Biomedical researchers at the University of California have built a camera that can image the brain of an alert mouse up to 3,000 frames per second. This let them record for the first time the passage of millisecond-long electrical pulses through neurons.
The new imaging technique combines two-photon fluorescence microscopy and all-optical laser scanning in a state-of-the-art microscope. The team used the microscope to film a two-dimensional slice through the neocortex of a mouse brain fast enough to trace electrical signals flowing through it.
With this capability, neuroscientists can now clock electrical signals as they propagate through the brain and ultimately look for transmission problems associated with disease.
One key advantage of the technique is that it will let neuroscientists track the hundreds to tens of thousands of inputs any given brain cell receives from other brain cells, including those that don’t trigger the cell to fire. These sub-threshold inputs—either exciting or inhibiting the neuron—gradually add up to a crescendo that triggers the cell to fire an action potential, passing information along to other neurons.
The typical method for recording electrical firing in the brain, via electrodes embedded in the tissue, detects only blips from a few neurons as the voltage changes. The new technique pinpoints the actual firing neuron and follows the path of the signal, millisecond by millisecond.
“In diseases, many things are happening, even before you can see neurons firing, like all the subthreshold events,” said Na Ji, a member of UC Berkeley’s Helen Wills Neuroscience Institute. “We’ve never looked at how a disease will change with subthreshold input. Now, we have a handle to address that.”
The team also developed a technique for imaging calcium signaling over much of an entire hemisphere of the mouse brain at once, one that uses a wide-field-of-view “mesoscope” with two-photon imaging and Bessel focus scanning. Calcium concentrations are linked with voltage changes as signals are transmitted through the brain.
This is the first time anyone has shown in three dimensions the neural activity of such a large volume of the brain at once, which is far beyond what electrodes can do. The imaging approach also lets researchers resolve the synapses of each neuron. (Synapses are where neurotransmitters are released by one neuron to excite or inhibit another.)
One of Ji’s goals is to understand how neurons interact across large areas of the brain and eventually locate diseased circuits linked to brain disorders.
The technique uses probes that can be pinned to specific types of cells and become fluorescent when the environment changes. To track voltage changes in neurons, for example, the team employed a sensor that becomes fluorescent when the cell membrane depolarizes, which occurs when voltage signal propagates along the cell membrane.
Researchers then illuminate these fluorescent probes with a two-photon laser, making them emit light, or fluoresce, if they have been activated. A microscope captures the emitted light; it is combined into a 2D image that shows where the voltage changes or the presence of a specific chemical, such as the signaling ion, calcium.
By rapidly scanning the laser over the brain, researchers obtain images of a single, thin layer of the neocortex. The team was able to conduct 1,000 to 3,000 full 2D scans of a single brain layer every second by replacing one of the laser’s two rotating mirrors with an optical mirror—a technique called free-space angular-chirp-enhanced delay (FACED).
The kilohertz imaging not only reveals millisecond changes in voltage, but also more slowly changing concentrations of calcium and glutamate, a neurotransmitter, as deep as 350 microns (one-third of a millimeter) from the brain’s surface.
To get rapid 3D images of the movement of calcium through neurons, they combined two-photon fluorescent microscopy with a different technique, Bessel focus scanning. To avoid time-consuming scans of every micron-thick layer of the neocortex, the Bessel focus of the two-photon laser is shaped from a point to a small cylinder, like a pencil, about 100 microns in length. This beam is scanned at six different depths through the brain, and the fluorescent images are combined to create a 3D image. This allows more rapid scanning with little loss of information because, in each pencil-like volume, typically only one neuron is active at any time. The mesoscope images an area about 5 mm in diameter, nearly a quarter of one hemisphere of the mouse brain, and 650 microns deep, close to the full depth of the neocortex, which is involved in complex information processing.
With conventional methods, biomedical engineers would have to scan 300 images to cover this volume, but with an elongated beam that collapses the volume onto a single plane, the new method only needs to scan six images. This means it has a fast-enough volumetric rate to look at its calcium activity.
The team is now working on combining four techniques—two-photon fluorescence microscopy, Bessel beam focusing, FACED and adaptive optics—to create high-speed, high sensitivity images deep in the neocortex, which is about 1 millimeter thick.