(A) Olfactory bulb neurons were densely labeled with Tetbow AAVs. The majority of M/T cells in the dorsal olfactory bulb were labeled. AON, anterior olfactory nucleus; CA, cortical amygdala; LEC, lateral entorhinal cortex; OB, olfactory bulb; OT, olfactory tubercle; PC, piriform cortex. (B) M/T cell axons were densely labeled in the lateral olfactory tract. An enlarged image of the red boxed area is shown below. A z-stack image of and an image of the x-z section along the orange line are shown. Using this sample, we compared the tracing performance of single-color vs. Tetbow labeling. (C) The definition of successful tracing. In the gray-scale image data, we judged that two axons were successfully dissected when they crossed-over at different z positions (success). We judged that axon tracing failed when the two axons crossed on the same z plane (failure). In Tetbow tracing (full color), we judged that two axons were successfully dissected when the color hues for the two axons were visually discernable (success). We judged that the tracing failed when the two axons crossed on the same z plane and the two colors were visually too alike to discriminate (failure). According to Figure 1H, an experimenter can discriminate between two different colors separated by Euclidean distance 0.1 at >90% accuracy. We did not consider the direction of the crossing axons when we made a decision of success vs. failure. (D) Cumulative plots showing successfully traced axon length. The maximum length of traced axons in the gray-scale image data was 155.229 μm. The maximum length of traced axons in Tetbow (full color) was 318.953 μm. Eight axons out of 100 were fully traced Tetbow (full color), whereas no axons could be fully traced in the gray-scale image. Thus, Tetbow labeling much improves the tracing performance for densely labeled axons. Scale bars are 100 μm (A), 1 mm (B, top), and 50 μm (B, middle and bottom).