To begin, take the caenorhabditis elegans slide prepared for imaging. Observe the slide under 4x to 20x magnification lens to locate nematodes. Remember their positions, and then switch to a 63x magnification lens for detailed imaging.
Capture an initial image to evaluate acquisition parameters. Monitor signal intensities using the intensity histogram, and increase excitation laser intensity if necessary. Next, using a 488 nanometer laser, bleach the region of the axon to be analyzed.
Compare the signal intensity before and after the bleaching step to determine the bleaching efficiency. Use two by two binning to enhance the signal intensity of individual transport events. Enhance the exposure time and use a temporal resolution of 300 to 700 milliseconds between consecutive time points to follow Rab3 transport events.
Record a time lapse of one to three minutes based on the signal intensity and frequency of transport events.
