For centuries scientists have been fascinated by the structure of the brain, all the more since the middle of the 19th century when advances in staining techniques enabled high-resolution light microscopic studies of brain cell morphology. Compared to studying cell morphology, it has been more difcult to experimentally investigate the dynamic nature of brain cells, including their morphological transformations, as well as the molecular and electrical signaling events underlying their specic functions. Still today, it remains challenging to study neural dynamics on the cellular level in intact brains of living animals. For many years, in vivo studies of brain cell dynamics relied solely on electrical recordings of neuronal activity, using either extracellular recordings of neuronal spike patterns or intracellular recordings of membrane potential

dynamics. Strong light-scattering of neural tissue generally precluded optical imaging with cellular and subcellular resolution in the intact brain (with few exceptions in favorable cases1). A major breakthrough in 1990 was the invention of two-photon excited uorescence laser scanning microscopy (TPLSM),2 which enabled optical studies of brain cell morphology and function in vivo.3,4 The success of TPLSM has been fostered by the parallel development of various novel staining techniques for in vivo labeling of brain cells with uorescent markers. Expression of variants of uorescent proteins by genetic means5,6 complements the in vivo imaging capabilities of TPLSM particularly well. The combination of high-contrast uorescence labeling of brain cells-neurons as well as glial cells-with high-resolution imaging in vivo has opened new research elds in neuroscience. It is now possible to watch brain cells “at work” so that neuroscientists can directly study cellular and molecular mechanisms underlying both normal brain function as well as brain diseases. Because of its great potential for gaining fundamental insights into how the brain develops, how it computes, and how it can adapt, in vivo two-photon imaging of brain cell dynamics has enormously expanded over the past decade, and it is still growing at a rapid pace.