New Microscopy Technique Provides Insights at Single-Neuron Level

A novel microscopy technique integrates new and existing approaches to help build a more cohesive picture of the brain, according to the scientists who developed it at Rockefeller University. Described in a paper published in Cell last week, hybrid multiplexed sculpted light microscopy (HyMS) captures cellular activity across large volumes of neural tissue, quickly and at new depths.

For decades, brain imaging has been plagued by trade-offs. Some techniques produce beautiful images but fail to record neural activity in real time. Others can keep up with the brain's speed but have poor spatial resolution. And although there are tactics that successfully combine rapidity and image quality, they typically capture only a small number of cells.

"This is in part because the limits that govern these tradeoffs have not been explored or pushed in a systematic and integrated manner," says Alipasha Vaziri, head of the laboratory of neurotechnology and biophysics. Hoping to end the era of trade-offs, Vaziri recently endeavored to improve upon two-photon (2p) microscopy, which has long been the gold standard for probing cellular activity in the brain. Yet, 2p has limitations, including that it requires point-by-point scanning of a given region, which results in slow imaging. To resolve this issue, Vaziri and his colleagues implemented a novel strategy that permits recording from multiple brain regions in parallel, while carefully controlling the size and shape of each spot recorded.

Another weakness of traditional 2p is that it measures only the cortex, neglecting structures buried deep within the organ, such as the hippocampus. "One of the biggest challenges in neuroscience is developing imaging techniques that measure the activity of deep brain regions while maintaining high resolution," says Vaziri. Taking up this challenge, he decided to make use of a newer technology: three-photon (3p) microscopy. Whereas 2P doesn't reach beyond the surface, or cortex, of a mouse brain, 3p penetrates deeper regions. HyMS, Vaziri's innovation, applies 2P and 3P concurrently, allowing researchers to generate a picture of rapid cellular activity across multiple layers of brain tissue.

In addition to its hybrid laser strategy, HyMS also integrates other recent technical and conceptual advancements in the field—a synergistic approach that Vaziri says guided the development of the technology. The goal, he says, was to maximize the amount of biological information that could be obtained through multi-photon excitation microscopy while minimizing the heat produced by this method. And when testing their new system, the scientists certainly obtained a lot of information.

HyMS boasts the highest frame rate of available 3p techniques, he says, which means it can capture biological changes at record speed. And whereas previous techniques scanned only a single plane of tissue, this technology can obtain information from the entire tissue sample and allows users to record from as many as 12,000 neurons at once. Another advantage of HyMS is its ability to simultaneously measure activity from brain areas at different depths. Since different layers of the brain constantly exchange signals, says Vaziri, tracking the interplay between these regions is key to understanding how the organ functions.