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Light Beads Microscopy Resolves Neurons Throughout the Brain

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NEW YORK, SEPT. 27, 2021 — Researchers from The Rockefeller University developed a microscopy method that allows scientists to capture detailed images of a vast number of cells across different depths in the brain at high speed and with high clarity.

The researchers demonstrated the capability of the method, light beads microscopy, by presenting functional video of the near-simultaneous activity of 1 million neurons across the mouse brain.

“Understanding the nature of the brain’s densely interconnected network requires developing novel imaging techniques that can capture the activity of neurons across vastly separated brain regions at high speed and single-cell resolution,” said Alipasha Vaziri in the Laboratory of Neurotechnology and Biophysics at Rockefeller. “Light beads microscopy will allow us to investigate biological questions in a way that had not been possible before.”

The present method of imaging neuronal activity uses a combination of two-photon scanning microscopy and fluorescent tags. It involves firing a focused laser pulse at a tagged target. A few nanoseconds after the pulse hits its mark, the tag emits fluorescent light that can be interpreted to give scientists an idea of the level of neuroactivity detected.

Two-photon microscopy on its own suffers a fundamental limitation. Neurobiologists need to record simultaneous interactions between the sensory, motor, and visual regions of the brain. To do so, there is a trade-off between resolution and speed. In the interests of high resolution, scientists must often sacrifice scale, or zoom out to view the larger structure at the cost of resolution. This can be overcome by snapping a series of high-resolution images from distant corners of the brain separately and later stitching them together, though with this approach, speed becomes an issue

“We need to capture many neurons at distant parts of the brain at the same time at high resolution,” Vaziri said. “These parameters are almost mutually exclusive.”

Light beads microscopy eliminates the “deadtime” between sequential laser pulses when no neuroactivity is recorded. The method breaks one strong pulse into 30 smaller sub-pulses — each at a different strength — that delve into 30 different depths of scattering mouse brain, but induce the same amount of fluorescence at each depth.

This is accomplished with a cavity of mirrors that staggers the firing of each pulse in time and ensures they can all reach their target depths via a single microscope focusing lens.

With this approach, only the time it takes fluorescent tags to flare limits the speed of recording. Broad swaths of the brain can be recorded in the time it would take a conventional two-photon system to capture a fraction of the data.

Because the method is an innovation that builds on two-photon microscopy, many labs already have obtained or can commercially obtain the technologies necessary to perform light beads microscopy as described in the researchers’ paper.

Vaziri is also developing a simplified self-contained module for more widespread use.

The research was published in Nature Methods (www.doi.org/10.1038/s41592-021-01239-8).

Photonics.com
Sep 2021

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