Electron microscopy studies on the precipitation of calcium carbonate with and without confinement A. Verch 1 , R. v. d. Locht 1 , I. Morrison 2 , Y.-Y. Kim 3 , F. C. Meldrum 3 and R. Kröger 1 1. Department of Physics, University of York, York, United Kingdom 2. Department of Biology, University of York, York, United Kingdom 3. School of Chemistry, University of Leeds, Leeds, United Kingdom email: andreas.verch@york.ac.uk Keywords: Crystallization, Atmospheric Scanning Electron Microscopy, Transmission Electron Microscopy The formation of calcium carbonate in organisms proceeds in many cases via an amorphous precursor, very often in a confined environment. Due to its low level of long-range order amorphous calcium carbonate (ACC) is extremely moldable and can adapt to a wide range of forms and shapes, which otherwise would not be accessible for crystalline materials in their typical equilibrium morphologies. Hence, a pathway via ACC is vital for many calcium carbonate forming organisms [1]. However, little is known about the influence of the confinement on the microstructure of calcium carbonate or the actual conversion of the amorphous phase into the crystalline form, like calcite. This is because the in-situ and time-resolved observation of the mineralization processes of single particles is even nowadays a very challenging task; for example, by means of X-ray analysis or Raman spectroscopy solely data averaged over a large volume are accessible. In this work we use a novel inverted atmospheric scanning electron microscope (JEOL Clairscope (tm)), which allows for an in-situ observation of the calcium carbonate precipitation and its subsequent development into matured crystals with a spatial resolution in the nanometer range. A 100 nm thick silicon nitride membrane separates the scanning electron microscope vacuum from the sample, which stays under atmospheric conditions during the experiment. This setup facilitates the investigation of processes at the surface of the membrane on the atmospheric side as the electrons have a penetration depth of only few μm into the aqueous solution. The crystallization experiments are performed in the absence and in the presence of polymeric additives such as poly(acrylic acid) or poly(styrene sulfonate-co-maleic acid). These experiments allow for example to determine crystal growth rates, e.g. a 5 mM concentration of calcium and carbonate ions results in growth rates of about 5 nm/s for the exposed CaCO 3 facets in the absence of additives. The appearance and disappearance of “bright spots” is observed in the vicinity of the monitored crystals indicating the possible formation and dissolution of amorphous calcium carbonate (Figure 1a). In the presence of many polymers amorphous calcium carbonate is stabilized. When poly(acrylic acid) is used it is even possible to trigger the transformation from ACC into crystalline calcium carbonate by the electron beam. The dissolution of the surrounding amorphous material for the benefit of the growing crystal creates a depletion zone (Figure 1), which facilitates the study of diffusion processes in the proximity of growing crystals. To investigate the influence of confinement on the growth of calcium carbonate crystals we analyze the microstructure of and defects in calcite nano-rods grown in 50 nm track-etch membranes [2]. High-resolution transmission electron microscopy (HR-TEM) and diffraction pattern (DP) analysis show that these rods have no preferred growth orientation. Series of electron diffraction patterns along these single crystalline calcite rods reveal that they show twists of up to 4° per μm in rod direction (Figure 2) demonstrating a remarkable elastic response of these nanostructure to the growth conditions. Our results help to get a better understanding of the mechanisms involved in the transformation of an amorphous material into the resulting crystals.