Abstract: A limitation of current methods for examining neural activity over long periods of time in the fly, Drosophila melanogaster, is the need to remove the head cuticle and the underlying tissue to gain optical access to the brain, a process that damages circulation and restricts the length of imaging time. Here, we developed a non-invasive preparation for structural and functional imaging of the fly brain through the intact head cuticle. We first showed that the head cuticle transmits long-wavelength laser light with surprisingly high efficiencies. In fact, the tissue that interferes with laser light during multiphoton imaging is the air sacs underneath the head cuticle. We developed a non-invasive imaging preparation that compresses the air sacs, and used it to image the mushroom body Kenyon cells and central complex ring neurons through the cuticle at cellular resolution using both 2-and 3-photon microscopy. We also performed non-invasive short-term and long-term functional imaging (for the first time for 12 consecutive hours) of odour-evoked calcium responses from the mushroom body Kenyon cells. Our results demonstrate that the non-invasive imaging preparation developed here extends the time limits of current in vivo imaging methods used in flies that require an invasive surgery, and opens up new ways to capture neural activity from the intact fly brain.