Knowledge Base

4 Check The DanioVision System

Before you start setting up EthoVision XT, you need to check the following:

Camera

The camera lens should have the correct aperture and must be in focus. See page 33 The camera is correctly configured. See page 28

Anti-condensation mechanism

You need to do this depending on the temperature inside the DanioVision chamber and the desired water temperature around the well-plate. See page 35

White Light stimulus

You need to do this when you want to use very low intensities for the DanioVision White Light. See page 36

Well plates and Petri dishes

The well plate or Petri dish must be inserted correctly. See page 37 Always control evaporation in the basin and in the well plate, especially when you carry out long trials (> 1 day). See page 40

Check The Camera Lens

Although the camera and lens are set up before shipment, the settings of the lens can shift during transport. Before you start to work with the DanioVision system, please check that the lens is set up correctly by doing the following: 1. The lens you use depends on the video resolution and frame rate you want to use. See the table on page 29. 2. Insert a well plate (o petri dish) in the basin. 3. Create a new EthoVision XT experiment (see create a daniovision

experiment on page 41) and open the Arena Settings to see the

camera image. When water is not present in the basin, the image of the well plate should look overexposed, like the following: 4. Check the focus (see Figure 9). If the camera image is not in focus, loosen the screw, adjust the focus and tighten the screw. The camera should be focused on the animals. The distance between animals and camera does not change much between types of well-plate and water level in the wells, however it is always a good idea to check the image before an experiment and adjust the focus until it is optimized. 5. Check the aperture (see Figure 9). The aperture has been adjusted in such a way that when the basin is filled with water, the video image is optimal for tracking (see

example of a good video image on page 126). However, in the

Detection Settings (page 64) check that detection of the larvae is good. If necessary, adjust the aperture to get a better contrast between larvae and background. 6. Make sure the camera is correctly configured (see page 28).

Notes

The more the aperture is closed, the larger the depth of field, which means that an object is 'sharp' at different distances from the lens. This is especially relevant when you work with a zoom lens and zoom in to focus on a smaller region of a well plate. On the other hand, closing the aperture decreases the contrast between animals and the background. Adjust the aperture until the image is optimized.

Figure 9 The 12-mm standard camera lens (left) and the 8-mm camera lens

(right). The 8-mm lens has a spacer ring (already installed if you purchased this lens from Noldus). See also Table 1 on page 29 for which camera settings apply to which lens.

Configure The Anti-condensation Mechanism

Anti-condensation switch

The DanioVision Observation Chamber is equipped with an anti- condensation mechanism to prevent water droplets forming on the lens during an experiment. A temperature sensor inside the DanioVision Observation Chamber checks whether the anti- condensation mechanism should be switched on or off when it is set to

Auto mode. Besides the Auto mode, you can also permanently switch

the anti-condensation mechanism On or Off. The switch to set the anti-

condensation mode is located behind the camera, at the right side.

Auto

Default and recommended when the DanioVision TCU is not used. The

anti-condensation mechanism is automatically switched on when the air temperature inside the chamber drops below 28 °C (82.4 °F) to

Figure 10 Position of the anti-condensation switch (labeled:

Anti-Moisture) inside the DanioVision Observation Chamber. prevent condensation on the lens. When the air temperature inside the chamber exceeds 28 °C, the anti-condensation mechanism is automatically turned off to prevent heating of the water in the wells. The value of 28 °C is a default setting of the embedded controller.

note If you use the DanioVision TCU, follow the instructions on the

Reference Manual - DanioVision Temperature Control Unit DV-TCU, and set the switch to either On or Off.

Off

The anti-condensation mechanism is permanently turned off. Use this mode when the air temperature inside the chamber is equal to or higher than the actual water temperature of the basin/well plate water (in most cases 28 °C). You can use this option in combination with the DanioVision TCU.

On

The anti-condensation mechanism is permanently turned on. Use this mode when the air temperature inside the chamber is lower than the water temperature in the wells. You can use this option in combination with the DanioVision TCU.

Configure The Daniovision White Light

In the DanioVision Observation Chamber it is possible to use very low light levels for the DanioVision White Light in addition to the default, standard white light levels. The standard light levels range from ±20 lux to ±10000 lux. The low light levels range from 0 to ± 20 lux. Both standard and low light can be controlled in the same way by EthoVision XT's Trial and Hardware Control. 1. Locate the switch, behind the camera, at the bottom-left of the black mini USB-IO Box. 2. Set the intensity of the White light. Right position H, standard high light level. Left position L, low light level.

Place Well Plates And Petri Dishes

Well plates (ANSI standard)

1. Put the back of the well plate against the bridge and place it on the basin floor.
2. Tilt the front of the well plate down to the floor. This way the water in the basin pushes the air from underneath the well plate. Make sure the well plate is accommodated correctly between the metal springs at the front and at the right side.

tip Drill a few holes on the sides of the well plate to allow air to

escape when placing the well plate.

Well plates (non-ANSI standard)

For smaller or larger well plates, place the bridge in a different position. The default position (ANSI standard) corresponds to the second hole on the side walls of the basin, when counting from the inner side of the observation chamber. 1. Remove the screws as indicated in the picture. 2. Slide the bridge by one or more positions. Check the video image in EthoVision XT, and make sure that the well plate is centered in the camera view. 3. Fix the screws in the corresponding holes.

Petri dishes

1. Put the Petri dish against the 90º angle of the bridge.
2. If necessary, remove the screws of the bridge (see above) and slide the bridge of a few positions until the dish is centered in the camera view. With DanioVision you can use Petri dish of diameter up to 90 mm. We recommend not to let water flow in the basin when using Petri dishes. Water may flow underneath the bottom of the dish, making it shift from its original position during a trial.

Control Evaporation

Wells on the sides of the well plate tend to show a higher rate of evaporation than wells in the center. Also, wells at the front have a higher evaporation rate than those at the back.

Our recommendations

In long experiments (> 24 h), evaporation may occur for both the wells in the plate and the basin. Wells: Check the wells every 24 hours and if necessary, refill them. Basin: Refill the basin every 2-3 days using the Basin Refill tool. For details, see the Reference Manual - Temperature Control Unit DV- TCU. The environmental conditions (temperature, relative humidity and air flow) and experimental conditions (lens heater on/off, White light intensity) influence the rate of evaporation (and condensation) of water from the basin and well plate. If you plan to carry out long experiments, always test evaporation at your environmental and experimental conditions, and plan refills to make sure that water drop never reaches a critical value.

tip You can use a special syringe to refill the basin without having to

open the DanioVision Observation Chamber. Contact Noldus for more information.

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Source: Reference Manual - DanioVision DVOC-0041