Recently, many super-resolution technologies have been demonstrated, significantly affecting biological studies by observation of cellular structures down to nanometer precision. However, current super-resolution techniques mostly rely on wavefront engineering or wide-field imaging of signal blinking or fluctuation, and thus imaging depths are limited due to tissue scattering or aberration. Here we present a technique that is capable of imaging through an intact Drosophila brain with 20-nm lateral resolution at approximately 200 mum depth. The spatial resolution is provided by molecular localization of a photoconvertible fluorescent protein Kaede, whose red form is found to exhibit blinking state. The deep-tissue observation is enabled by optical sectioning of spinning disk microscopy, as well as reduced scattering from optical clearing. Together these techniques are readily available for many biologists, providing three-dimensional resolution of densely entangled dendritic fibers in a complete Drosophila brain. The method paves the way toward whole-brain neural network studies and is applicable to other high-resolution bioimaging.
