Supplemental Data A Map of Olfactory Representations in the Drosophila Mushroom Bodies

Gene expression patterns are revealed by GFP reporter. Brains are counterstained with DiD, a fluorescent membrane probe penetrates readily into thick tissue to label all neuropil structures. Because of near infrared emission, DiD is excellent for multicolor labeling together with GFP expression. We routinely image DiD-labeled brains using a 40x C-Aprochromat water-immersion objective lens (N.A. value 1.2, working distance 220µm). This objective has an extended working distance, which allows imaging of the whole thickness of the Drosophila brains (140-180µm thick). To overcome the limited field of view, each brain was scanned in two parallel stacks of confocal images with some overlaps in the middle for each brain hemisphere (S1A). We then stitch the two image stacks into a single dataset with the 3D Montage