014