Whole-brain imaging and characterization of Drosophila brains based on one-, two-, and three-photon excitations

To study functional connectome, optical microscopy provides the advantages of in vivo observation, molecular specificity, high-speed acquisition, and sub-micrometer spatial resolution. Now, the most complete single-neuron-based anatomical connectome is built upon Drosophila ; thus it will be a milestone to achieve whole-brain observation with sub-cellular resolution in living Drosophila . Surprisingly, two-photon microscopy cannot penetrate through the 200-μm-thick brain, due to the extraordinarily strong aberration/scattering from tracheae. Here we achieve whole- Drosophila -brain observation by degassing the brain or by using three-photon microscopy at 1300-nm, while only the latter provides in vivo feasibility, reduced aberration/scattering and exceptional optical sectioning capability. Furthermore, by comparing one-photon (488-nm), two-photon (920-nm), and three-photon (1300-nm) excitations in the brain, we not only demonstrate first quantitative reduction of both scattering and aberration in trachea-filled tissues, but unravel that the contribution of aberration exceeds scattering at long wavelengths. Our work paves the way toward constructing functional connectome in a living Drosophila .