Two-Photon-Microscope Smashes Speed Records

A new microscope breaks a long-standing speed limit, recording footage of brain activity 15 times faster than scientists once believed possible. It gathers data quickly enough to record neurons’ voltage spikes and release of chemical messengers over large areas, moni­toring hundreds of synapses simul­taneously – a giant leap for the powerful imaging technique of two-photon micro­scopy.

The SLAP microscope is a new kind of two-photon microscopy that can capture fleeting neural activity at unprecedented spatiotemporal resolution. (Source: M. Staley, HHMI)

The trick lies not in bending the laws of physics, but in using knowledge about a sample to compress the same information into fewer measure­ments. Scientists at the Howard Hughes Medical Institute’s Janelia Research Campus have used the new micro­scope to watch patterns of neuro­transmitter release onto mouse neurons. Until now, it’s been impossible to capture these milli­second-timescale patterns in the brains of living animals.

Scientists use two-photon imaging to peer inside opaque samples like living brains that are impene­trable with regular light microscopy. These micro­scopes use a laser to excite fluorescent molecules and then measure the light emitted. 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. That, in theory, limits how fast one can capture an image, says Kaspar Podgorski, a fellow at Janelia. “You’d think that’d be a funda­mental limit – the number of pixels multiplied by the minimum time per pixel,” he says. “But we’ve broken this limit by compressing the measure­ments.” Previously, that kind of speed could only be achieved over tiny areas.

The new tool – Scanned Line Angular Projection micro­scopy, or SLAP – makes the time-consuming data-collection part more efficient in a few ways. It compresses multiple pixels into one measure­ment and scans only pixels in areas of interest, thanks to a device that can control which parts of the image are illu­minated. 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.

Much like a CT 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 – sort of like solving a giant Sudoku puzzle.

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, an important neuro­transmitter, is released onto different parts of mouse neurons. In the mouse visual cortex, for example, they identi­fied regions on neurons’ dendrites where many synapses seem to be active at the same time. And they tracked neural activity patterns migrating across the mouse’s cortex as an object moved across its visual field.

Podgorski’s ultimate goal is image all of the signals coming into a single neuron, to 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. Once we have that, we won’t be limited by the micro­scope anymore,” he says. His team is upgrading the scope’s scanners to increase its speed. They’re also seeking ways to track other neuro­transmitters so they can fully tap into the symphony of neural communi­cation. (Source: HHMI)

Reference: A. Kazemipour et al.: Kilohertz frame-rate two-photon tomography, Nat. Meth. 16, 778 (2019); DOI: 10.1038/s41592-019-0493-9

Link: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, USA

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