Abstract.SignificanceStimulated emission depletion (STED) microscopy has been used to address a wide range of neurobiological questions in optically well-accessible samples, such as cell culture or brain slices. However, the application of STED to deeply embedded structures in the brain of living animals remains technically challenging.AimIn previous work, we established chronic STED imaging in the hippocampusin vivobut the gain in spatial resolution was restricted to the lateral plane. In our study, we report on extending the gain in STED resolution into the optical axis to visualize dendritic spines in the hippocampusin vivo.ApproachOur approach is based on a spatial light modulator to shape the focal STED light intensity in all three dimensions and a conically shaped window that is compatible with an objective that has a long working distance and a high numerical aperture. We corrected distortions of the laser wavefront to optimize the shape of the bottle beam of the STED laser.ResultsWe show how the new window design improves the STED point spread function and the spatial resolution using nanobeads. We then demonstrate the beneficial effects for 3D-STED microscopy of dendritic spines, visualized with an unprecedented level of detail in the hippocampus of a living mouse.ConclusionsWe present a methodology to improve the axial resolution for STED microscopy in the deeply embedded hippocampusin vivo, facilitating longitudinal studies of neuroanatomical plasticity at the nanoscale in a wide range of (patho-)physiological contexts.