Figure 3: Millimeter-scale imaging of a Drosophila leg at single-neuron resolution.

(a) Schematic of XNH imaging strategy. Ten partially-overlapping XNH scans (Supplementary Data Table 2) were used to capture a front leg’s coxa, trochanter, femur, and half of the tibia, plus the prothoracic neuromere (T1) of the VNC that controls this leg’s movements. The final leg segment, the tarsus, contains no muscles and was not imaged. (b) Photograph of a leg sample after heavy-metal staining, resin embedding, and mounting for XNH imaging. (c) Rendering of reconstructed leg segments, sensory neurons, motor neurons, and muscle fibers. Individual neurons were reconstructed from their target structures in the leg (sensory receptors or muscle fibers) into the VNC. (d-g) Cross-sections through the coxa and femur at locations indicated by dotted lines in (c). Color code is the same as in (c). (d) The arrangement of neurons, muscle fibers, and fat cells in the coxa. (e) Detailed view of nerve from (d). (f) The six long tendon muscle fibers in the femur are organized as a group of two and a group of four, which attach to the long tendon at different locations (see (c)). Asterisk indicates the femoral chordotonal organ, a proprioceptive sensory structure (see Extended Data Fig. 3g)³¹. (g) Detailed view of nerve from (f). Arrowheads indicate swellings of motor neuron axons, likely sites of neuromuscular junctions. Visible here are two swellings from the only motor neuron that innervates these two fibers (light green, see (c)). A different motor neuron (dark green, see (c)) branches off of the leg nerve, traveling past the proximal fibers to innervate the distal fibers. (h) Muscle fibers of the femur. The 97 fibers identified in the femur using XNH is nearly triple that reported previously using fluorescence microscopy³⁸.