To begin, record a timelapse of endogenous GFP Rab3 in the asynaptic zone of the DA9 axon in a live Caenorhabditis elegans. Import the timelapse data file into Fiji. Then check for movement or slight drift of the animal or axon segment during image acquisition.
Use the StackReg plugin with a rigid body transformation option to correct for any movement or drifting. Employ the segmented line tool and adjust the line width to match the axon diameter. Double click with the left mouse button on the segmented line icon to draw a line along the axon segment for analysis.
Now using the Fiji plugin KymoResliceWide, generate the kymograph, setting the maximum intensity value across the width of the line in its parameter settings. Using the straight line tool, trace transport events in the kymograph. trace individual transport events and save each line to the ROI manager.
In Fiji, select measurement parameters such as area, bounding rectangle, mean gray value, and Feret's diameter. Then paste the results table into a spreadsheet for calculation. Multiply the width by the resolution of the camera to get the run length in micrometers.
To determine the duration of a movement event, multiply the height by the acquisition time between consecutive time points. Calculate pausing time as the run duration between two consecutive movements where velocity is zero. Normalize the total events to the total length of the kymograph for event number per minute and axon length segment.
Using the Ferret angle tool, categorize events into anterograde and retrograde transport. For kymographs generated by drawing the segmented line from proximal to distal, use the Feret angle to determine the directionality of each event.
