We developed a multiphoton imaging method to capture neural structure and activity in behaving flies through the intact cuticles. Our measurements show that the fly head cuticle has surprisingly high transmission at wavelengths > 900 nm, and the difficulty of through-cuticle imaging is due to the air sacs and/or fat tissue underneath the head cuticle. By compressing or removing the air sacs, we performed multiphoton imaging of the fly brain through the intact cuticle. Our anatomical and functional imaging results show that 2- and 3-photon imaging are comparable in superficial regions such as the mushroom body, but 3-photon imaging is superior in deeper regions such as the central complex and beyond. We further demonstrated 2-photon through-cuticle functional imaging of odor-evoked calcium responses from the mushroom body g-lobes in behaving flies short-term and long-term. The through-cuticle imaging method developed here extends the time limits of in vivo imaging in flies and opens new ways to capture neural structure and activity from the fly brain.
12A limitation of current methods for examining neural activity over long periods of time in the 13 fly, Drosophila melanogaster, is the need to remove the head cuticle and the underlying tissue 14 to gain optical access to the brain, a process that damages circulation and restricts the length 15 of imaging time. Here, we developed a non-invasive preparation for structural and functional 16 imaging of the fly brain through the intact head cuticle. We first showed that the head cuticle 17 transmits long-wavelength laser light with surprisingly high efficiencies. In fact, the tissue 18 that interferes with laser light during multiphoton imaging is the air sacs underneath the 19 head cuticle. We developed a non-invasive imaging preparation that compresses the air sacs, 20 and used it to image the mushroom body Kenyon cells and central complex ring neurons 21 through the cuticle at cellular resolution using both 2-and 3-photon microscopy. We also 22 performed non-invasive short-term and long-term functional imaging (for the first time for 23 12 consecutive hours) of odour-evoked calcium responses from the mushroom body Kenyon 24 cells. Our results demonstrate that the non-invasive imaging preparation developed here 25 extends the time limits of current in vivo imaging methods used in flies that require an 26 invasive surgery, and opens up new ways to capture neural activity from the intact fly brain.27
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