Figure 1. The leg-tracker apparatus and its properties.

(a) Schematic of the leg-tracker apparatus. A fly is mounted by wire tether to a clear sphere supported by flowing air. Fictive motion of the fly is recorded by tracking sensors. The dye spots on the leg fluoresce when excited by the HeNe laser and a second sphere re-collimates the image of the dye spots that are tracked from below by two cameras. (b) Photograph of a fly with bits of 221ox and julox dyes (arrowheads) glued alternately on each leg. (c) Cartoon illustrating the positioning of the dye spots. Dye was placed upon the femurs of the fore and hind limbs and upon the tibia of the middle legs. (d) Imaging of dye spots and detection of their positions. Top panel shows differential detection of alternating legs marked with the two dyes. Because the dyed segments on the front and hind legs do not cross, leg identity can be readily inferred in single frames (bottom panel). (e) Diagram of the absorption wavelengths of the Drosophila rhodopsins (top). Diagram of the absorption (dashed lines) and emission (solid lines) wavelengths of the 221ox and julox dyes and their chemical structures (middle). The dyes are compatible with green fluorescent protein (GFP). Emission wavelengths of the HeNe laser, vertical cavity surface emitting laser (VCSEL) of the tracking sensors and Ti:Sapphire lasers are depicted as vertical lines (bottom). Also shown are the transmission properties of the rig dichroic filters. (f) Representative data showing the 15 data vectors (3 for ball motion and 12 for leg positions) being recorded over 7,200 s (top). Magnification of a 120s data slice (middle). Higher magnifications of two typical behaviours (bottom). Red, black and green traces represent the ball’s pitch, yaw and roll components, respectively. Grey and blue traces are the x and y coordinates, respectively, of each leg. Plots are in arbitrary units, with ball motion vectors and leg-position components respectively comparable. Instrument output is calibrated. (g) Analysis of swing and stance phases during the second half of the running bout magnified in f. Blue lines correspond to the last six data vectors in f. Colour bar indicates the number of legs in swing phase at each frame, that is, the gait index—with Te indicating tetrapod gait (green) and Tr indicating tripod gait (orange).