Field Emission Scanning Electron Microscopy (FE-SEM) is an advanced type of microscopy that uses a field emission gun as the source of electrons. Consequently, FE-SEM uses a smaller beam size than conventional scanning electron microscopy (SEM), resulting in a high degree of spatial resolution. In addition, this microscopy modality allows the use of very low voltages, which helps to minimize the charging effect, being particularly delicate with the most electron beam-sensitive samples. In this context, FE-SEM is an invaluable tool for the study of very fine cell surface features. By combining the advantages of FE-SEM with colloidal gold-labeling, comprehensive quantitative and qualitative studies of the localization of surface molecules of interest can be performed. This chapter outlines the main technical and methodological considerations related to cell sample preparation, colloidal gold-labeling of surface molecules, and FE-SEM image acquisition and processing with particular focus on the study of sperm cells. In conclusion, the high degree of polarization and the presence of pivotal membrane molecules of the sperm cell render it an exceptionally intriguing type of cell to analyze using FE-SEM and colloidal gold-labeling.