# Physical setup of an arena

Think carefully about the kind of enclosure to use in your experiments. Consider both the background of the arena, as well as its size and position.

Location

Place the computer and all electrical equipment other then the cameras in a room separate from the test room. Computers and electrical devices produce ultrasound, which may affect the behavior of the subjects.

For specific tests, see also the EthoVision XT 19 - Application Manual. See Manuals

tip Watch the video Set Up Your Test Environment, which you can find in the EthoVision XT video tutorial (Help > Video Tutorial).

Background

▪The animals you are tracking should be able to move freely and naturally.

▪The background must be fixed in relation to the camera. Make sure you firmly anchor the entire setup when it is completed. If the arena moves, you must update the background image.

▪The background should be made of non-reflective surfaces. If this is unavoidable (e.g. the water in a Morris water maze) use indirect illumination (e.g. bounce light of the ceiling) and non-reflective surfaces (e.g. black pool sides). For details, see Lighting setup.

▪There should be maximum contrast between the background and the animal. For a Morris water maze:

▪If you use dark animals, color the water with white tempera paint.

▪If you use white animals, add 300 g of tempera black nontoxic powdered paint to a 45 liter pool.

Dimensions of the arena

▪The arena should have no 'depth', because the camera can only see in two dimensions. If the animal can move towards or away from the camera, like when climbing on a shelter, its apparent size will vary. This may interfere with subject detection and tracking.

▪If you want to track animals in a three-dimensional environment like an aquarium, then make sure this is as shallow as possible, or only calculate variables like In zone, not Distance moved or Distance to a zone. For true 3D movement analysis, contact your nearest Noldus office.

▪The maximum arena size (in pixels) is limited by the image resolution (W x H) of the digital video file you are using, or the resolution of the live camera image set in the Experiment Settings. Your subject must be at least three pixels wide in order for EthoVision XT to distinguish it from system noise. Therefore, the ratio of your maximum subject size to the maximum arena size is given by the limiting resolution (usually H) divided by three. With digital; cameras and a vertical resolution of 1024 pixels, this is approximately about 300 times the animals' size. In practice the limit is lower, depending on various factors including the animal’s contrast with the background. Especially when tracking small insects, for more robust tracking, make sure that the subject is always at least 10-20 pixels large.

Maximum size of the arena

When you have video of resolution L × H (in pixels), and you want to track a subject of l × w (length x width, in mm), check the following:

▪Calculate the ratio maximum subject size to maximum arena size = H/3.

▪Calculate the maximum arena size = w × (H/3)

If the arena was bigger than that limit, the subject would often be less than three pixels wide and EthoVision XT would not be able to distinguish it from random noise.

Maximum size of multiple arenas in one image

If you use multiple arenas, the calculation applies to the total distance across all arenas, thus if you have four arenas in a square, the maximum width of each one is half what it would be if you had only one arena.