TY - JOUR TI - Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle AU - Aragon, Max Jameson AU - Mok, Aaron T AU - Shea, Jamien AU - Wang, Mengran AU - Kim, Haein AU - Barkdull, Nathan AU - Xu, Chris AU - Yapici, Nilay A2 - Bhalla, Upinder Singh A2 - VijayRaghavan, K VL - 11 PY - 2022 DA - 2022/01/24 SP - e69094 C1 - eLife 2022;11:e69094 DO - 10.7554/eLife.69094 UR - https://doi.org/10.7554/eLife.69094 AB - We developed a multiphoton imaging method to capture neural structure and activity in behaving flies through the intact cuticle. Our measurements showed that the fly head cuticle has surprisingly high transmission at wavelengths >900nm, 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 γ-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. KW - multiphoton imaging KW - olfaction KW - behavior JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -