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Two-Photon Scope Captures Millisecond-Timescale Neuron Patterns In Vivo

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ASHBURN, Va., Aug. 9, 2019 — A new two-photon microscope from scientists at Howard Hughes Medical Institute’s Janelia Research Campus can record footage of brain activity 15 times faster than once believed possible, the team said, revealing voltage changes and neurotransmitter release over large areas and monitoring hundreds of synapses simultaneously.

The new tool, called scanned line angular projection microscopy, or SLAP, makes data collection more efficient by compressing multiple pixels into one measurement and scanning only the pixels in the areas of interest. A device controls which parts of the image are illuminated, and thus which parts are scanned. A high-resolution picture of the sample, captured before the two-photon imaging begins, guides the scope and allows scientists to decompress the data to create detailed videos.

The SLAP microscope is a new kind of two-photon microscopy that can capture fleeting neural activity at unprecedented spatiotemporal resolution. Courtesy of Matt Staley/HHMI/Janelia Research Campus.
The SLAP microscope is a new kind of two-photon microscopy that can capture fleeting neural activity at unprecedented spatiotemporal resolution. Courtesy of Matt Staley.

Much like a computed tomography scanner, which builds up an image by scanning a patient from different angles, SLAP sweeps a beam of light across a sample along four different planes. Instead of recording each pixel in the beam’s path as an individual data point, the scope compresses the points in that line together into one number. Then, computer programs unscramble the lines of pixels to get data for every point in the sample. SLAP can computationally recover high-resolution images attaining voxel rates of over 1 billion hertz (Hz) in structured samples.

In classic two-photon microscopy, each measurement takes a few nanoseconds. Making a video requires taking measurements for every pixel in the image in every frame. This, in theory, should limit how fast one can capture an image, research fellow Kaspar Podgorski said. However, the new microscope from Podgorski’s team surpasses these limits, achieving a speed that could previously be achieved only over tiny areas.

SLAP two-photon microscope from HHMI/Janelia Research Campus captures neurotransmitter release in vivo.
As shown in this artist’s impression, the HHMI team labeled individual neurons in a mouse’s brain with a fluorescent protein that makes synapses flash when activated (red stars). The SLAP microscope records those flashes along four different axes (red lines). Then, a computer program reconstructs which synapses were active at any given time based on how the signals on the four axes overlap. Courtesy of Ella Marushchenko.

In the time it takes SLAP to scan the whole sample, a traditional scope going pixel-by-pixel would cover just a small fraction of an image. This speed allowed Podgorski’s team to watch in detail how glutamate, a neurotransmitter, is released onto different parts of mouse neurons.

The team’s ultimate goal is to image all of the signals coming into a single neuron, so they can better understand how neurons transform incoming signals into outgoing signals. This current scope is “only a step along the way — but we’re already building a second generation,” Podgorski said.

Scientists use two-photon imaging to look inside opaque samples, such as living brains, that are impenetrable with regular light microscopy. Until the introduction of SLAP, it had not been possible to capture the patterns of neurotransmitter release onto neurons in the brains of living animals at a millisecond timescale, according to the HHMI team.
 
The research was published in Nature Materials (https://doi.org/10.1038/s41592-019-0493-9).   

Photonics.com
Aug 2019
Research & TechnologyeducationAmericasHoward Hughes Medical InstituteJanelia Research CampusMicroscopytwo-photon microscopyfluorescence imagingimagingBiophotonicsmedicalneural imagingsuperresolutionlight sourcesoptics

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