Lighting - Introduction
Lighting has the greatest affect on the quality of your data. This section provides some definitions and introductions to common lighting terms which can help you set up lighting for your assay.
Selecting the optimal lighting strategy for your specific purpose is important. The most common ones are mentioned below.
Direct Frontal Lighting
Light is applied from the same direction as the camera is positioned at. Light reflects from the arena and the objects in the arena directly back into the camera. This allows imaging of features on top of the object/animal. Contrast may be reduced in comparison to background illumination. A diffuse and homogeneous lighting is recommended to avoid reflections.
Diffusing a light source can be accomplished in different ways:
- Using a diffusing material between the light and the arena. This could be a sanded or opaque sheet of acrylic or any other diffusing material.
- Diffusion by reflection (indirect lighting) can be accomplished by bouncing the light off one or several surfaces (e.g. the walls and ceiling of a room).
Double sided satiated (also referred to as "white frosted" or "white snow") acrylic with a translucency between 70% and 80% is optimal for diffusing LEDs.
This lighting schema is in between direct lighting and dark field lighting. The light source illuminates the arena at an angle which will reduce direct reflections of glossy or reflective surfaces (e.g. water). The angle can be shallow or steep, both will change the visual appearance and properties significantly.
Backlight Illumination (Bright Field Illumination)
The light source, usually a light panel, is located below the arena (the floor needs to be transparent). Objects within the arena appear as silhouette and the contrast is very high but no surface information. For an optimal result the object should be a bit away from the light panel to reduce "illuminated flanks".
Dark Field Lighting
The light source is located at a very shallow angle, almost perpendicular or actually perpendicular to the camera. Vertical edges are bright. Also, objects embedded in liquids that have a different refractive index (e.g. a fish in water) will appear bright.
Brightness of an image is determined by:
- ambient lighting intensity
- aperture of the lens (large number = less light)
- exposure time (the longer the brighter the image)
- the sensitivity of your camera sensor
IR light is not visible for most animals. Using IR light (850nm or 940nm possible) allows having visual light present for the animals but one can block the camera from detecting it (using a filter) thus making lighting conditions in the video more constant, helping the performance of your image processing algorithms.
Additionally, by not having (or turning off) the visual light, using IR allows continual 'in the dark' recording, e.g. in the night phase of an experiment.
- Using IR light results in a grayscale image
- IR of this wavelength is not thermal (NIR)
- Some objects look different in IR compared to visual light. E.g some black inks are white in IR
- IR is not visible for humans, always protect your eyes if you use high IR intensities
- Camera sensors are more sensitive to IR with a wavelength of 850nm than to IR with longer wavelengths
- For high resolution applications make sure the camera lens is suitable for IR light
- IR light produces slightly less reflections on glossy surfaces (e.g. water)
If you use IR light, you should use an IR longpass filter which blocks visual (white) light. You should not mix IR and visual light as the chromatic aberration is significant. An scenes illuminated with white light have a different focus point than the same scene images with infrared (if you focus with white light and then switch to IR the scene will be not as sharp as before, the same applies in the other direction).
Why is my video flickering (this is often visible in high frame rate recordings and in recordings with a low exposure time.
Flickering videos (or horizontal lines or bands) arise from insufficient light sources. Most consumer light bulbs flicker at the frequency of the main power supply. Depending on their construction, consumer fluorescent lamps or LED lamps may also flicker. Low-quality LED-dimmers are particularly problematic as they modulate with rather low frequencies.
Horizontal lines or bands are observed with Rolling Shutter cameras. The clearest expression manifests in the part of the image being black and the other part being bright (horizontal split).
Tips for removing flicker include:
- Use flicker free light sources such as LEDs driven with constant current
- Increasing the exposure time can help for low frame rate recordings (
Flashing or Strobing LED Lights
Flashing LED lights in synchronization with the camera shutter (exposure) allows you to run the same LED brighter (at higher intensity) than continuous operation of the LED (thermal reasons). Also, this mechanism can be used to reduce the thermal footprint by only switching the LED light on when an actual image is taken.
More complex arrangements are used if multiple light sources are flashed alternatively for generating different images (e.g. a fluorescent image and a bright field image ). This can also be used if a semi opaque structure, such as a bee-hive, should be illuminated and recorded from both sides (see case study). Illumination on one side would reduce the contrast of the image on the other side.