The principle of the 3-D worm tracker (3DWT).

<p>(A) Experimental scheme of the stereoscopic recording. Two imaging assemblies at right angles with the same focal point are used to synchronously record images of <i>C. elegans</i> from two perpendicular directions. (B) Stereomatching of skeletons. The two X-Z and Y-Z skeletons, extracted from the two projection images of the worm, are combined using stereomatching to reconstruct the 3-D skeleton. (C) Volume rendering of the 3-D skeleton. The 3-D volume of the worm is rendered by assigning particles that are centered on the 3D skeleton points.</p

Volume-rendered images in three views.

<p>(A) 3-D posture of a worm showing bends in various directions (gray arrows) with only slight changes in the moving direction (green arrow) during crawling. (B) 3-D posture of another worm showing a significant change in the moving direction (green arrow). The asterisk in each image indicates the head of the worm.</p

3-D Worm Tracker for Freely Moving <em>C. elegans</em>

<div><p>The manner in which the nervous system regulates animal behaviors in natural environments is a fundamental issue in biology. To address this question, <i>C. elegans</i> has been widely used as a model animal for the analysis of various animal behaviors. Previous behavioral assays have been limited to two-dimensional (2-D) environments, confining the worm motion to a planar substrate that does not reflect three-dimensional (3-D) natural environments such as rotting fruits or soil. Here, we develop a 3-D worm tracker (3DWT) for freely moving <i>C. elegans</i> in 3-D environments, based on a stereoscopic configuration. The 3DWT provides us with a quantitative trajectory, including the position and movement direction of the worm in 3-D. The 3DWT is also capable of recording and visualizing postures of the moving worm in 3-D, which are more complex than those in 2-D. Our 3DWT affords new opportunities for understanding the nervous system function that regulates animal behaviors in natural 3-D environments.</p> </div

Bending vector analysis of worm motion.

<p>(A) Schematic of the bending vector in a 3-D skeleton. The arrows indicate the bending vector at a skeletal point, P_i. (B) Variable bending vectors of a second skeletal point from the head of a representative worm during crawling. (C) The X-Y projections of bending vectors along the whole body for 5.5 s of crawling. The colors of arrows represent the directions of the bending vectors by linearly converting the components of normalized bending vectors (−1.0∼1.0) to 8-bit colors (0∼255): the X-component is displayed in red (R), the Y-component in green (G), and the Z-component in blue (B).</p

3-D trajectory and velocity of a worm.

<p>(A) 3-D trajectory of the worm. Red, blue, and green arrows are movement directions of a worm for 12 s, 14 s, and 12 s, respectively. (B) The x-, y-, and z-components of the worm's velocity.</p