Lsm 510 vis

A Leica M205 Stereo Microscope was used for documenting live images, videos, and NBT/BCIP developed whole-mount in situ specimens. Zeiss LSM-510 VIS or LSM-700 Upright confocal microscopes were used to capture fluorescent whole-mount in situ specimens and image projections. To quantify the average size of each protonephridial unit and mitotic activity, individual worm was imaged and tiled on a Perkin Elmer Ultraview spinning disk microscope. Stitching and mitotic activity quantification was performed in FiJi using standard plugins (Schindelin et al., 2012 (link)). Worm area, protonephridial size and number were measured/counted using a custom signal to noise thresholding and seeded region grow plugins. Batching was performed using macros. Movement speed quantification was performed on video sequences (acquired at 17.5 Hz) using a custom thresholding plugin and Mtrack2 (Klopfenstein and Vale, 2004 (link)). For each tracked object, the initial position was subtracted from the final to determine an average translocation velocity. Average velocities were computed by weighting track averages by the length of the track. Plugins and macros are available at https://github.com/jouyun.