biostudies-literatureHsu KJMinistry of Science and Technology, Taiwan1627-1637https://www.ebi.ac.uk/biostudies/studies/S-EPMC6484994biostudies-literatureUnknown10(4)<i>Drosophila</i> 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-<i>Drosophila</i>-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 <i>Drosophila</i>.Biomedical optics expressOptical properties of adult <i>Drosophila</i> brains in one-, two-, and three-photon microscopy.PMC6484994MOST 104-2218-E-007-022-MY2MOST-105-2628-M-002-010-MY4MOST-107-2321-B-002-009Hsu KJLin YYChu SWChiang ASfalseOptical properties of adult <i>Drosophila</i> brains in one-, two-, and three-photon microscopy.<i>Drosophila</i> 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-<i>Drosophila</i>-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 <i>Drosophila</i>.2019-01-01T00:00:00Z2019 Apr2026-07-12T03:24:01.4Z2026-07-12T03:13:15.209ZS-EPMC64849943108669710.1364/BOE.10.00162710.1364/boe.10.001627