Hardware: FTIR-Based Imaging Method

Since frustrated total internal reflection (FTIR) is used for imaging the contact surface of the target and the surface medium, this technique is called FTIR-based Imaging Method (FIM). For this purpose, infrared (IR) LEDs are placed around an acrylic glass plate. Because of different optical densities of air and acrylic glass, the light enters the glass and is completely reflected at the glass/air boundary (total internal reflection; TIR). For larval locomotion experiments a moist surface (e.g. agar) is placed on the glass. Again, the light enters the agar and is completely reflected at the agar/air and agar/glass boundary. The reflection angles change according to the refractive indices of the media. Given animals touching the agar, refracted light is now below the so-called critical angle: It is no longer totally reflected and can be captured by a camera from underneath.

The illumination intensity is controlled using a micro-controller (pulse-width modulation). A detailed explanation, how to build a FIM setup is given in the construction section . You can also order a preassembled setup in the order section.

Software: FIMTrack

To facilitate 2D locomotion quantification (i.e. tracking) we developed FIMTrack. FIMTrack processes images recorded using a FIM setup to calculate posture and motion related parameters. Although this tracking program is designed for Drosophila larvae, it is also tested for C. elegans worms and planaria.

FIMTrack is available for all operating systems (MS Windows, Mac OS and Linux). A user-friendly graphical interface allows easy interaction with the program. Beside the calculation of the trajectories, there is another module to get detailed informations about the results (called Results Viewer). This module includes the table with all calculated features, images with highlighted posture and motion related features and a plotting functionality. Furthermore, advanced manual tracking can be done easily. For more details see the FIMTrack manual in the downloads section.

Calculated Features

Features calculated by FIMTrack can be divided into primary features (based on the contour), secondary features (based on the primary features) and tertiary features (based on primary features and time). In addition, several stimulus related points, lines and regions can be set via drag and drop which are used to calculate stimulus-related features such as distance to stimulus etc.

Primary Features

  • Momentum
  • Heat and tail points
  • Several spine points
  • Spine point radii
  • Area
  • Perimeter

Secondary Features

  • Body bending angle
  • Spine length
  • Is left/right bended indicator
  • Is coiled indicator
  • Distance to stimulus
  • Is in stimulus region indicator

Tertiary Features

  • Movement direction
  • Velocity
  • Acceleration
  • Accumulated distance
  • Distance to origin
  • Bearing direction
  • Stop/go phase indicator