Abstract
The highest level of three-dimensional (3D) resolution in stimulated emission depletion (STED) nanoscopy involves harnessing a 4Pi architecture using two opposing objectives. This protocol describes the construction and alignment of a 4Pi-STED nanoscope, commonly referred to as an isoSTED nanoscope. It guides interested researchers through assembling optomechanical components, configuring electronic and control devices, aligning the optical beam path, and assessing the instrument's performance. Designed for adept optical instrument builders, this protocol offers a detailed roadmap for constructing an isoSTED nanoscope with adaptive optics (AO) in approximately 12 months. With this finely calibrated instrument, researchers can achieve 3D biological images with isotropic sub-50-nm resolution in thick samples up to 35 µm in depth.