Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle

Abstract

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

Set lasers to image

“…"But 3 photon has also been super useful in fruit flies. " In flies, for example, the 3P lasers are able to make it through the insect's thick cuticle 7,8 .…”

PhotoGal4: A Versatile Light-Dependent Switch for Spatiotemporal Control of Gene Expression in Drosophila Explants

“…For instance, surgical windows in head and thorax of living fruit flies are becoming common practice for central nervous system imaging in freely walking flies ( Chen et al., 2018 ; Huang et al., 2018 ; Simpson and Looger, 2018 ). Similarly, adaptation of multiphoton microscopy to live flies is facilitating high-definition imaging of deep structures such as the brain mushroom bodies ( Aragon et al., 2019 ). Thus, we believe that the combination of these technologies with PhotoGal4 will capture the imagination of the community and stimulate exciting new ideas to expand the horizons and applications of our system in the future.…”

Automated long-term two-photon imaging in head-fixed walking Drosophila

“…A preparation for repeated imaging over up to 50 days through a transparent window allowed functional imaging in an epifluorescence widefield microscope after anesthetizing and reintroducing the fly into the setup for each experiment 9 . Three-photon imaging of neural activity in intact walking flies over 12 hours was performed for bright and large neurons close to the surface of the brain 10 .…”