Human brains consist of around 86 billion neurons that connect and communicate with each other, forming an intricate network. Understanding how the human brain functions and is structured is a long-term goal in neuroscience. A step toward understanding human brains is understanding how a mouse brain works. While a mouse brain consists of between 8 to 14 million neurons, it can function as a proving ground to refine the technology and analytical methods used to monitor neurons and ultimately understand behavior.
A History of Looking at Small Things
Humans have had a long history of magnifying small things. Lenses date back to 700 BC in Ancient Egypt and Mesopotamia. Made from quartz, the ancient Assyrians used these early lenses as magnifying glasses, decorations, and tools to start fires. Similarly, the Romans used glass spheres to magnify small text.
The first compound microscope was invented in the late 16th century when a father and son found that objects were magnified when they looked through a tube with a lens on the top and bottom. Animalcules—meaning “tiny animal” in Latin—was what Antonie van Leeuwenhoek called the bacteria he saw in the microscope he had made. He gathered them from varied sources of liquid such as pond water, rainwater, well water, and even the human mouth. Today he is known as “the Father of Microbiology.”
Microscopes introduced the world to the biology of smaller life. Microscopes have illuminated how bacteria found everywhere can affect plants, animals, human life, and even how disease spreads. Over the last few decades, microscopy has advanced to let us not just peek into our miniature world but also to help us watch and understand how large and complex systems, including our brain, function in real time.
Two Photons Are Better than One
In the realm of brain imaging, microscopy often uses unique molecules called fluorophores. A fluorophore is mostly inert save for one property: it can fluoresce. A fluorophore will absorb light of one color (or frequency) and then re-emit that light as a different color. For example, the fluorophore known as GFP (green fluorescent protein) will emit green light when exposed to blue light. Biologists can tease apart the structure and function of a live brain by strategically choosing between different fluorophores and putting them in selected parts of a brain.
Microscopes are used to detect when and where a fluorophore emits light. Microscopes can be divided into two groups: one-photon (or single-photon, 1P) and two-photon (2P). Traditional microscopes, like those used in high school science labs, are one-photon microscopes.
With one-photon microscopy, a single particle of light, a photon, is used to cause a fluorophore to emit light. In two-photon microscopy, two photons simultaneously hit a fluorophore and cause it to emit light. A critical difference is that the photons used with two-photon microscopy have half the energy of those used with one-photon microscopy.
Using lower-energy photons in two-photon microscopy has several advantages. A fluorophore has a limited life. Over repeated exposure, it will eventually change its shape and be unable to fluoresce anymore. This phenomenon is known as photobleaching. The lower-energy photons of two-photon microscopy ensure that fewer fluorophores get enough energy to stop working compared to one-photon microscopy. With one-photon microscopy, each photon is like a bullet with a lot of energy and destructive ability. It might work for a few snapshots, but you’ll quickly do a lot of damage. In contrast, two-photon microscopy is like using foam darts. You can continue monitoring a brain without worrying about doing much damage.
Another advantage of two-photon microscopy is imaging depth. There is a limit to how far light can penetrate a tissue. The high-energy photons of one-photon microscopy have the potential to work well up to about 200 microns (for reference, a hair is around 70 microns). A lack of imaging depth is the reason that conventional one-photon microscopy typically involves cutting things into slices and putting them on a glass slide. In contrast, two-photon microscopy works well to about 1,000 microns.
Microscopy of live brains in rodents started with attaching a mouse to a microscope. Similarly, the first miniaturized microscopes were attached to immobilized rodents. While sleeping mice are potentially appropriate for researching areas like smell responses, you won’t learn anything about how a mouse thinks about pathfinding or any behavior that requires moving around.
In 2013, Ghosh and colleagues reported on their development and use of a miniaturized microscope integrated into freely moving animals. They were able to visualize and measure activity and blood microcirculation in the brain of a freely moving adult mouse as it performed activities. Their microscope used one-photon microscopy and brought along the associated technical limitations, including fluorophore photobleaching and a limited ability to examine deep into the brain.
Tiny Two-Photon Microscopes
Researchers have made multiple attempts to miniaturize a two-photon microscope that can be mounted on a freely moving mouse without it being too heavy or disrupting the mouse’s movements. Early attempts had to make sacrifices in terms of resolution, depth, or even the stiffness of the tether.
Weijian Zong, an optical engineer at the Kavli Institute for Systems Neuroscience, and his team recently published their work in Cell describing the development of a tiny two-photon microscope called MINI2P. The microscope weighs just 2.4 grams, about as much as a dime. It’s so light that mice can freely explore their environment without getting tired of “wearing” the microscope.
MINI2P comes with all the benefits of two-photon microscopy on a freely moving animal, including decreased photobleaching of fluorophores and increased imaging depth. Compared to previous attempts at two-photon microscopy, MINI2P can also image an order of magnitude more cells while attached to a mouse.
Zong has provided a complete material list, instructional videos, and a discussion of how to analyze collected data. The materials cost around $5,000 (not including the much more expensive light source). The Kavli Institute has also held workshops to help researchers build their own MINI2P (bring-your-own-materials). Imagine spending an afternoon assembling a miniature two-photon microscope with Ikea-like instructions.
Advancing Live Imaging
MINI2P continues the quest to miniaturize microscopy, permitting the visualization of structure and activity in the brains of freely moving animals. The development of new technologies will undoubtedly make more of the brain accessible to researchers. For example, the MINI2P microscope uses a lens system called GRIN, which involves implanting a highly specialized lens into the brain. However, researchers have found that a competing system that inserts a relatively inexpensive cylindrical glass plug permits more of the brain to be imaged and allows imaging at a greater depth.
Microscopes have gotten smaller, and the trend will undoubtedly continue through the discovery and use of new techniques to create new microscopes. It’s not clear yet what the technology will look like when fully realized but work like Zong’s propels us toward the goal of watching a brain think in real time.
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