3D FIB-SEM tomography of the interface retinal tissue on a subretinal electronic chip B. Schroeppel 1 , W. Nisch 1 , A. Stett 1 and C. Burkhardt 1 1. Natural and Medical Science Institute (NMI) at the University of Tuebingen, Reutlingen, Germany birgit.schroeppel@nmi.de Keywords: FIB-SEM tomography, biological technical interface For successful application of micro implants, coupling and connection of the biological system to the technical surface of the micro implant is of crucial relevance. Beside any chemical modifications applied to the technical interface and any material induced biological responses of the tissue to the micro implant surface, gaining morphological information of the biological technical interface with high spatial resolution is an important issue. On the example of a subretinal micro electrode implant which is designed for electric stimulation of retinal tissue to substitute for degenerated photoreceptors [1], a method is presented which combines classical biological and material science sample preparation methods with 3D focused ion beam (FIB) – scanning electron microscopy (SEM) tomography imaging method yielding morphological information of the interface retinal tissue to micro electrode implant with high spatial resolution. In the present contribution, conventional biological preparation methods for tissue based samples such as chemical fixation with glutaraldehyde, staining with osmium tetroxide and uranly acetate, dehydration and embedding in epoxy resin are used on the biological technical system consisting of the chip of the subretinal micro electrode implant and of retinal tissue of a mouse. This yields to a compact and fixed sample well manageable for further preparation steps. A difficulty of the sample system present in this study is the biological technical interface being hidden some hundreds of microns inside the sample. The thickness of the chip on one hand and the thickness of the retinal tissue make the access to the interface quite complicated. By utilizing mechanical preparation methods commonly applied to material science samples such as grinding and polishing, a well defined thin section is obtained providing a sample with the chip-retina interface being accessible to FIB–SEM analysis over a wide range of the interface and not only in a limited cross section. Additionally, this preparation procedure makes the interface accessible to light microscopy methods. The resulting light microscopic images aid in defining the area of interest for the subsequent FIB-SEM analysis by correlating transmission light microscope images and reflected-light microscope images to the sample surface images obtained with the SEM. For FIB-SEM analysis, a single stimulation electrode with the adjacent retinal tissue is chosen by light microscopy as the area of interest. Using a crossbeam instrument (Zeiss Auriga 40) equipped with a gallium FIB and a low voltage SEM, FIB-SEM serial sectioning tomography [2] is accomplished. Thereby, the gallium FIB produces a series of blockface cross sections containing the biological technical interface of interest. Each of these cross sections is imaged by the low keV SEM using the energy selected backscattered (EsB) electron detector for image acquisition which yields to images with exceptionally high contrast due to the staining. Additionally, the images show almost no FIB induced artefacts such as curtaining because of the missing topographical information in backscattered electron images. Using typical voxel sizes in the range of 10 x 10 x 10nm, morphological information with high spatial resolution is obtained in the volume of interest. The resulting stack of 2D images is utilized for 3D reconstruction by appropriate software. The open source software ImageJ [3] equipped with the plugins StackReg [4], VolumeJ [5] and 3D Viewer [7] was chosen for image processing and 3D reconstruction of the chip-retina interface. The resulting 3D reconstruction helps to analyse morphological aspects of the interface e.g. the distance of the retinal tissue to the micro electrode. 3D reconstruction of biological technical interfaces is helpful to gain morphological insights of these interfaces. The combination of classical biological and dedicated mechanical preparation methods with 3D FIB/SEM serial sectioning tomography allows size specific nanoanalytics of the complex device containing both soft organic matter and hard material and possessing a biological technical interface of interest considerably hidden inside the sample.