Knowledge Base

2 System setup

2 System Setup

Test Environment

Track3D can be used with wind tunnels or any other test chamber with at least one transparent wall.

Wind tunnel

For studies of insect orientation and flight in relation to visual and odor stimuli, a wind tunnel is the optimal set up. Wind tunnels with glass walls and metal frames are not optimal for video tracking as they cause reflections. Noldus offers a complete solution with a high-quality and flexible wind tunnel, optimized for video tracking with Track3D. Our wind tunnel consists of a tracking chamber and an air flow production system. The tracking chamber is made of polycarbonate with a transparent top wall. The black infrared-absorbing walls combined with the infrared LED arrays make it ideal for tracking insects in the dark (provided that the insect body or wings reflect infrared). However, you can also track insects in daylight. Figure 3 Standard wind tunnel. Left: Air flow production system and chamber for the stimulus source. Right: tracking chamber. 2 System setup In our standard wind tunnel, the tracking chamber measures 160 x 60 x 60 cm (L x W x H), but other sizes are possible. Please contact us for more information. The air supply system consists of a fan and an air lamination diaphragm. You can set different air speeds, up to approx. 40 cm/s (with an air flow of approx. 450 m3/h. The multi-layer air lamination diaphragm ensures that air speed and direction are uniform throughout the tracking chamber. Below you find an example of the relationship between flow (in m3/h) and air speed: Note that these figures may vary slightly between wind tunnels. See the documentation that comes with your wind tunnel. For details, see Spitzen, J.; Spoor, C.W.; Grieco, F.; ter Braak, C.; Beeuwkes, J. et al. (2013). A 3D analysis of flight behavior of anopheles Figure 4 The fan with air speed control box. 2 System setup gambiae sensu stricto malaria mosquitoes in response to human odor and heat. PLoS one, 8(5), e62995, doi:10.1371/journal.pone.0062995.

tip Watch the video:

www.youtube.com/watch?feature=player_embedded&v=AEfK2IInWi0

Fish tanks

You can use a wide range of fish tanks to reliably track fish in 3D. The fish tank you use in your experiments determines the shape and size of the calibration object. Contact Noldus if you plan to use a new type of tank. Useful web sites: www.aquaticsolutions.it/index.php www.aquatichabitats.com

Large enclosures

Large enclosures require custom-made calibration frames. Contact Noldus for information. Figure 5 Tracking a bat (red dots) and a moth (blue dots) in a large enclosure. 2 System setup

Lighting

Infrared LED arrays

Use Infrared LED arrays for tracking insects in dim light or in the dark. For tracking mosquitoes in a Noldus standard wind tunnel, we recommend to use at least four LED arrays. Place the LED arrays on a frame, and point them to the downwind mesh screen. Check on the video image that shadows inside the tracking chamber are minimized. Also try to minimize the amount of light hitting the Plexiglas wall. Good lighting is crucial for a good tracking result. A good image of a wind tunnel under infrared lighting may look like the following: Figure 6 Four infrared LED arrays placed at the downwind side a wind tunnel. Figure 7 Camera view of the tracking chamber. 2 System setup

Infrared backlight units

For tracking fish we recommend to use backlight units. This has the advantage that it dramatically reduces reflections and thereby improves tracking. Place the fish tank directly on one unit. Place another backlight unit behind the fish tank.

Video Equipment

Digital cameras

If you have digital cameras connected though Gigabit Ethernet (GigE), connect each camera to the labeled Ethernet port on the computer. Each camera is dedicated to a specific Ethernet port. If you switch cameras, EthoVision XT no longer detect them. Take note of which camera (serial number) is associated with which Ethernet port. For how to install and connect digital GigE Vision cameras, watch the

EthoVision XT video tutorial Set Up the Cameras. To open the tutorial,

in EthoVision XT choose Help > Video Tutorial. Figure 8 Sketch of a setup for zebrafish tracking. The two white boxes represent Infrared backlight units. 2 System setup

Analog video cameras

If you have analog video cameras, connect them

via gen-lock. That is, the Video output of one

camera (master) is split into two signals using a T-connector.

One video signal goes to the Gen-lock In input of

the other camera (slave). The other signal goes to one of the inputs of the encoder board.

