Emma Yasinski, a summer intern in the scientific communications department at Max Planck Florida Institute for Neuroscience met with Long Yan, Ph.D., who handles the 2-photon microscopes to learn about how scientists here apply the technique to their work. Her experience is below.
Dark Rooms at Max Plank Florida Institute for Neuroscience
Nearly every room in the Max Planck Florida Institute for Neuroscience is bathed in natural light. Floor-to-ceiling windows embrace the institute on all sides, drenching the pristine, white laboratories with Florida sunshine, providing scientists with fanciful views of palm trees throughout the workday. But there are some rooms in the institute with no windows at all. These are the rooms where Long Yan, Ph.D., spends most of his time.
I cautiously approached room 3310, where Yan had suggested I meet him. The door was made of solid, light-colored wood, and held a red “DANGER Class IV laser” sign on the outside. The sign included a picture of a black starburst, meant to represent a laser, which appeared to be shooting out of nothing directly below the word “danger.” Softly, I knocked. A moment later, Yan opened the door. His teenaged student slipped out, and I slipped in to a room untainted by the midday sun. Aside from a few red and green dots on the sides of his computer, the only light source was an ethereal glow emanating from the screen behind Yan’s head.
To the left was a long metal table, pushed up against the wall, covered with screwdrivers, hammers, and loose wires scattered like a modern-day Gepetto’s workbench. Yan sat straight-backed, hands in his lap, legs crossed, and smiled at me from his chair. The computer screen in front of him lit his gray “Max Planck” t-shirt and cargo shorts.
The view of palm trees through the other windows may be impressive, but the scientists here take more pride in another view- through the lenses of 20 powerful microscopes. Through these microscopes, they can see intricate details about the ways in which brain cells interact with each other. In order to clearly view the samples, the microscopes all exist in windowless rooms, where scientists must complete their work in the dark. Yan, who was originally planning to practice medicine in China, now holds degrees in chemistry, electrical engineering, and biomedical engineering. In an effort to combine the aspects of his unique background, he found his way to here, where he is in charge of maintaining the microscopes as well as helping researchers develop new techniques for visualizing different brain activities.
2-photon microscopy is the backbone of most of the innovative imaging techniques developed at Max Planck Florida. It gives researchers the power to view structures deep within a sample, or even inside the brain of a living, breathing animal. While 2-photon technology has enhanced microscopy in all fields, it has played a particularly large role in neuroscience.
Traditional fluorescent microscopy, known as confocal microscopy, uses a single, continuous laser beam to light up a structure or molecule of interest in a sample that has been tagged with fluorescent dye. When a photon from the laser hits the dye, it glows, sending light back through the microscope to the researcher’s eye. I looked over Yan’s shoulder as he pointed to a cartoon diagram of a laser beam bouncing off of a sample on his computer screen. Depending on the dye they choose, researchers can identify signs of disease, concentrations of molecules, or different structures within cells.
The laser must be a strong enough for one of its photons to activate the fluorescent dye. Most dyes require a laser with an ultraviolet wavelength, which, in some samples can be problematic. The structure of interest must be at the surface of sample, or else the ultraviolet light will bounce off of areas between it and the structure, making it difficult to see. The strength of the light can also damage the surrounding tissue, so it cannot easily be used in living animals.
Yan explained that many researchers rely on 2-photon microscopy to avoid the issues associated with ultraviolet lasers. 2-photon microscopes use an infrared laser with half the energy needed to excite the dye. The laser pulses, and the structure will only fluoresce when it receives energy from two photons at the same time. If one photon hits the structure, it will not have enough energy to make the structure fluoresce. It’s rare for two photons to hit a structure at exactly the same time, so it only happens in the precise area where the laser is focused. Thus, the structures between the laser and the structure of interest are less likely to obstruct the scientist’s view, allowing researchers to see deeper than just the surface of the sample.
Since Winfried Denk, Ph.D., now a director at the Max Planck Institute for Neurobiology in Germany invented the 2-photon microscope in 1990, researchers across the world have been using the technology. With Yan’s help, researchers at Max Planck Florida are constantly developing new strategies to get more information from the microscopes. In one laboratory, researchers are placing the microscopes directly on top of small areas of a ferret’s brain in order to visualize how neurons interact and form networks together. Without the 2-photon technique, researchers would not be able to see anything below the surface of the ferret’s brain. The technology aided Max Planck Florida’s Scientific Director, David Fitzpatrick Ph.D. and Postdoctoral Researcher, Gordon Smith Ph.D., in identifying unique patterns of network activity during that take place during the animal’s development in their recent paper published in Nature Neuroscience this past January 2015.
As Yan described how researchers at Max Planck are using different types of molecular tags to visualize different processes, two urgent knocks thumped on the door. “I know, give me 5 more minutes,” said Yan. The hurried scientist said “Okay as long as you’re aware.” And closed the door. Before he left, I asked Yan one last question- what are the limitations of 2-photon microscopy? “It can only go one millimeter, which means it cannot study my brain,” he laughed. “My brain is much bigger.”