Lighting setup
Ideal light conditions
A good lighting setup is vital to get a good image that EthoVision can use to accurately track your animal. This is particularly important when you want to do nose-tail tracking or track multiple animals with color markers. To produce a good image, the lighting must satisfy three criteria:
▪It must be bright enough. Stand-alone video cameras (which we recommend) can sometimes work at 0.1 lux or darker. At higher light levels you can work with a smaller aperture. See also Using EthoVision XT in near darkness. Please note that poor lighting gives more noise in the image which enlarges the video file size.
▪It must be even. That is, a diffuse light source, such as a shaded fluorescent tube or globe-type incandescent bulb. Spotlights can produce changing shadows and reflections.
▪There must be no reflection of the light source visible in the image (see below).
tip Watch the video Set Up Your Test Environment, which you can find in the EthoVision XT video tutorial (Help > Video Tutorial).
If you have reflections in your image, EthoVision may confuse those reflections with your subject, and track the reflections rather than your animal. Preventing reflections is particularly important when you want to do nose-tail tracking. There are a number of measures that you can take to reduce this problem:
▪Surfaces within the arena should be (whenever possible) a dark color and matte texture.
▪Lighting should be from a diffuse source, such as a shaded fluorescent tube or globe-type incandescent bulb.
▪The light source should not be in direct line-of-sight of the camera (that is, it should be indirect lighting). You can achieve this by bouncing the light off the ceiling or walls (which should be a pale color and matte texture) and, if necessary, placing a shield in between the light source and lens.
▪If your arena is transparent (for example, a glass olfactometer), you can use backlighting. This will eliminate reflections, but you must take special care that the lighting is even and diffuse. Place the light source underneath the setup and attach a sheet of white paper to the bottom to create diffuse back light. It is important to avoid light coming from above as much as possible, otherwise there is no effect of back light.
▪You could also try a polarizing filter in front of the camera lens. The filter will remove reflections that are incident to the reflective surface at 32° - 37°.
▪If possible, draw your arena in such a way that bright reflecting rims of mazes are excluded.
▪To remove reflections brighter or darker than your animal, set appropriate thresholds in the Detection Settings.
▪To remove reflections larger than your animal, enter an appropriate value for the Maximum Subject size in the Detection Settings. Note that if your animal enters the area of the reflection, EthoVision might no longer be able to track it if you use this setting.
Lighting a Morris water maze needs to be carried out particularly carefully because the water can give large and variable reflections. Some of these can be excluded by the software settings listed above, and the following lighting setup should give good results:
Place four (or more) bulbs round the pool, below the level of the water surface or at the ceiling. 'Globe' type bulbs are ideal (twice the diameter as standard incandescent light bulbs). They should be close enough to the pool wall so that there is no direct line of sight between the bulbs and the camera lens. The light is reflected off the walls and ceiling, so that it only reaches both the lens and water surface indirectly.
▪Take special care when dealing with water and electricity in the same room. The lights should be placed so that water cannot splash on them, ideally they should be suitable for outdoor use (double-insulated) and if connected to an electrical outlet in the same room as the water maze they should be connected through a circuit-breaker.
▪Ask a photographer for advice on the lighting of your water maze.
When using water tanks like in the Porsolt forced swim test, a problem is often that there is a lot of reflection, especially just above the water surface. Furthermore, detection of the animal can be hindered by the water's meniscus.
Try to make a set-up with as much contrast between animals and background as possible. For instance, when you have white rats, preferably use a completely black background. Back light (that is, light from behind the subjects so you only see the animals' contour) generally gives very good results. A solution to remove the water's meniscus in the video image is also to place the test cylinders in a separate tank with water (for example an aquarium) and then make recordings. The water level in the tank should be higher than in the cylinders.
For more information on the Porsolt forced swim test, see the EthoVision XT 19 - Application Manual.
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. See Light in 24-hour experiments
Below: Sensitivity to infrared light of a standard monochrome camera and a specialist IR camera.
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 cameras 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 gets 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.
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. See Manuals
▪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.