Camera position - wind tunnel

Place the cameras in such a way that the image of the tracking chamber in your video is maximized. Make sure that the cameras are firmly attached to the walls/ceiling. To achieve good accuracy in 3D positioning of the subject, make sure that the angle formed by the camera axes is no less than 40 degrees. An angle of 90 degrees is optimal, but often cannot be obtained, for example when the markers of the calibration frame cannot be seen from one camera. If the angle is less than 30 degrees, inaccuracy in the direction of the cameras is about 4-5 times greater than it would be if Figure 9 Gen-lock camera connections. Left: master camera (video output split in two). Right: slave camera. 2 System setup the cameras are perpendicular to each other, keeping the distance between cameras equal.

note There has to be enough space between the cameras and the test

chamber in order to obtain a proper image on the screen.

Camera position - fish tank

The optimal position is: one camera at the top of the water tank (pointing below) and one at the front (see Figure 8 on page 15). Always keep the cameras perpendicular to the water tank walls. This minimizes distortions due to the water refractive index. Figure 10 The angle formed by the camera long axes should be greater than 40 degrees. 2 System setup

The Calibration Object

Standard calibration frame

For wind tunnels, the calibration object is a black frame provided with white round markers on two planes. The frame must be placed inside the tracking chamber in such a way that all or most of the markers are fully visible to both cameras. The standard calibration frame can be of two sizes, depending on the size of the tracking chamber: 40x40x40 cm (15.75x15.75x15.75 inches). This is suitable for chambers wider than 42 cm (16.5 inches). 60x60x60 cm (23.6x23.6x23.6 inches). This is suitable for larger chambers. Figure 11 A standard calibration frame for wind tunnels. 2 System setup Two or more objects can be connected and combined to create a larger calibration object.

Custom calibration frames

Calibration objects for water tanks and for tracking chambers other then wind tunnels can be made upon request.

Notes

The X,Y,Z coordinates of the center of each marker in the real world must be known in order to have a reliable calibration. When projected on the video monitor, the markers should not be very close to each other or to bright objects in the image (minimum distance 10 pixels). When doing calibration, It is handy to have a sketch of the calibration object with markers numbered. An example is stored in

the Calibration Coordinates.xls excel file on your installation disk

under Documentation. Figure 12 You can connect two calibration frames to calibrate large tracking chambers. You can click all markers or some of them when doing calibration. 2 System setup

Typical Setups

General recommendations

We advise to place the EthoVision XT/Track3D computer in a room separate from the test room. Alternatively, hang curtains around the test apparatus and the cameras. This prevents that lights in the test room, like that of the computer screen, interfere with the software detection, and ensure that the behavior of the animals is not influenced by movement and light changes outside the test environment. Using an infrared-pass filter mounted on the camera lens is not always necessary. However, in some situations external light sources interfere with subject detection and tracking by the software. If those light sources cannot be switched off, use this kind of filter to block visible light. As a result, the cameras only see the infrared light coming from the IR arrays or backlight units.

Track3D setup for wind tunnels

Figure 13 A typical setup for recording insects in a wind tunnel. 1 - PC with EthoVision XT, MediaRecorder (optional) and Track3D add-on. 2 - Two cameras fixed to the ceiling of the test room, connected to the PC. 3 - A number of infrared LED arrays for illuminating the tracking chamber. 4 - wind tunnel with ventilation unit and tracking chamber with transparent top plate. 5 - Calibration frame for the wind tunnel. 6 - Curtains hanging between computer and wind tunnel. 2 System setup When using infrared lights, it is important that infrared light is absorbed by the materials of the tracking chamber and of the objects around it. This will reduce reflections that can interfere with tracking. Contact Noldus for information on infrared-absorbing materials that you can use to optimize the background.

Track3D setup for fish tanks

Avoid placing the fish tank under reflecting objects or light sources other than the backlight units. Figure 14 Typical setup for fish in small tanks. 1 - PC with EthoVision XT and Track3D add-on. 2 - Two digital cameras connected to the PC via Gigabit Ethernet. 3 - Two infrared backlight units. 4 - Calibration frame for fish tanks. 5 - Fish tank.

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Source: Track3D 2 - Reference Manual (March 2025)