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As light travels deeper into the brain, it scatters. Three-photon microscopy, the current state of the art, reduces the resulting background fluorescence and can, in principle, image much deeper than earlier techniques—but to do that, it uses average laser power limits that cause overheating and damage living tissue.

Instead of accepting that trade-off, a Rockefeller team has now redesigned the entire imaging system from the ground up. In a paper published in Nature Methods, the team describes a microscopy platform called Three-photon Ultra-Deep Imaging (TrUDI), which pushes three-photon imaging close to its theoretical limits, in part by reengineering the rate and energy of laser pulses, how they are delivered and scanned at the sample—all within the safe limits of sample power exposure.

The result is a roughly 250-fold increase in imaging efficiency, allowing researchers to image through the entire mouse cortex and record neural activity in deep brain regions, such as the entire depth range of the hippocampus and striatum, without surgically removing the overlying tissue.

"Often in technology, people stick with a certain laser or parameter for historical reasons, and everyone just accepts those limitations," says Alipasha Vaziri, head of the Laboratory of Neurotechnology and Biophysics.

"We wanted to take a clean-slate approach.

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