Troubleshooting tracking errors

Checking assets

You can do this by putting your well plate/tank(s) into the system, running a script that loads the asset for your well plate, and selecting the ‘arenas’ button from the live video page. Things to check include:

• Have you loaded the correct asset? Individual systems can have slight variations in camera alignment. It is important to use an asset that has been built for the system on which it will be used (e.g., you cannot download an asset from the website and use that). Assets are built taking the calibration of the individual system into account. More information about calibrations can be found here.
• Do the arenas line up with the wells/tank(s) correctly? This can be corrected in the asset building script. More information about asset building can be found here for the MWP unit, the AD unit or the LT unit.
• Are the arenas the correct size? Too large and you can pick up the shadow of animals in adjacent arenas or the reflection of an animal within the arena, this will cause the crosshair to jump between the animal in the well and the shadow/reflection. Too small and the animal can be lost when moving outside of the arena and will result in missing data.

Checking tracking settings

We have settings for some model organisms on our website here. These are robust and have been tested in various labs, globally. We anticipate that these should work. It is good to first test with some empty arenas (or just water if aquatic) and arenas with animals. Then run a short script to look at the tracking:

• Do you have tracking in empty wells? If so, then the tracking settings are too sensitive.
• Are you not tracking animals that you can see moving in wells? If so, then the tracking settings are not sensitive enough. Please note that no crosshair does not always mean bad tracking. If you have the correct tracking settings, it is most likely that the animal simply has not moved during the autoreference and the trial period. You can check this by looking at the autoreference image (see 'checking the autoreference' section below). You may see some flickering of the crosshair, this is also not something to worry about as the system will record the distance travelled between the flickers.

More details about the tracking settings commands can be found on the Tracking settings - Z2S page.

Checking the autoreference

An autoreference is required for tracking. It removes the animal from the background image. To do this effectively the animals should move during the autoreference period. For very inactive animals, a longer autoreference period may be required. You can check an autoreference whilst running an experiment by selecting “Assets directory” from the Homepage and taking a look at the “autoref” image. There should be empty arena(s). If you can see whole animals/large parts of an animal, the system will lose tracking when the organism passes that point since it will not be able to differentiate the organism from the background. More information about autoreferences can be found here.

Tracking errors developing over time

For the first experiments, in particular longer experiments (1 hour plus), it is good to keep an eye on the live feed periodically to ensure that tracking remains good. Using time-lapse video with track traces is a useful tool for checking tracking errors which can develop over time. Here are some of the most common reasons that tracking issues develop and how to mitigate them:

• Strong vibrations. Make sure your system is placed on a solid bench away from excess traffic. Very strong vibrations near the system can shift a well-plate outside of the arenas and generate bad tracking. If using a very strong vibration stimuli within the MWP system, it is recommended to secure the plate to the stand. Instructions on how to do this can be found here.
• Evaporation/meniscus developing. This is prominent in multi-well plates. As water evaporates or waste products build up over time, a dark ring (meniscus) can develop within the wells. This can generate bad tracking since the image now looks different to the autoreference. Placing a lid on the plate will reduce the speed of evaporation. Taking regular, repeat autoreferences during long experiments will mitigate bad tracking developing over time.
• Condensation. If the temperature between the plate/tank(s) and Zantiks system vary/differ from each other, condensation can build up and disappear on surfaces over time. This can cause tracking errors since the image has changed from that of the autoreference. To mitigate this, ensure that the Zantiks system is at the correct temperature before starting. If using plates or petri dishes, consider keeping them in an incubator so that they are also at the correct temperature. Ensure that you are using the temperature control system when using the MWP, both the standard and PELTIER temperature control systems will help to maintain a constant temperature within the systems and prevent condensation issues.

Water splashing within the system

When working with aquatic species, water can be spilled from tanks or multi-well plates. Spilled water within the systems, both above and below the tanks, creates imaging problems in terms of bright spots where there is water, dark lines on the edge of water puddles, and salty deposits which obscures the lens. Additionally, as puddles of water dry over time, the image changes from the autoreference. Reducing the water levels within tanks/wells will reduce the risk of spills. Mop up any water spills with a paper towel or a soft cloth. Ensure that the surfaces both above and below the tank are cleaned regularly with a damp cloth.

Animals are brighter and darker than the background

This is an issue which can occur in the AD or LT systems for aquatic organisms in tanks. Side lighting when the animal is close to the source of infra red light (i.e., bottom of the tank) makes them appear bright against the background. When further from the source of light (i.e., off the bottom of the tank) they appear dark against the background. 

To mitigate this you can insert the louvered sheet (supplied with AD and LT systems) between the tank and the screen. This will make the animals always appear dark, so changes to the script would not be required.

If problems persist, try change the detector mode to DELTA_TARGETS to track both bright and dark targets.

SET(DETECTOR_MODE,DELTA_TARGETS)
SET(AUTOREF_MODE,MOVEMENT)

Commands to set detector modes

There are 3 SET commands which can be used in your script in order to alter to way the system 'finds' and tracks your animals. One of these commands is used at a time and the system's default setting is SET(DETECTOR_MODE,DARK_TARGETS). The command should be placed at the top of your script prior to the ACTION MAIN.

SET(DETECTOR_MODE,DARK_TARGETS)
SET(DETECTOR_MODE,LIGHT_TARGETS)
SET(DETECTOR_MODE,DELTA_TARGETS)

SET(DETECTOR_MODE,DARK_TARGETS) - tracks dark targets (animals) against a lighter background. This is the default setting for tracking.

SET(DETECTOR_MODE,LIGHT_TARGETS) - tracks animals lighter than the background. 

SET(DETECTOR_MODE,DELTA_TARGETS) - allows the system to track animals both brighter and darker than the background. For this to work, the autoref mode should also be set to MOVEMENT.

If you are having any issues with tracking which are not covered by this troubleshooting guide or are in any way unsure, please contact us. We are keen to help you get reliable tracking.