Optical properties of adult Drosophila brains in one-, two-, and three-photon microscopy.

Drosophila is widely used in connectome studies due to its small brain size, sophisticated genetic tools, and the most complete single-neuron-based anatomical brain map. Surprisingly, even the brain thickness is only 200-μm, common Ti:sapphire-based two-photon excitation cannot penetrate, possibly due to light aberration/scattering of trachea. Here we quantitatively characterized scattering and light distortion of trachea-filled tissues, and found that trachea-induced light distortion dominates at long wavelength by comparing one-photon (488-nm), two-photon (920-nm), and three-photon (1300-nm) excitations. Whole-Drosophila-brain imaging is achieved by reducing tracheal light aberration/scattering via brain-degassing or long-wavelength excitation at 1300-nm. Our work paves the way toward constructing whole-brain connectome in a living Drosophila.