This article describes a protocol that details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immunogold labelling of proteins, and visualization by Field Emission Scanning Electron Microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high resolution microscopy. The nuclear isolation step is performed by enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of nuclei by centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs), and associated cytoskeletal structures. Samples, once processed for FESEM, can be stored under vacuum for weeks, allowing considerable time for image acquisition.