Track-A-Worm 2.0: A Software Suite for Quantifying Properties of C. elegans Locomotion, Bending, Sleep, and Action Potentials

Spline generation and bending property quantification. a, Conversion of a grayscale image (left) to a binary image (middle), followed by spline fitting to the binary image (right). The spline is generated by placing 13 markers at equal intervals along the longitudinal axis of the worm's body, with the marker at the head tip shown in red and the centroid in orange. b, Bend trace of a worm recorded over 30 s. c, Bending frequency spectrum of the same worm, with the dominant bending frequency and RMS (root mean square) bend angle from the Results section displayed within the graph. d, Quantification of the maximum bend. Left, Diagram illustrating the definition of the maximum bend, which is the maximum bending angle of a spline marker point in the ventral and dorsal directions relative to a straight line fitted to the two subsequent spline markers. Right, A bend trace with x and y coordinates marking the times and bending angles of alternative peaks and troughs. The coordinates are determined by clicking on the peaks and troughs, using a 20° threshold.

Analysis of locomotion directionality, speed, and body curvatures. a, Directionality is determined by examining the relationship between the velocity vector and the head vector. The velocity vector connects the centroids of the current (#2) and previous (#1) frames, while the head vector connects the current centroid to the current head point (a or b). If the head vector projects onto the positive side of the velocity vector, the worm is moving forward; otherwise, it is moving backward. b, Method for quantifying worm amplitude. A rectangle is drawn parallel to the velocity vector, just large enough to enclose all spline markers. The width of the rectangle is the worm's amplitude. c, Reconstructed worm travel paths based on the positions of the centroid (left) and spline marker 1 (right). d, Plots of forward and backward locomotion speeds over time shown for interpolated data. e, Method for quantifying body curvatures. Circles are fitted to worm segments, with curvature values calculated as the worm length (L) divided by the radius r of each fitted circle (L/r).

Quantification of sleep duration in freely moving worms using SleepTracker. The actogram shows the locomotor activity of a worm developing from the L4 to young adult stages. Images were captured at a rate of 1 frame every 10 s. Sleep duration, highlighted by the rectangular box, is defined as the period from the start of three consecutive motionless frames to the end of the last three motionless frames. The worm is considered motionless if the difference between the centroid positions of two consecutive frames is <10 µm. Spikes within the sleep period are classified as active events. Extended Data Figure 4-1 illustrates the design of the PDMS chamber.

Quantification of action potential (AP) properties using AP Analyzer with two different approaches. a, AP threshold is the membrane potential at a user-defined pre-AP peak time for APs lacking a preupstroke inflection point. b, AP threshold is automatically detected for APs with a preupstroke inflection point. The representative APs are from a body-wall muscle cell of a wild-type C. elegans (a) and a suprachiasmatic nucleus (SCN) neuron of a wild-type CBA/CaJ mouse (b). The RMP is defined as the 20 ms average preceding the user-selected time point for non-inflected APs, and as the lowest 3 ms average within that 20 ms window for inflected APs. The rise time is defined as the time from AP threshold to AP peak, the decay time from AP peak back to the membrane potential matching the AP threshold, and afterhyperpolarization (AHP) as the actual membrane voltage. c, Voltage phase plot of an SCN neuron AP from a mouse, illustrating the quantification of AP maximum and minimum slopes, corresponding to the points where the membrane potential increases and decreases most rapidly, respectively.