We report the investigation of the interfaces between microneedle arrays and cell cultures in patch-on-chip systems by using Focused Ion Beam (FIB) preparation and Scanning Electron Microscopy (SEM). First, FIB preparations of micro chips are made to determine the size and shape of the designed microneedles. In this essay, we investigate the cell-substrate interaction, especially the cell adhesion, and the microneedle's potential cell penetration. For this purpose, cross-sectional preparation of these hard/soft hybrid structures is performed by the FIB technology. By applying the FIB technology followed by high-resolution imaging with SEM, new insights into the cell-substrate interface can be received. One can clearly distinguish between cells that are only in contact with microneedles and cells that are penetrated by microneedles. A stack of slice images is collected by the application of the slice-and-view setup during FIB preparation and is used for threedimensional reconstruction of cells and micro-needles. SCANNING 34: 221-229, 2012. © 2011 Wiley Periodicals, Inc.