High-Speed Microscopy Tracks Millisecond Voltage Changes in Neurons of Awake Mice

University of California, Berkeley, researchers have built a microscope that can image the brain of an alert mouse 1000 times a second and record the passage of millisecond electrical pulses through neurons. The new imaging technique combines two-photon fluorescence microscopy and all-optical laser scanning in a microscope that can image a 2D slice through the neocortex of the mouse brain up to 3000 times per second. According to the researchers, that’s fast enough to trace electrical signals flowing through brain circuits.

To track voltage changes in neurons, the researchers used a sensor that becomes fluorescent when the cell membrane depolarizes as a voltage signal propagates along the membrane. The scientists illuminated the sensors with a two-photon laser, which made them fluoresce when activated. The emitted light was captured by a microscope and combined into a 2D image that showed the location of the voltage change or the presence of a specific chemical, such as the signaling ion, calcium.

By rapidly scanning the laser over the brain, the researchers were able to obtain images of a single thin layer of the neocortex. The team was able to conduct 1000 to 3000 full 2D scans of a single brain layer every second by replacing one of the laser’s two rotating mirrors with an optical mirror. This technique, called free-space angular-chirp-enhanced delay (FACED), was developed at the University of Hong Kong.

The kilohertz imaging revealed millisecond changes in voltage as well as more slowly changing concentrations of calcium and glutamate, a neurotransmitter, as deep as 350 µm from the brain’s surface.

To obtain rapid 3D images of the movement of calcium through neurons, the researchers combined two-photon fluorescence microscopy with Bessel focus scanning to enable a wide-field-of-view “mesoscope” that enabled them to image calcium signaling over much of an entire hemisphere of the mouse brain at once. To avoid time-consuming scans of every μm-thick layer of the neocortex, the excitation focus of the two-photon laser was shaped from a point to a small cylinder, like a pencil, about 100 μm in length. This pencil-like beam was scanned at six different depths through the brain, and the fluorescent images were combined to create a 3D image.

This approach allowed more rapid scanning with minimal loss of information because in each pencil-like volume, typically only one neuron was active at any time. The mesoscope was able to image an area about 5 mm in diameter — nearly a quarter of one hemisphere of the mouse brain — and 650 μm deep, close to the full depth of the neocortex.

“Using conventional methods, we would have to scan 300 images to cover this volume, but with an elongated beam that collapses the volume onto a single plane, we only need to scan six images, which means that now we can have a fast enough volumetric rate to look at its calcium activity,” professor Na Ji said.

Ji and her group are now working on combining four techniques — two-photon fluorescence microscopy, Bessel beam focusing, FACED, and adaptive optics — to achieve high-speed, high-sensitivity images deep in the neocortex, which is about 1 mm thick.

“As a way to understand the brain, my dream is to combine these microscopy techniques to get submicron spatial resolution so we can see the synapses, millisecond time resolution for the voltage imaging, and see all of this deep in the brain,” Ji said. “What is complicated and challenging about the brain is that, if you only do one single optical section, in a way you don’t get a complete picture, because a neural network is very much three-dimensional.

“In brain disorders, including neurodegenerative disease, it’s not just a single neuron or a few neurons that get sick,” Ji said. “So, if you really want to understand these illnesses, you want to be able to look at as many neurons as possible over different brain regions. With this method, we can get a much more global picture of what is happening in the brain.”

The research was published in Nature Methods (www.doi.org/10.1038/s41592-020-0762-7).

When a neuron fires, calcium flows into the cell in a wave that sweeps along the cell body. Images of this infragranular neuron were obtained three times per second by two-dimensional scanning with a Bessel focus. Redder structures are deeper in the mouse cortex. Courtesy of UC Berkeley/Na Ji.