Anat Akiva 1 Netta Vidavsky 1 Lia Addadi 1 Steve Weiner 1 Andreas Schertel 2
1Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel
2Global Applications Support, Carl Zeiss Microscopy GmbH, Oberkochen, Germany

Understanding the ultrastructure of intact biological tissues at different scales (from whole tissue organization to cellular and subcellular compartments) can lead to far-reaching mechanistic insights. By using conventional imaging methods (such as SEM, TEM and microCT), a compromise between the imaging resolution and the sample size has to be made. Serial FIB milling and block face SEM imaging (FIB-SEM) enable high resolution imaging of tissues at up to 5 nm resolution, with the ability to detect large volumes (dozens of micrometers). Conventional FIB-SEM imaging requires intense sample processing that may damage or modify the specimens during preparation (fixation, dehydration, staining etc). This procedure is also time consuming (>1 week). The recently developed cryo-FIB-SEM technique (Schertel et al, 2013) allows 3D imaging of high pressure frozen biological samples in conditions that are very close to their native state, avoiding any chemical procedures. Cryo-FIB-SEM workflow is extremely fast, requiring less than an hour from organism sacrifice, to the first cryo-FIB-SEM results.

By utilizing the cryo-FIB-SEM technique, we show the ultrastructure of cellular, sub-cellular and extracellular compartments of two highly studied biological model systems; the sea urchin embryo and the zebrafish larva. The large volume of imaging reveals intra and extra cellular compartments in the tissue in their biological context. By combining simultaneous detection of secondary and backscattered electrons, we locate and characterize mineralized elements embedded in the tissues, and show their interactions with their environment. By correlating the backscattered electron signal with secondary electron gray level data, we characterize different features and organelles inside the tissue. Cryo-FIB-SEM technique is advantageous for 3D imaging of biological systems in which tissue dehydration and processing may cause morphological changes. In addition, cryo preservation is highly beneficial in cases where sensitive or transient moieties are present inside the tissue. In the future, cryo-FIB-SEM could be combined in a correlated manner with other imaging or analytical methods, such as fluorescence, cryo-EDX and cryo-STEM.

[1] A. Schertel, N. Snaidero, H. M. Han, T. Ruhwedel, M. Laue, M. Grabenbauer, W. Möbius, 2013, J.Struct.l Biol., 184, 355-360

[2] N. Vidavsky, A. Masic, A. Schertel, S. Weiner, L. Addadi, 2015, J. Struct.l Biol., 192, 358-365

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