Fig. 1. Generation of binarized movies of medaka behavior during the O–O test using the UMATracker program. Generation of binarized movies of medaka behavior.

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Fig. 1. Generation of binarized movies of medaka behavior during the O–O test using the UMATracker program. Generation of binarized movies of medaka behavior during the O–O test using the UMATracker program. (A) An original image recorded by the video camera. The rotating stripes were at the edge of a purple circle (arrowhead), and medaka swam within the inner circle (i.e. the cylindrical tank; arrow). The dark circle at the center is the 50 ml tube that was used to prevent shortcut swimming during the OMR. The irradiated light appeared as red to the human eye, but was recorded as purple by the IR-recording video camera. (B) Background subtraction. All objects that did not move from the beginning to the end of the movie were regarded as background and subtracted from each frame, which retained the medaka, rotating stripes, and noise items (e.g. feces at the bottom of the tank or reflection at the surface). (C) Conversion to gray scale. The RGB image was converted to gray scale prior to binarization. (D) Binarization. By setting an appropriate threshold, only medaka should become white, while other items should become black. In this image, the rotating stripes and light fluctuations remained as noise, which had to be eliminated in the subsequent steps. (E) Definition of the tracking area. The noise from the rotating stripes was excluded. (F) Noise reduction I. The Median filter removed small dots without affecting big dots. (G) Noise reduction II. The Erosion filter reduced the size of all dots. Only the largest dot (i.e. medaka, in most cases) remained after this filter was applied. (H) Noise reduction III. The Dilation filter recovered the volume of the remaining dots. This step was dispensable but helpful in detecting the remaining noise manually. If such remaining noise was detected in this step, the Median and/or Erosion filters were applied again, because any noise may disrupt tracking. The goal of this binarization was to obtain a movie in which a single white dot was moving on a black background. Megumi Matsuo et al. Biology Open 2018;7:bio033175 © 2018. Published by The Company of Biologists Ltd