Activity Recording Setup

1. Soak specimen plate in 1% bleach for 1 h, then rinse repeatedly with hot tap water, then deionized water, then egg water.

2. In the afternoon or evening of the first recording day, transfer larvae to specimen plate wells. Use the baby tube to gently strain out larvae, then invert the tube and wash the larvae into clean egg water in a 100-mm Petri dish.

3. Fill the specimen plate wells with egg water. Use a large-bore pipet to transfer larvae to individual wells. Avoid transferring water with the fish. Touch the tip of the pipet to the water surface and encourage the fish to swim down with gentle tapping (see Note 11).

Fig. 2. Control panels for the image analysis software.

4. Set up the camera and illumination system in a light-tight, refrigerated incubator maintained at a constant 24°C. The light source is located outside the incubator, with fiberoptic leads passing in through a light baffle. Set the illuminator at 50% power. Humidify the incubator by bubbling air through a large open reservoir of water.

5. Place the specimen plate directly on the reflecting surface of the mirror, and position the diffuse axial illuminator over the specimen plate.

6. Launch Optimate and the "Swimming" macro (Fig. 2, left), and click on "Acquire" to view the digitized video image (Fig. 1A). Focus the camera so that the array of occupied wells fills the image field.

7. Set the camera controller: "Automatic gain control" on; "Autoenhance" off; "Boost" off; "Detail" at minimum; "Shading correction" normal.

8. With the "Swimming" macro in "Acquire" mode, use the four "Shading Correction" dials to even out any detectable variations in background shading from center to periphery, side to side or up and down. To fine-tune the image shading, click on "Freeze" to capture a still image, then on "Monochrome Threshold" to bring up the "Threshold" dialog window (Fig. 2, right). Set the lower threshold to 0, and then scan the upper threshold up and down to visualize any shading variation in the image. If shading variations exist, close the "Threshold" window, click on "Acquire," and repeat these steps until variations are minimized (see Note 12).

9. Set a threshold range that, as much as possible, includes all the fish and excludes all the background (see Note 13).

10. Set sampling parameters in the "Images from Camera" and "Process Cycle" sections of the "Swimming" macro window (Fig. 2, left). "Images/Cycle," 38; "Delay," 600 ms; "Cycle Length," 240 s (see Note 14).

11. Freeze the image and click on "Region of Interest," then use the cursor to draw a rectangle that encompasses all the wells. Set the number of "Rows" and "Columns" of wells. Click on "Test Layout" to superimpose a grid produced by dividing the region of interest into the specified rows and columns onto the image. Repeat these steps if every cell in the grid does not encompass one and only one well.

12. Create an ASCII data file by clicking on the "?" button, selecting a folder, and entering a file name with a .txt extension. If a file with the same name and path already exists, a dialog that asks whether to "Replace the existing file," "Append the new data," or "Cancel" pops up.

13. Click on "Go" to start automated image capture and analysis. A window with a "Stop" button will pop up.

14. Once each day, check the swimming paths that are superimposed on the image at the end of a cycle (Fig. 1C). If any problems appear (see Note 15), click on the "Stop" button, which works only during the image capture phase of the cycle, close and reopen the "Swimming" macro, optimize the image, and repeat steps 9 through 13.

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