Under physiological conditions and in the pathogenesis of diabetes mellitus systemic influences play a substantial role for function and survival of cells of the islet of Langerhans. Therefore, in vivo studies to understand islet biology are indispensible and imaging techniques are increasingly used for this purpose. Among the diverse imaging modalities currently only laser scanning microscopy (LSM) allows resolution and visualization of individual cells and cellular processes. To overcome limited tissue penetration and working distance of LSM and enable in vivo investigations of islet cell physiology, various experimental approaches have been developed. Especially, the recently developed imaging platforms have significantly improved the possibility to study islets at a cellular level in vivo, and provided novel insight into islet biology in health and disease. The various approaches, their applications, and reported results, as well as their limitations are reviewed in this article.