Technical Aspects of Ion Milling and Electron Imaging of Epoxy Embedded Samples for FIB/SEM Tomography

Abstract

FIB/SEM tomography is a relatively new imaging technique for 3D investigation of biological tissue. It uses a dualbeam FIB/SEM instrument to alternately image a sample surface with an electron beam and mill off successive slices with an ion beam, collecting a series of images representing the investigated volume. This technique was employed to study alumina nanoparticles, brain tissue and tissue engineered cartilage embedded in epoxy. Hydrocarbon deposition of contamination layers as well as suboptimal sample geometry, were shown to be issues for imaging experiments. It was important to ensure that sample blocks were cut smoothly, and that the material of interest was concentrated at an edge. The alumina nanoparticle sample was used as a model specimen to investigate the volume of origin of signals detected for image formation at different acceleration voltages. This was shown to have a reasonable correspondance with Monte Carlo simulation results. Imaging and Slice and View experiments on the biological samples showed that FIB/SEM tomography can resolve structures below 10nm in size, and that detailed 3D models from properly stained tissue are obtainable. In summary FIB/SEM tomography constitutes a valuable 3D imaging technique for biological samples.