Femtonics’ research and development manager, Gergely Szalay, and applications specialist Tamás Tompa demonstrate new functions on the FEMTO3D Atlas microscope

Sounding out the brain: acoustic signals focus light on neurons

A new innovation is allowing neuroscientists to follow the activity of thousands of neurons in a three-dimensional brain region.

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Botond Roska investigates the computations that brain circuits execute on a moment-by-moment basis. His approach is conceptually straightforward — but technically demanding. Experiments in his lab at the Institute of Molecular and Clinical Ophthalmology Basel in Switzerland involve recording the activity of single cortical neurons — plus the activity of up to 200 neighbouring neurons that provide the cell with synaptic inputs.

His group scans these neurons using two-photon (2P) imaging. However, conventional 2P imaging systems operate only in a 2D plane, but neuronal networks are dispersed in three-dimensional space. The problem lies in the way most 2P systems focus the laser, scanning continuously along lines or planes. Moving beyond a plane typically requires mechanical movement of the lens, which slows down sampling rates considerably.

When Roska’s team scanned the 3D network plane-by-plane using 2P imaging, they had to sequentially characterize all the presynaptic neurons’ activity, then infer how these qualities could yield the properties of the target neuron. When they published the results in 2015, they included a supplementary figure showing how an alternative type of imaging could be used to do these experiments.

This other technology, called random-access 3D microscopy, allows a 2P laser to move almost instantaneously between many points within a region. “What you can now do”, Roska says, “is look simultaneously at all 150-200 presynaptic neurons that give input to your postsynaptic neuron and what they do at a given moment. You can really start to analyse how the brain computes in real time.”

Sound and vision

This 3D scanning is achieved by inserting an upgrade containing acousto-optical (AO) deflectors in the laser path of a 2P microscope. An AO deflector is essentially a transparent crystal with a refractive index that can be modulated by passing soundwaves through it. Changing the frequency of those soundwaves changes where the laser focuses, allowing it to be directed anywhere in a 3D space in microseconds (see ‘3D imaging microscope design’).

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