In vivo STED microscopy: A roadmap to nanoscale imaging in the living mouse

Superresolution microscopy techniques are now widely used, but their application in living animals remains a challenging task. The first superresolution imaging in a live vertebrate was demonstrated with STED microscopy in the visual cortex of an anaesthetized mouse. Here, we explain the requirements for a simple but robust in vivo STED microscope as well as the surgical preparation of the cranial window and the mounting of the mouse in detail. We have developed a mounting stage with a heating plate to keep the mouse body temperature stable and that can be adjusted to the optical axis of the microscope. We have optimised the design to avoid inducing thermal drift, which is critical for nanoscale imaging. STED microscopy with a resolution of 60 nm requires special cranial window preparation to avoid motion artefacts. We have implemented a drain tube to reduce the fluid between the glass window and the surface of the brain, which has been identified as the main cause for the motion artefacts. Together, these advances in the preparation allow the use of a simple intraperitoneal anaesthesia and make the previously used venous infusion and artificial respiration obsolete.