Deep brain imaging with three-photon microscopy and a swim from the US to China.
Watching active neurons in the brain has become possible thanks to two-photon microscopy and a range of genetically encoded sensors. Two-photon microscopy was developed by Winfried Denk and Watt Webb, scientists who both loom large in the life of Chris Xu, an applied physicist at Cornell University. Xu has now completed an approximately 20-year path to apply three-photon microscopy for deep brain imaging. At a depth of one millimeter, he and his team, including Dimitre Ouzounov, recorded activity of a neuronal population at single-cell resolution. Xu says that researchers familiar with two-photon imaging will find the technique straightforward, and neuroscientists without optical physics expertise will be able to use it, too.
The three-photon advantage in microscopy mirrors the advantage of two photons over one photon: the ability to image deeper in the brain. The methods all complement one another, says Xu, and all are needed for large-scale recording from the brain: one-photon microscopy for imaging transparent samples; three-photon approaches for imaging deep into tissue and for densely labeled brain tissue; and two-photon microscopy to address the other applications in between these two classes. But brain tissue is a light-scattering nightmare.
Imaging deeper into tissues with wavelengths beyond 1,300 nanometers, it is hard to excite green fluorescent proteins. Only a handful of fluorescent dyes and proteins are compatible with longer wavelengths. These issues motivated Xu to explore how to harness three-photon excitation for brain imaging. When calculated on the back of the envelope, the task is a “no brainer,” he says. In practice, it was more of a brain squeeze. He was missing a type of laser that did not exist. So he developed and built it, using his experience in fiber optics.
The resulting excitation source generates a powerful pulse in the long-wavelength window needed for three-photon imaging. Xu worked with two laser manufacturers to mature the excitation source, and some commercial well-performing, compact systems have emerged, which will help disseminate three-photon microscopy, he says. He and his team keep improving performance and robustness of the excitation source, and they are working on better imaging speed by applying adaptive optics. “I think another order of magnitude improvement is possible within the next couple of years,” he says, that will open up approaches for functional imaging in the intact mouse brain at depths that were previously impossible to reach.
After graduating from Fudan University, Xu completed his PhD in multiphoton imaging with Cornell University researcher Watt Webb. Joining the Webb group was his “luckiest break,” and it made him the scientist he is today, says Xu. When Webb identifies an important research direction, he moves fearlessly into it. “By doing so, he is able to always come out as a leader and pioneer of new fields, and never a follower,” Xu says.
Xu did postdoctoral work with Denk, who was at AT&T Bell Laboratories and is now director at the Max Planck Institute of Neurobiology. Denk recruited Xu to Bell Labs, which Xu calls “a dream place.” At the time, Denk had started brain imaging, as had David Tank, also at Bell Labs. Tank is now at Princeton University and a current collaborator of Xu's. Denk and Tank established two-photon microscopy in neuroscience. At Bell, Xu worked on making long-haul fiber-optic transmission systems more efficient; yet labs working on brain imaging experiments were all around him. When the telecommunications industry contracted, Xu explored academia. Webb alerted him to an opening at Cornell “almost as if he knew what I was thinking about,” says Xu, who joined the faculty in 2002. He continued fiber-optic research but then began combining it with imaging the brain, the organ that had fascinated him since high school.
Everyone is a brand new book to read.
Xu loves to apply physics to problem solving in telecommunications, neuroscience or other areas. To do so involves listening to the important challenges and being flexible when solving them. His ideas stem from the way he sees connections between different fields, which takes broad training and an open mind, he says. He has had great mentors and says he is still learning about how to be one himself since there is no winning formula to follow. “Everyone is a brand new book to read,” he says. “I try to give my people as much freedom as possible.”
When Xu is not in the lab or mentoring, he swims. His secret goal is that in his lifetime he can cumulatively swim the approximately 6,000-mile distance between the US and China. And, he says, “I am well on my way.”
Ouzounov, D.G. et al. In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain. Nat. Methods 14, 388–390 (2017).
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Marx, V. Chris Xu. Nat Methods 14, 327 (2017). https://doi.org/10.1038/nmeth.4231