2.6.2. Instantaneous velocity measurement
The instantaneous velocity was analyzed by Fiji (ImageJ) and Origin software. First, ordinal click ImageJ-Process-Subtract Background could decrease the background noise of original images and highlight the fluorescence signal. Then, click ImageJ-Analysis-Set Scale to set the scale bar. After that, the program files of KymographClear Plugin and TrackMate Plugin downloaded from ImageJ’s official website should be directly imported into the plugins folder within ImageJ software, and ImageJ was restarted for subsequent operation.
Importing the refined image, pulling down the right arrow ”More Tools” menu, selecting KymographClear 2.0.0, and sequentially operating Make a segmented line Tool [F3] to manually select the region of interest (ROI) area, SplineTool [F4] to smooth freehand lines, Kymograph generation Tool [F5] to record the kymograph of specific axons. After randomly circling a piece of the background area, the background value was obtained by the Background Tool shortcut key [F6], which was automatically stored in the Kymograph folder to calibrate the fluorescent signal. In turn opening KymographDirect 2.1 software, importing Color Coded Directions named picture in Kymograph folder, clicking Tools-Parameters, inputting and affirming Time Per Frame (ms) and Pixel Size (μm), then adjusting Intensity threshold to ensure that moving particle tracks were tagged, finally, right-clicking upper-right Particle velocities-Times graph to save as a text file.[18] Later data processing was done in Origin software.
Another analysis method also started at importing the refined image. Firstly, selecting Plugins-Tracking-TrackMate to enter the calibration parameter setting window, input the value of Pixel Width (μm) and Time Interval (s). Then, the default settings were applied, such as LoG Detector, HyperStack Displayer and Simple LAP Tracker, and the parameters were adjusted empirically to properly select the target particles. Finally, clicking Analysis in the Display options window, the exported features of all Tracks were saved as a text file. Data analysis and visualization were done in Origin software. The detailed operation could refer to the relevant reference.[19]