How to track protists in three dimensions

We present an apparatus optimized for tracking swimming micro-organisms in the size range of 10-1000 mu m, in three dimensions (3Ds), far from surfaces, and with negligible background convective fluid motion. Charge coupled device cameras attached to two long working distance microscopes synchronously image the sample from two perpendicular directions, with narrow band dark-field or bright-field illumination chosen to avoid triggering a phototactic response. The images from the two cameras can be combined to yield 3D tracks of the organism. Using additional, highly directional broad-spectrum illumination with millisecond timing control the phototactic trajectories in 3D of organisms ranging from Chlamydomonas to Volvox can be studied in detail. Surface-mediated hydrodynamic interactions can also be investigated without convective interference. Minimal modifications to the apparatus allow for studies of chemotaxis and other taxes.