Knowledge Base

Detection Settings

What Do You Want to Do?

• Track one subject per well/enclosure. See below.
• Track two or more subjects in the same enclosure. See the Multiple Subjects in the Same Enclosure section.
• Use Activity. See the Activity Analysis section.

One Subject Per Enclosure

Choose Setup > Detection Settings > Detection Settings 1.

The DanioVision Detection Method

The template experiment contains a Detection Settings profile with DanioVision selected as method. The DanioVision detection method is optimized for detecting small dark objects over a white/gray background, like in DanioVision experiments.

The difference with other methods is that DanioVision detects the subject based on the level of contrast within the well where the subject is. You can view this method as Dynamic Subtraction applied to each well independently. This method should detect all subjects even when there are differences in lighting between wells.

This method does use a reference image, just like Dynamic Subtraction, however you do not have to choose one. The software does that for you by taking the first video frame available.

After comparing the reference image with the current video image, DanioVision selects the pixels within the well that are more likely to be part of the subject. How "choosy" the software is depends on the Sensitivity. With a low value of Sensitivity the software considers pixels over a wider range of grey values as candidates. With high values, it picks pixels over a narrow range of grey values.

If some subjects are completely still, the software may not find them. If that occurs, gently tap the well plate to make the animals move. Once all the subjects are detected, adjust your settings.

Important: If you have upgraded EthoVision XT to version 18 while you have a DanioVision system older than DVOC-0040, the DanioVision method does not give optimal detection of the larvae with images from your camera. If that is the case, adjust the Sensitivity (see below) or use another detection method in the Advanced section of the Detection Settings pane. For details, see the EthoVision XT Help.

Procedure

1. Place the well plate with the larvae in the DanioVision chamber and close the chamber door.
2. Click the Auto-detect button. Use the Auto-detect button every time you change a well plate.
3. The Auto-detect window with a timer appears. Wait until it has disappeared. All larvae should be detected.

What If Some Larvae Are Not Detected or the Software Tracks Noise?

Do one or more of the following:

• Open the Advanced section and under Method move the Sensitivity slider. Moving the slider to the left results in detecting more of the body of the larvae, but also more noise. Moving the slider to the right results in detecting less noise, but also gives less accurate detection of the larvae.
• If you see erratic tracks and spikes, it means that small groups of pixels at the border of the well are sometimes detected as the larva, and there is too much noise (orange pixels). Move the Sensitivity slider to the right.
• If some larvae are not detected while they are in the corner or border of a well, check that the arenas are drawn correctly. Click the Show/Hide button on the toolbar, select Arena Features, and then select the check box in front of Arenas. Check that the arenas cover the entire wells. The subject is not detected when it swims in a region not covered by the arena.
• Try to maximize the contrast by opening the aperture of the camera lens. However, make sure the light intensity is equal for all wells (that is, the center and outer wells).
• Certain compounds dissolved in the water can affect the pigmentation in the larvae, thereby decreasing contrast and making detection more difficult. Make sure that EthoVision XT can still detect all animals after treatment.

If detection remains unsatisfactory:

• It could be that the software tracks other objects outside the well. Restrict the arenas only to the part where the larvae swim.
• Try the Dynamic Subtraction method. For an explanation of the options, see Configure Detection Settings in the EthoVision XT Help.

Notes

• The Dynamic Subtraction method may also work fine with zebrafish larvae.
• If your experiment is set to track three body points (snout, center, and tail), you can choose the Adult Fish/For Occlusions method.
• If the experiment is set to center-point detection only, choose either DanioVision or Dynamic Subtraction, with the Other Species method to track the center point of the subjects.

Activity Analysis

Use Activity to quantify overall activity of your subjects. In the Experiment Settings, select Activity Analysis. In the Detection Settings pane, open the Activity section. Activity is shown by purple pixels in the video window. Set the Activity threshold so that movements of the animals are detected and some noise is left.

For more information on activity, see Activity Settings in the EthoVision XT Help. Also watch the video tutorial on Activity Analysis. For more general information on Detection Settings, see Configure Detection Settings in the EthoVision XT Help.

Multiple Subjects in the Same Enclosure

Prerequisites

In the Experiment Settings, Number of Subjects must be set to 2 or higher.

Procedure

1. Choose Setup > Detection Settings > Detection Settings 1.
2. Click Automated Setup. One subject is detected per well. Play the video to check detection.
3. Click Advanced, then Method, and select DanioVision.

Note that subjects are not individually marked, so identity swaps do occur.

Source: DanioVision DVOC-0050 Reference Manual - Detection Settings, Noldus Information Technology