2. Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids MI USA
3. Department of Genetics, Michigan State University, East Lansing MI USA
4. Mager Scientific, Dexter MI USA
In our initial attempts to use video-tracking to measure basal slowing and thrashing, we encountered two main obstacles: using a camera mounted on a dissecting microscope resulted in too small of a field of view, such that worms could enter and exit the field during the assay, and using a brightfield illuminator made it difficult to have sufficient contrast between the worms and the bacteria. To overcome these limitations we developed a microscope-free system for measuring movement (see Fig 1A for image). To expand the field of view, we have utilized a macro lens (Navitar Zoom 7000) attached directly to a FireWire camera (Allied Vision Technologies Stingray F-504B 2/3” CCD Monochrome Camera), both mounted on a weighted boom stand. To achieve increased contrast, we employed a LED darkfield base illuminator (Nikon Model P-DF).
For those who have not measured basal slowing before, we provide our standard protocol. Approximately 50 worms (day 3 of adulthood) are washed in 1.5 ml of M9 buffer to remove any residual bacteria, and allowed to sink to the bottom by gravity. The 50 worms are then transferred in 250 µl of liquid to 60 mm NGM plates that are either unseeded or seeded completely with OP50 (seeded plates are covered completely with bacteria and allowed to dry and grow for 48 hours). The 250 µl of liquid is spread out over the plate and the plate is left uncovered to facilitate drying. After 5 minutes of acclimation, videos of the largest square that would fit inside the edges of a plate were recorded for 1 min using and the MATLAB image acquisition tool (see Fig 1B for typical zoom). Videos were recorded in 8-bit at 2452×2056 resolution with a frame rate of 9fps (540 frames for the 1 minute video).
For analysis, videos were imported to imageJ as AVI files using the wrMTrck plugin. This plugin is publically available at http://www.phage.dk/plugins. The website also includes detailed instructions for thresholding and analysis. Briefly, videos are thresholded and, after defining min/max pixel size for a worm, quantification of crawling speed on seeded plates and unseeded plates is performed. Basal slowing is then calculated as the difference in rate of movement on unseeded plate versus seeded plates divided by the rate of movement on unseeded plates (See Fig 1C for typical data). Thrashing rate, the rate of movement in liquid, can also be measured using this set-up. For this assay, approximately 50 worms are transferred to an unseeded 60 mm NGM plate to which 1 ml of M9 buffer is added. Videos are recorded and analyzed as with basal slowing although we typically zoom in further to measure the thrashing rate.