1.1  “Load”: Load a movie file (.avi or .mov).

  1.2  “Save”: Preserve the created kymograph as an image data.

2.1  “Trimming”: Crop unnecessary top and bottom regions from of the movie. The specified value represents the width of the regions in pixels.

2.1.1  If you checked “Show Progress”, several intermediate videos during generating kymograph are

      shown.

  2.2  “Generate”: Create a grayscale kymograph and a color kymograph.

2.3  “Rotational Kymograph”: Create a “detailed kymograph” by the rotational watershed method.

     2.3.1  “Rotation (degree)”: Set the interval of the rotation angles for the rotational watershed method. For example, if its value is set at 10, 36 watershed images are generated and then integrated into the detailed kymograph.

     2.3.2  “Denoise”: Set theseverity of the denoising process which removes small fragment traces generated

            by the watershed method.

3.1  “Target”: If “Original” is selected, traces are extracted from the grayscale kymograph. If “Rotation” is selected, traces are extracted from the detailed kymograph.

  3.2  “Detecting Method”: Specify the method for automatic trace detection. If “Watershed” is selected, one-dimensional watershed is used. If “Thresholding” is selected, a simple thresholding operation is used.

 3.3  “Filtering”: Remove unnecessary traces by setting various parameters on trace direction and length, i.e., Displacement (µm), Observed Time (sec), Velocity(µm/s, plus for right direction, minus for left direction).

    3.3.1  “Jaggy Ratio”:  Remove unnatural zigzag lines.

    3.3.2  “Filtering”: Activate the filtering operation with the parameters specified above.

    3.3.3  “Masking”: Superpose traces on Kymograph.

 

  4.1  “Target size”: Set the size (pixel) of brightest point for LoG filter.

  4.2  “Observation width”: Set the physical width of the video frame (µm).

  4.3  “Observation time”: Set observation time length of movie (sec).