A fruit fly is held in place inside the flight simulator. The "sun" behind it is actually an LED display.

Image Credit: Dr. Michael Dickinson

Traditional fluorescent microscopy uses a single high-energy photon (shown in purple) to boost an electron to a higher-energy state, indicated by the horizontal lines. After a brief period, the excited electron falls back down to its low-energy state (shown in turquoise), emitting a photon that is picked up by a detector. Two-photon microscopy instead uses two lower-energy photons (orange) to send the electron to its excited state via a midway “virtual state”. The electron then falls back to its low-energy state, emitting a photon as usual.

Image Credit: Alberto Diaspro, et al. Multi-photon excitation microscopy. via Wikimedia (CC BY 2.0)

When you think of fruit flies, many words likely come to mind: buzzing, hovering, annoying...butprobably isn’t one of them. As it turns out, these tiny insects are known to travel up toper evening in search of food. Since they often live in barren deserts, Dr. Ysabel Giraldo reasoned that theyhave some way of keeping a straight course—there’s just no way they could survive otherwise. It’s been shown that without the presence of external cues, most insects and animals—humans included—tend to wander in circles, so Giraldo wanted to uncover the secret to the fruit fly’s navigation. “Even though there have been so many studies on, surprisingly no one really knew much about how fruit flies navigate,” she says.Working as a postdoctoral scholar in the lab of Dr. Michael Dickinson at the California Institute of Technology, Giraldo used an unusual kind of flight simulator to study how fruit flies—formally known as—react to a bright spot, which was very similar to the sun. She found that the flies used this simulated sun to keep track of their motion , much like a backpacker might. “If I’m walking for an hour or two and always keep the sun on my right, I will walk in a pretty straight line,” she says. “The same is true for Drosophila.” In fact, the fruit flies were even able to remember their initial bearing with respect to the sun after 6 hours! Upon further investigation, she and her colleagues were able to show that recently discovered “compass neurons” are responsible for this behavior.Theflight simulator was developed about a decade ago in the Dickinson lab and consists of a series of LED panels with the fly held in place at the center. The fly’s wings can move, but the fly itself does not. Instead, high-speed IR cameras pick up the fly’s wingbeats, which can reach 200 beats per second. This information is sent to a sophisticated software program that uses the differences between the right and the left wing angles to calculate whether the fly is going straight ahead, or turning left or right. If the fly is turning, the simulated sun on the LED screen moves accordingly. Giraldo explains, “If you’re sitting in a train and the train next to you starts moving, you may think you’re also moving. But really, that’s just the visual motion tricking your brain.” In a similar fashion, the fly senses motion even when there is none.She found that when the flies were introduced to a bright dot, simulating the sun, they adopted a random orientation with respect to it—and kept that orientation for hours, directing them in a straight path. Remarkably, even when the fly was removed from the arena for up to six hours, when it was reintroduced the fruit flies once again adopted the same heading, indicating that they have some directional memory.Once they had identified the simulated sun as a guide for, Giraldo and her fellow researchers took a closer look at the mechanisms at play. She had known about work done in recent years involving so-called “compass neurons”, which had been shown to help fruit flies keep track of their headings. However, while compass neurons had been investigated from a neurobiological point of view, she says it hadn’t been applied to “real-world ecological problems” like navigation. Even so, she says, “We had a good hunch that they were probably involved.”In fact, the researchers were able to watch these neurons, formally known as E-PG neurons, in real time as the flies reacted to their virtual environment. By genetically modifying, they tagged the neurons with a calcium indicator, which reacted to neural activity by emitting fluorescence. To catch these changes in activity, they cut tiny holes into the insects’ heads and placed them under a microscope.Naturally, this part of the experiment introduced its own challenges. To begin with, microscopes use photons to probe tiny structures, but these light particles impart their own energy and tend to heat up the surrounding tissue. For an insect as tiny as the fruit fly, this carries serious repercussions in terms of biological function. Additionally, researchers need to worry about light scattering as it interacts with the tissue, blurring the overall image.To counteract these issues, Giraldo’s group used an increasingly popular tool, a two-photon microscope. Traditional fluorescence or excitation microscopy uses a stream of photons to boost the electrons in specific atoms to their excited (high-energy) states. Since the electrons don’t typically remain excited for long, they fall back down fairly quickly, releasing a photon. (This type of fluorescence is known as stimulated emission , and is also involved in the function of lasers!)In two-photon microscopy , researchers use two lower-energy (longer wavelength) photons to excite the calcium indicator, rather than a single high-energy one. Since the probing photons carry less energy, they don’t heat up the surrounding tissue as much, and their longer wavelengths make them less susceptible to scattering. Two-photon microscopy also allows researchers to limit the excitation to the few cubic microns within the microscope’s focal point, permitting the use of lower laser powers. Ivo Ros, one of the coauthors on the study, used this technique to study the activity pattern illuminated by the calcium indicator when the compass neurons became active, allowing them to get a glimpse insidebrain.They found, much as they expected, that the compass neurons were in fact highly active as the flies noticed the “sun” and used it to keep track of their headings. When they silenced the neurons (again, using genetic tools), things went a little differently: instead of keeping a constant bearing with respect to the simulated sun, the flies reverted to the much simpler reflex of flying directly towards the light.Given these experimental results, it’s pretty clear that fruit flies can take advantage of sunny days to keep a straight course. But what do they do when it’s overcast, or after sunset? It turns out that sun navigation is just one of the many tools they have for navigation. For example, it has been documented that they use vertical stripes—like trees—as short-distance bearings, although Giraldo and coathors’ work demonstrated that this likely uses a different mechanism from sun navigation. Incredibly, they can also use specialized cells in their eyes to detect the polarization of light, which is correlated with the sun’s (or moon’s) position in the sky. Giraldo says, “We don’t know how those cues interact or if they prefer one cue over another.” Whatever the case may be,are clearly in the possession of a remarkably sophisticated navigational toolkit.In reality, of course, flies are faced with a variety of stimuli that aren’t present in a lab setting: olfactory, auditory, sensory, and even much more detailed visual cues. As Giraldo mentions, the more complicated environments they encounter in the real world make it “necessarily more difficult for flies to make decisions while flying around.” Nevertheless, she is reasonably certain that the behavior she observed in the lab replicates natural behavior.Although this research is exciting, it’s clear that there’s more work to be done. In addition to the unknowns in how various competing stimuli are prioritized by the fly, it’s also unclear how the compass neurons fit into the overall circuitry of’s brain complex. In future studies, Giraldo hopes to take a more holistic approach to these neurons and study the overall networks they are part of.So next time a fruit fly shows up as an uninvited guest at your picnic, maybe take a moment before you shoo it away to appreciate the complex toolkit it used to find its way to you.