About twenty years ago, nanotechnology began to be applied to biomedical issues giving rise to the research field called nanomedicine. Thus, the study of the interactions between nanomaterials and the biological environment became of primary importance in order to design safe and effective nanoconstructs suitable for diagnostic and/or therapeutic purposes. Consequently, imaging techniques have increasingly been used in the production, characterisation and preclinical/clinical application of nanomedical tools. This work aims at making an overview of the microscopy and imaging techniques in vivo and in vitro in their application to nanomedical investigation, and to stress their contribution to this developing research field.