Steve Weiner, Lia Addadi and Ron Shahar *

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel 76100

** Koret School of Veterinary Medicine, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel 76100

Biological tissues are composed of cells, extracellular matrices, minerals and water, and they are often organized hierarchically at many length scales. Furthermore, some of the tissue constituents are graded and contain interphases. It is therefore extremely challenging to image such tissues, and this complexity clearly requires the use of multiple methods, preferably in a correlative manner and if possible in vivo. The key to preparing such tissues for imaging is cryo-fixation, which not only retains the important water component, but preserves the organic components and the often highly unstable mineral phases.

In vivo options for 3D imaging are at present limited to confocal light microscopy. A powerful approach applied to the study of bone formation in the zebrafish is correlative confocal imaging, Raman spectroscopy and fluorescence imaging. This provides chemical information from well-defined small volumes within the living tissue (1). We have also used correlative microCT-fluorescence to image the mineral distributions in fresh leaves, and by incorporating a phase contrast component in the microCT images, the leaf anatomy can also be imaged (2). MicroCT has also been used to image the 3D structure of unfixed and unstained periodontal ligament; the soft tissue that binds vertebrate teeth to the bone of the mandible (3). Cryo-SEM surface images can also be combined with fluorescence imaging and cryo-EDS mapping, as has been demonstrated in the study of ion containing vesicles in foraminifera; protozoans that produce mineralized shells (4). One of the most challenging mineralized tissues to study in 3D is bone. We have used FIB SEM in the serial surface imaging mode to study the structures of the matrices of different chemically fixed bone types, after first removing the mineral. This approach has revealed the presence of a previously unrecognized second material in all the bones we have studied (5). FIB SEM can now be used in a cryo-mode. This enables the 3D imaging of cells, matrices and minerals in a fully hydrated state (vitrified ice) from volumes as large as tens of microns cubed at a resolution of a few nanometers. We have used cryo-FIB SEM to study ion uptake and transport into certain cells of the sea urchin embryo (6), as well as ion pathways to the fin bones of zebrafish larvae. Cryo-FIB SEM can in principle be combined in a correlative manner with EDS, and may thus prove to be one of the most versatile and effective methods for imaging mineralized tissues.  These advanced techniques, alone and in combination, open an exciting array of opportunities to advance our understanding of the structure of biological tissues in 3 dimensions’

1.       Akiva, A.,  Malkinson, G., Masic, A., Kerschnitzki, M., Bennet, M., Fratzl, P., Addadi, L., Weiner, S. and Yaniv, K. 2015. On the pathway of mineral deposition in larval zebrafish caudal fin bone. Bone 75, 192-200.

2.       Pierantoni, M., Tenne, R., Brumfeld, V., Kiss, V., Oron, D., Addadi, L. and Weiner, S. 2017. Plants and light manipulation: the integrated mineral system in okra leaves. Advanced Science 4, 1600416.

3.       Naveh, G.R.S., Brumfeld, V., Shahar, R. and Weiner, S. 2013. Tooth periodontal ligament: direct 3D microCT visualization of the collagen network and how the network changes when the tooth is loaded. J. Structural Biol. 181, 108-115

4.       Mor Khalifa, G., Kirchenbuechler, D., Koifman, N., Kleinerman, O., Talmon, Y., Elbaum, M., Addadi, L, Weiner, S. and Erez, J. 2016. Biomineralization pathways in a foraminifer revealed using a novel correlative cryo-fluorescence-SEM-EDS technique. J. Struct. Biol. 196, 487-495.

5.       Reznikov, N., Shahar, R. and Weiner, S. 2014. Bone hierarchical structure in three dimensions. Acta Biomaterialia 10, 3815-3826.

6.       Vidavsky, N., Addadi, S., Schertel, A., Ben-Ezra, D., Shpigel, M., Addadi, L. and Weiner, S. 2016. Calcium transport into the cells of the sea urchin larva: implications for spicule formation. Proc. Natl. Acad. Sci. USA. 201612017.