Implementation of an adaptive-optics assisted isoSTED nanoscope

In stimulated emission depletion (STED) nanoscopy, high 3D resolution requires harnessing a 4Pi architecture using two opposing objectives. Here, we provide the step-by-step process for the construction and alignment of a 4Pi-STED nanoscope, commonly referred to as an 'isoSTED nanoscope'. The procedure guides interested researchers through the assembly of the optomechanical components, the configuration of the electronic and control devices, the alignment of the optical beam path and the assessment of the instrument's performance. The protocol offers a detailed roadmap for constructing an isoSTED nanoscope with adaptive optics in about 12 months and is designed for users with expertise in optical instrumentation builds. Once the instrument is finely calibrated, researchers can expect to achieve 3D biological images with isotropic sub-50-nm resolution in thick samples ≤35 µm in depth.