Flying insects use visual cues to stabilize their heading in a Windstream. As such, visual stabilization of upwind tracking directly aids in odor tracking. Using an olfactory magnetic tether apparatus or OMT, we can examine whether visual cues directly influence odor tracking behavior independent of wind cues.
The procedure begins with optimizing the fly illumination by adjusting the intensity of an infrared LED to get a clear image of the fly. Too little illumination will result in a dim image and too much will result in an oversaturated image once the fly is properly imaged. Analysis software measures body angle in real time visually dragging a fly into a food odor plume results in consistent heading into the spatial location of the plume.
But only when the visual panorama contains strong visual cues. Testing the same fly in a visual arena of equal mean luminance, but with no visual edges results in poor plume tracking as the fly loses its spatial orientation. Hi, I'm Brian DeMars from the laboratory of Dr.Mark Fry in the Department of Physiological Science at the University of California Los Angeles.
Today we will show you how to acquire and optimize video images for analyzing body angle in an olfactory magnetic tether apparatus. We will also describe two experiments to examine the influence of visual cues on odor tracking. We use this procedure in our laboratory to study visual and olfactory mediated flight control and fruit flies.
So let's get started. Optimizing the video image is crucial for the quality of the acquired data to obtain cleaner images. First place a freshly tethered fly in the arena using a pair of forceps.
Next, position the fire wire board camera under the clear acrylic vacuum chamber such that the camera points directly upward through the chamber through the glass vacuum tube to the ventral aspect of the tethered fly. Be sure that there is no infrared filter coating on the lens of the video system. To illuminate the fly, use a circular array of infrared LEDs, which are positioned just under the LED panels.
Focus each LED on the fly's body individually for uniform illumination to reduce glare from the panel system and to minimize visual cues observed by the fly. It is useful to coat all reflective surfaces within the camera view with flat black paint room lights should be switched off to reduce glare and brightness modulations detectable by the fly's eye. Next, visually inspect the camera.
The fly should be bright white against a black background. If the image is blurred, the lens may need to be focused. If the fly is dark, the LED array may be unfocused or insufficiently powered for proper illumination.
Finally, acquire video with custom subroutines written in MATLAB using the image acquisition toolbox. One simple experiment in the OMT is to observe a starved fly locating and actively tracking a plume of attractive food odor. These experiments are typically run in a random block format to minimize any bias in experimental order.
By manipulating the visual surroundings, you can influence the fly's ability to track an odor. To do this experiment, place a freshly tethered fly in the arena using a pair of forceps before every experiment using the visual display, rotate the fly for several revolutions to ensure smooth motion around the entire 360 degree YA axis. Discard the fly if it does not spin smoothly.
If the problem persists, the magnets may be misaligned. Next, present the fly with the following experimental conditions. In random order a high contrast striped pattern with water vapor emanating from the odor port.
A high contrast striped pattern with an attractive odor vapor. A single vertical stripe offset 90 degrees from the odor port with water vapor and a single 90 degree offset stripe with odor vapor 32nd intervals work well for experiments where flies are challenged to locate the odor source on their own. Acquire video at 30 hertz for the duration of each experimental condition and use MATLAB to analyze the images offline When using high odor concentrations, residual odor may be released from the odor port after the odor is switched off to minimize any effects.
This may have spin the fly for a few seconds with a rotating panorama to allow the system to flush out any residual odor. It also helps to visually rotate the fly between experimental treatments. This keeps the flies actively engaged in the experiment and keeps them from remaining in one spot.
A second basic experiment in the OMT is to visually drag a fly into an odor plume instantaneously change the visual conditions and then measure its ability to remain in the plume. Experiments are done in a random block format to minimize any bias in experimental order. Using forceps, place a freshly tethered fly in the arena.
Next, as a diagnostic check, visually rotate the fly several times around the arena. This ensures that the animal has the capacity to orient in every direction. Before each condition, turn on the experimental odor, either water or vinegar, and visually drag the fly into the plume by oscillating a small vertical stripe at the position of the plume.
Now that the fly is oriented toward the plume, remove the oscillating stripe. Then present the following visual conditions. High contrast panoramic stripes with water vapor, uniform background with water vapor, high contrast panoramic stripes with odor vapor and uniform background with odor vapor.
Acquire video at 30 hertz for the duration of each experimental condition. Then use custom software routines written in MATLAB to analyze the images offline. When optimizing the OMT video image, remember that it should have a clear view of the illuminated fly on a dark black background.
Overly bright images can be improved by reducing background interference, reducing the intensity of the infrared LEDs, or by turning off the room lights. If the fly is properly illuminated. Adjusting the focus of the infrared LEDs or modulating their intensity may fix the problem.
When challenging a fly to find an odor plume on its own, it should never localize a water vapor plume to a significant degree, but it should always localize an odor plume in the presence of rich panoramic visual cues. A second test is to visually drag a fly directly into an odor plume and challenge it to maintain its heading within the plume against a variety of stationary visual backgrounds. If the fly is dragged into a water vapor plume, it should quickly turn away.
If the fly is dragged into an odor plume and there are sufficient visual cues to mediate robust tracking, the fly will stay in the plume. We've just shown you how to optimize the acquired video image for an olfactory magnetic tether apparatus. We've also described two experimental protocols for examining the influence of visual cues on odor tracking.
When doing these experiments, it's important to remember that optimizing the video image is crucial for the quality of the acquired data. Also, no matter the experimental protocol, it's important to keep your flies happy and actively engaged. So that's it.
Thanks for watching and good luck with your experiments.
