Knowledge Base

Using EthoVision XT in Near Darkness

Nocturnal animals like rats and mice are active in the dark. It is, therefore, a logical choice to measure their behavior in the dark. You can use infrared (IR) lights to illuminate the arena and a camera that is sensitive in the infrared. Most animals, including rats and mice, do not see infrared (IR) or near-infrared (NIR) light. Infrared illumination is perceived by them (and also by humans) as being total darkness.

Camera

Infrared light (IR) is light of a longer wavelength than visible light. Generally, to track with EthoVision XT, near-infrared light is used, with a wavelength of 700-900 nm (near the visible range of 400-700 nm). Color video cameras are insensitive to infrared but standard monochrome cameras have reasonable sensitivity to near IR. In particular, the Basler monochrome GigE cameras are IR sensitive. Normally they do not require an IR-pass filter unless you want to track the animals in both dark and light conditions during the same trials, for example in multi-day trials.

Optimizing Image Brightness

Depending on how bright your IR setup is, you might need to:

• Open the aperture on the lens all the way (do this first).
• Adjust the camera's Gain and/or the Exposure setting in EthoVision XT.
• A high Gain increases brightness but also noise in the image.
• A longer Exposure time may not be compatible with the frame rate set in the camera. Check carefully in Adjust the Camera Exposure Time.

TIP: Watch the video Set Up the Cameras, which you can find in the EthoVision XT video tutorial (Help > Video Tutorial).

Light Source

As your light source, you can use IR illuminators, which basically are LED lamps that produce infrared rather than visible light. Using an IR illuminator is just like using a lamp, except that neither you nor your animals can see its light. Check the image from your camera to see what the illumination actually looks like. Just as with visible light, you want uniform lighting throughout your arenas.

You may find that objects that look light in visible light appear dark in the IR image, and vice versa. Likewise, objects that appear transparent may be opaque, or opaque objects (especially some kinds of acrylic) may be transparent in the IR image. You can record through acrylic, unless it has been colored or coated with pigments that absorb IR light. You can use, for example, a sheet of acrylic to cover the top of a cage or arena.

If you use an IR illuminator from overhead, you may get spots of reflective glare, just as with normal lighting. Moving the illuminators to the sides, rather than overhead, can eliminate the glare. To eliminate shadows, you may get the best results by using two or more illuminators.

• You can obtain IR LED illuminators from Noldus Information Technology.
• You can also use an IR lightbox as your light source. An IR lightbox provides a uniform source of IR illumination from beneath your animal. Unlike the case with an IR illuminator, the camera will see a dark silhouette of the animal against a light background, which is easy to track in EthoVision. An IR lightbox is also an excellent solution if your animals are highly variable or mottled in color, for example Long Evans rats, and eliminates any concern about glare.

Light in 24-Hour Experiments

If you want to go from light to dark conditions (or vice versa) during a single trial (e.g., recording across a 24-hour circadian cycle), EthoVision needs consistent lighting. This can be accomplished by using an IR-pass filter on your camera and by preventing any variation in the amount of IR illumination.

Should you require an IR-pass filter, take note of the lens characteristics. The filter should match the diameter of the lens, and simply screws on to the front of the lens. The filter prevents the camera from seeing visible light — only IR light gets through. The filter is not necessary under constant lighting conditions, but you need it if the visible light level changes during the trial. The IR illumination must remain on for the entire duration of the trial.

Some IR illuminators have the feature of automatically turning off when there is visible light (for example, IR illuminators designed for security applications usually turn off during the day). If your illuminator has this function, it needs to be disabled. Consult the documentation for your illuminator to see how this is done. If it is not possible to prevent the automatic turning off, you can cover the illuminator's light sensor with tape so that it will not detect light.

The normal light in the room should not affect the detection setting unless it also emits IR light. Many light fixtures will give off some IR illumination in addition to visible light. Incandescent and halogen lights especially will produce a great deal of infrared light. Preferably use fluorescent lights which produce much less infrared light. If there is an intensity adjustment on your IR illuminator, turn it up as high as possible. Increasing the amount of IR illumination will minimize the impact of any infrared light from your visible lights. Sunlight also has an IR component, so if you have windows, they should be covered. When watching the camera image, ideally you should see no change in the image when the visible lights turn on or off. It might be that your image is slightly out of focus, but that is no problem for tracking in EthoVision.

Notes:

• If you have light colored animals, use the darkest non-reflective bedding possible for improved contrast. Be aware that some dark materials may appear white under infrared light. See also the section Recommended Materials in the PhenoTyper - EthoVision XT 19 - Reference Manual.
• If you have not eliminated all infrared light from the visible light in the test room, and you are using Static subtraction as your detection method, the entire arena may be detected as the animal after the visible lighting changes. Choose Dynamic subtraction and the animal will be detected after a few frames.

Source: EthoVision XT 19 Help, Noldus Information Technology