Imaging Neuronal Structure in Antennae and Brains

Serial optical sections of the antennae and brains were obtained at 0.84-μm (brains) or 0.57-μm (antennae) intervals with an FV-1000D laser-scanning confocal microscope (Olympus, Tokyo, Japan) equipped with a silicone-oil immersion 30x (brains) or 60x (antennae) Plan-Apochromat objective lens (NA = 1.05 and 1.3, respectively). For three-dimensional (3D) image reconstruction, confocal image datasets were processed with the 3D-reconstruction software FluoRender (http://www.fluorender.org; Wan et al., 2009 (link)). For the projection analysis and FLP-out image analysis, signals of cells that were not relevant to the traced neurons were erased manually from the original images with FluoRender for clarity (Figures 1, 3). For the images of axonal projections of labeled JO neurons in the brain, cleaned image stacks were aligned to a template brain with non-rigid registration using the Computational Morphometry Toolkit (CMTK; Jefferis et al., 2007 (link)). To visualize the somata locations of single JO-A neurons that innervate specific subarea(s), the location of each somata was mapped manually onto the somata array of JO neurons according to the corresponding confocal image (Figure 5). The size, contrast, and brightness of the images were adjusted using Photoshop CS5 or later (Adobe Systems, San Jose, CA).