# Choosing the optimal reference image in a series of trials

Background information

The background image is the image that the software compares with the incoming images from a camera (or video file) in order to find the subject. A good background image is critical for obtaining high quality tracking data. See The reference image

When you acquire multiple trials in a series, a reference image is used at the start of each new trial. Choosing the reference image is important especially when there is a long temporal gap between the end of one trial and the start of the next one. Follow the guidelines below.

This topic applies when:

▪You use one of the detection methods Dynamic subtraction, Differencing, or DanioVision.

▪You Acquire a series of trials (Batch acquisition), or carry out trials manually one after the other.

Procedure

1.Create your series of trials in the Trial List. See Prepare the list of trials

2.In the Detection Settings, choose Automated Setup or choose the detection method.

3.Under Method, click the Background button. Obtain the reference image for the current video (or live image). See The reference image

4.In the Background Image window, under Acquisition Settings, choose one of the following:

▪Use saved reference image. The saved reference image is the image that you obtained in the previous step. The software will use this image at the start of each trial. Choose this option if you are 100% sure that the background does not change with time (or between video files). For example, when there is no bedding material, the background is lit uniformly, and the camera is always exactly in the same position relative to the arena.

▪Use dynamic reference image. As explained in How the reference image is updated in Dynamic subtraction, a dynamic reference image keeps changing during a trial. With this option, for each new trial, the software will use the most recent dynamic reference image available, that is, the one that was created at the end of the previous trial. Choose this option if the live (or video file) image at the end of a trial is comparable to that at the start of the next trial. For example, when a trial starts a few seconds/minutes after the end of the previous trial.

▪Use first frame of each trial. With this option, the software takes the first video frame available from the camera (or video file) and uses it as the reference image. Choose this option whenever you suspect that the video image at the end of a trial is likely to differ from that at the start of the next trial. For example when lighting conditions change, or the bedding material looks different between two trials. When you specify long inter-trial intervals, that is often the best option. See Trials and inter-trial intervals

Notes

▪Consider that the video image could also change between trials if the camera moves slightly relative to the camera. For example if you place a Petri dish under the camera not exactly on the same spot each time. This effect is more important when you track small animals. Any shift in the reference image, even a few pixels large, could affect detection in the next trial.

▪You should not use the option Use first frame of each trial when the subject is already in the arena at the start of each trial. If you do so, EthoVision XT misses the subject in the first frames and finds it only when it moves away from the starting point.