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]