<p>Understanding brain function and pathology requires observation of cellular dynamics within intact neural circuits. Although two-photon microscopy revolutionized mammalian in vivo brain imaging, its limitation to upper cortical layers has restricted access to many important brain regions. Three-photon microscopy overcomes this constraint, enabling minimally invasive yet high-resolution visualization of the deep cortical and subcortical structures that are crucial for higher-order brain functions. This emerging technology opens new avenues for investigating fundamental aspects of neuroscience, from circuit dynamics to disease mechanisms. Here, we examine how three-photon microscopy has started to transform our ability to investigate neural circuits, glial biology, and oncological and neuroimmune interactions in previously inaccessible brain regions, primarily in the mouse, but also in other model organisms. We discuss current technical challenges, recent innovations and future applications that promise to bring us greater understanding of the living brain.</p>