Knowledge Base

Calibrate the Arena

Aim

To convert the subject's coordinates expressed in pixels to real-world coordinates. Avoid barrel and spherical distortion by using correct lenses. Place the camera above the middle of the arena, and perpendicular to the arena surface. If you use Track3D, do not perform calibration within EthoVision XT; instead, make sure that you set Pixel calibration to ON.

Prerequisites

• You grabbed a background image.
• You know the real-world size of the arena. Measure this distance at the height of the animal, not at the top of the arena.

Procedure

1. In the Arenas and Zones section, click 1. Draw Scale to Calibrate.
2. Click the mouse button on the first point in the video image, hold the button down, and drag to the second point in the image. Then release the mouse button.
3. In the Calibration Distance window, type in the real-world distance between the two points.

Notes

• Repeat the steps above for all calibration lines. EthoVision XT uses the average of the scale factors obtained from each calibration line. This factor is used to analyze movement of the subject in all directions. Draw calibration lines in both directions, horizontal and vertical.
• For each calibration line, EthoVision XT calculates a scale factor (real-world distance entered divided by the length of the line in pixels). If this factor differs by more than 20% from the average of the other lines, a message is shown: "Calibration scale substantially larger or smaller than other existing calibration scales." Click No to edit the real-world distance, or click No and then Cancel to re-draw the last calibration line. Click Yes to accept the value. Be aware that very different scale factors may produce false results.
• If you enter a real-world distance smaller than 10 mm or greater than 10 m, a message is shown. Click Yes to confirm the value or No to edit it.
• If you have clicked Cancel and want to repeat the calibration procedure, click again 1. Draw Scale to Calibrate.

Calibrating with the Noldus Calibration Card

1. Open the Grab Background Image dialogue (also accessible via Arena Settings > Arenas and Zones > right-click on Background > Grab Background Image).
2. Place the Noldus Calibration Card in view inside the arena. Make sure that the black rectangular outline within the card is clearly visible in the camera view. If you are using a dark arena, you can place a piece of white paper behind the card.
3. Click Calibrate. The Calibration dialogue will open.
4. Click Calibrate to accept or decline the result.
5. If calibration using the Noldus Calibration Card fails, you can use the manual calibration procedure described above.

Draw the Arena

Aim

To define an area of interest so that EthoVision XT ignores all activity outside this area, so only movement inside the arena is tracked. With multiple arenas within one video image, you can track more animals simultaneously and separately. Each arena is an independent replicate.

Prerequisites

• You have calibrated the video image.

Procedure

1. Click 2. Select Shape and Draw Arena in the Arenas and Zones section. When working with multiple arenas, click the layer for the arena you want to draw.
2. Select a shape on the Arena Settings toolbar at the top of the window by clicking it or by pressing the corresponding shortcut key. To split an arena into quadrants automatically (for example, in a Morris water maze or an open field), choose Create Subdivided 3-Point Circle or Create Subdivided Rectangle.
3. Create a shape that covers the region on the screen in which you want EthoVision XT to track the subject. Make sure the arrow point of the arena label is inside the shape you just created.

Arena Settings

Add Subdivided Arena

If you have chosen one of the subdivided options, the Add Subdivided Circle (or Rectangle) window appears.

• For circular arenas, enter the number of sectors (for example, 4 for quadrants in a Morris water maze). Default = 2, maximum = 50.
• For rectangular arenas, enter the number of rows and columns. A total of rows x columns equal zones will be defined.

In the Arenas and Zones section, a new zone group named Subdivision Zones is added, listing the new zones.

Notes

• If you do not draw an outline, the whole background image is considered as the arena.
• If you have multiple arenas with the same size and shape, you do not have to draw each arena separately. You just draw one arena and duplicate it.
• The shape is filled with the color visible next to the arena name in the Arenas and Zones section. To change this color, click the color next to the name.
• To create a circle or a square, select the Ellipse or Rectangle tool respectively, and hold Shift down while you draw the shape.
• To export the image of the arena, click the camera button on the toolbar. Give the export file a name and select a picture format (*.png, *.jpg, *.bmp, *.gif). The image contains all layers that are currently visible.
• For an experiment in DanioVision mode, EthoVision XT can detect the arenas automatically. See the DanioVision DVOC-0041 Reference Manual.
• You cannot delete an arena that has been used for acquisition; it has become read-only. To edit it for future tracks, duplicate the arena, edit the copy, and use these settings for acquisition.

Make a Subdivided Arena

When you draw an arena using the subdivided tools, note the following:

If the Auto-Label Zone is active, all sectors are automatically defined as zones and labeled with S1, S2, etc. A layer named Subdivision is added in the Arenas and Zones section together with its zones.

When the Auto-Label Zone is active, the Auto-Label Zone button is highlighted in orange.

If the Auto-Label Zone button is not highlighted, the arena is still divided in sectors, but the resulting zones are not labeled. Click the Add Zone Label button to label the zones.

To activate or deactivate Auto-Label Zone, click 3. Select Shape and Draw Zones (Optional) in the Arenas and Zones section and click the Auto-Label Zone button.