Redesigned Three-Photon Microscope Images Neuronal Activity 2.1 mm Deep in Mouse Brain

Light scatters as it moves deeper into brain tissue, so at depth it becomes impossible to tell neurons apart from background fluorescence. Three-photon microscopy produces less of this background than the more widely used two-photon microscopy and should, in principle, see much deeper. In practice, though, it needs high pulse energies, and the laser power the brain can safely absorb before overheating and tissue damage has limited its reach.

A team at Rockefeller University has now rebuilt the entire imaging system from scratch rather than working within that constraint. In a paper published in Nature Methods, the researchers describe Three-photon Ultra-Deep Imaging (TrUDI), a platform that brings three-photon imaging close to its theoretical limits while staying within safe power exposure for the sample.

“Often in technology, people stick with a certain laser or parameter for historical reasons, and everyone just accepts those limitations,” says senior author Alipasha Vaziri. “We wanted to take a clean slate approach. By not taking anything for granted and asking how a system fully optimized for maximizing the depth reach of three-photon imaging would look like from scratch, we found that what people previously thought to be limiting factors could be overcome.”

Two-photon microscopy reaches about 0.8 mm into the brain. Three-photon microscopy was meant to extend that, but it has not reached its potential.

“In any multi-photon excitation scheme, there is a depth limit at which the background and the signal become equal. For three-photon excitation in the mouse brain, theoretically that depth limit should be on the order of 2.3 millimeters,” Vaziri says. “But the maximum depth at which functional recording has been previously achieved was about 1.2 millimeters.”

The team first used computer models to work out the theoretical limits of deep-brain imaging and what a microscope would need to approach them. Based on those calculations, they optimized every part of the system. Most notably, they delivered fewer but far more energetic laser pulses at the same average power and redesigned how the microscope focuses and collects light. They also developed an excitation scheme that switches the laser on only while scanning over identified neurons, which cuts laser exposure by up to 80 percent during long sessions.

“When you look at individual parameters, an optical tweak might only give you an improvement of a factor of four or five, which is easy to overlook when you are up against an exponentially hard problem,” Vaziri says. “But if you have enough of these optimizations and each of these improvements is multiplicative, then you suddenly have something that is 200-fold better.”

In mice, TrUDI recorded neuronal population activity across cortical depths and into deep regions of the hippocampus and striatum, reaching up to 2.1 millimeters in the intact brain. For the first time, the team captured activity across multiple interconnected hippocampal regions and deep within the striatum without surgically removing the cortex. Tissue analyses found no inflammation, neuron loss or DNA damage under the imaging conditions used.

“There are regions of the striatum that are inaccessible, and there are still a number of deep brain nuclei which are much deeper that still require invasive approaches,” Vaziri says. “But our work brings a series of brain regions that have been previously accessible to imaging only through invasive means within reach, without that kind of invasiveness.”