The brain is undoubtedly one of the most complex structures in the known universe.
Continued advances in our understanding of the brain and our ability to effectively treat a range of neurological diseases depend on probing the brain’s neural microcircuits in ever greater detail.
A small electrode, eight millionths of a meter in diameter, is used to locally inject a cloud of electrical charge into a liquid placed on top of the diamond chip. The fluorescence of the diamond reflects the diffusion of this charge through the liquid in real time. GIF: Provided
One class of methods for studying neural circuits is called voltage imaging. These techniques allow us to see the voltage generated by the neurons that fire our brains, which tell us how networks of neurons develop, function and change over time.
Today, voltage imaging of cultured neurons is performed using dense arrays of electrodes on which cells are cultured (or grown), or by applying light-emitting dyes that optically respond to voltage changes on the surface of the cell
But the level of detail we can see with these techniques is restricted.
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Smaller electrodes cannot reliably distinguish individual neurons, some 20 millionths of a meter in diameter, let alone the dense network of nanoscale connections that form between them, and for more than two decades have made significant technological advances in this area.
In addition, each electrode requires its own cable connection and amplifier, placing significant limitations on the number of electrodes that can be measured simultaneously.
Dyes can overcome these limitations by imaging the wireless voltage as light; this means that complex electronics can be located away from the cells inside a camera.
The result is high resolution over large areas, capable of distinguishing each individual neuron in a large network. But there are limitations here as well, the voltage responses of state-of-the-art dyes are slow and unstable.
Because the voltage in a conductive solution varies uniformly, the brightness of the light emitted by the diamond chip follows with an almost instantaneous response. Here, the surface of the diamond has been patterned into a series of nanopillars to increase the detected light signal. GIF: Provided
Our recent research published in Nature Photonics explores a new type of scalable, high-speed, high-resolution voltage imaging platform designed to overcome these limitations: a voltage imaging microscope of diamond
Developed by a team of physicists from the University of Melbourne and RMIT University, the device uses a diamond-based sensor that converts voltage signals on its surface directly into optical signals; this means we can see the electrical activity as it happens.
The conversion uses the properties of an atom-scale defect in the crystal structure of diamond known as a nitrogen vacancy (NV).
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NV defects can be engineered by bombarding the diamond with a beam of nitrogen ions using a special type of particle accelerator. Fabrication of the sensor begins by using this process to create an ultrathin layer of high NV defect density near the surface of the diamond.
You can think of each NV defect as a bucket containing up to two electrons. When this bucket is empty, the NV defect is dark. With an electron, the NV defect emits orange light when illuminated by a laser; this property is known as fluorescence. With two electrons, the color of the fluorescence becomes red.
A previously discovered property of NV defects is that the number of electrons they contain, and the resulting fluorescence, can be controlled with a voltage. Unlike dyes, the voltage response of an NV defect is very fast and stable.
Our research aims to overcome the challenge of making this effect sufficiently sensitive to neural activity in the image.
A prototype of a diamond voltage imaging microscope built by physicists at the University of Melbourne. A small electrode is suspended above the diamond chip to test the performance of the device. A green laser shining from below provides fluorescence excitation to the chip. Image: supplied
On the surface of the diamond, the crystalline structure ends with a layer one atom thick, made up of hydrogen and oxygen atoms. The NV defects closest to the surface are the most sensitive to stress changes outside the diamond, but they are also very sensitive to the atomic composition of the surface layer.
Too much hydrogen and the NVs are so dark that the optical signals we are looking for cannot be seen. Too little hydrogen and the NVs are so bright that the small signals we’re looking for are completely wiped out.
So there is a “Golden Rites” zone for the voltage picture, where the surface has the right amount of hydrogen.
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To reach this area, our team developed an electrochemical method to remove hydrogen in a controlled manner. By doing this, we have managed to achieve strain sensitivities two orders of magnitude better than those previously reported.
We tested our sensor in salt water using a microscopic wire 10 times thinner than a human hair. By applying a current, the wire can produce a small cloud of charge in the water above the diamond. The formation and subsequent diffusion of this charge cloud produces small stresses on the surface of the diamond.
By capturing these voltages using high-speed recording of NV fluorescence, we can determine the speed, sensitivity, and resolution of our diamond imaging chip.
We were able to further increase the sensitivity by patterning the diamond surface into “nanopillars”: conical structures with NV centers embedded in their tips. These pillars channel the light emitted by the NVs into the camera, dramatically increasing the amount of signal we can collect.
An electron microscope image showing the surface of the diamond chip, which has been patterned into an array of fluorescent diamond nanopillars, or “optrodes,” using processes similar to those used in the manufacture of computer chips. The shape of each optrode channels the light they emit into a camera while providing an anchor for the growing neurons. Image: supplied
With the development of the diamond voltage imaging microscope to detect neuronal activity, the next step is recording the activity of neurons cultured in vitro: these are experiments with cells grown outside their normal biological context, also known as test tube or petri dish. experiments
What sets this technology apart from existing state-of-the-art in vitro techniques is the combination of high spatial resolution (on the order of a millionth of a meter or less), large spatial scale (a few millimeters in each direction, which for a network of neurons in mammals is quite vast) and total stability over time.
No other existing system can offer these three qualities simultaneously, and it is this combination that will enable our Melbourne-made technology to make a valuable contribution to the work of neuroscientists and neuropharmacologists worldwide.
Our system will help these researchers pursue fundamental insights and the next generation of treatments for neurological and neurodegenerative diseases.
Banner: Developed by physicists at the University of Melbourne, the diamond voltage imaging microscope offers a new way to image voltage changes in liquids, such as those produced by firing neurons, with high spatial and temporal/supplied resolution .