Cardiovascular disease is the leading cause of death and morbidity worldwide. Improving vascular prevention and therapy based on a refined mechanistic pervasion of atherosclerosis as the underlying pathology could limit the effect of vascular disease in aging societies. During the past decades, microscopy has contributed greatly to a better understanding of vascular physiology and pathology by allowing imaging of living specimen with subcellular resolution and high specificity. An important advance has been accomplished through the application of multiphoton microscopy in the vascular domain, a technological development that enabled multidimensional and dynamic imaging deep into the cellular architecture of intact tissue under physiological conditions. To identify and validate new targets for treating atherosclerosis, novel imaging strategies with nanoscale resolution will be essential to visualize molecular processes in intracellular and extracellular compartments. This review will discuss the current use of 2-photon microscopy and will provide an overview and outlook on options for introducing nanoscopic optical imaging modalities in atherosclerosis research.