Growth cones stall and collapse during axon outgrowth in Caenorhabditis elegans
Karla M. Knobel, Erik M. Jorgensen and Michael J. Bastiani
Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA
Confocal microscopy
We used a BioRad MRC 600 laser scanning confocal microscope equipped with a 1% neutral density filter to image living growth cones. We identified candidate worms by scoring the recent division of their intestinal nuclei using DIC optics. To locate migrating growth cones, we scanned selected animals at low magnification (60¥, aperture 2/3 open). Selected growth cones were centered in the field of view and the scanning box size reduced to a 256 square pixel field. We increased the image zoom from 1.0 to 3.0-3.5 (magnification 60x to 180-210x) and reduced the confocal aperture. The start and stop of the Z-series were set using the motor drive. Generally, 1 mm steps between each slow scan were used with Kalman set at 1 (each Z-series averaged 3 steps). Changes in step size were modified to correspond with the aperture setting. Z-series were taken at 2 minute intervals. Bio-rad confocal 'pic' files were converted into Quicktime movies using the 4D-Turnaround program designed by Charles Thomas and licensed from the Integrated Microscopy Resource Center at the University of Wisconsin (Thomas et al., 1996).
