Inspired by technologies used in aerospace engineering, scientists have developed a way to capture the brain’s intricate structures using X-rays without destroying the samples.
Dendrodendritic synapses imaged using volume EM (bottom panels) after X-ray ptychography imaging of the same sample (top panels). Credit: Carles Bosch Piñol, Ana Diaz, Adrian Wanner and Andreas Schaefer, Nature Methods.
In recent years, technological innovation has transformed the field of neuroscience, as Andreas Schaefer has witnessed. “The brain was like a black box, its intricate inner structures shielded by dense layers of complexity, which were nearly impossible to image without invasive techniques and damage,” he says.
“But now we’re able to look within the brain to map this complexity and detail every connection. It’s transformed our understanding of the brain and how it functions, from how we perceive the world around us, to the diseases that impact our mental health.”
Andreas leads a team in the Crick’s Sensory Circuits and Neurotechnology Laboratory, and they are pushing this even further, forming an international team of imaging experts with scientists at the Paul Scherrer Institute and the European Synchrotron.
Together they’ve developed a new imaging protocol, published today in Nature Methods, to capture mouse brain cell connections in precise detail. The images acquired using this technique, called X-ray ptychography, allowed the scientists to see how nerve cells connect without needing to cut the fragile biological samples.
At the best resolution achieved with X-ray ptychography imaging (top), dendrodendritic synapses were visible (magenta arrowheads). The same sample was then imaged using volume EM (FIB-SEM method) to confirm the accuracy in detecting synapses based on X-ray ptychography alone (bottom).
The team's work is built on the foundations of a gold standard imaging technology, volume electron microscopy (volume EM), which paved the way for scientists to create maps called connectomes, of entire brains, first in larvae and then the adult fruit fly. This type of imaging involves cutting 10s of nm thin slices thin slices (tens of thousands per mm of tissue), imaging each slice and then building the images back into their 3D structure.
But, compared to electrons, X-rays have the potential to penetrate even deeper into the matter, so the international team set out to investigate if this type of imaging would be suitable for capturing the fine details of the nerve cells within tissue without the need to slice the sample.
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Building on standard volume EM sample preparation protocols, they tested a new step – embedding the stained tissue using a resin developed in the aerospace industry to prevent spaceships from being damaged by radiation. As lead for the study Carles Bosch Piñol explains, “This resin allowed us to expose the samples to up to 20 times more radiation than common volume EM preparation. And that radiation was necessary for our images to reach the resolution needed to delineate neuronal circuits.”
The X-ray ptychography images were captured in a ‘particle accelerator’ facility called a synchrotron, which uses magnetic and electric fields to propel electrons at very high speeds. They achieved a resolution of 38nm, which was enough to show multiple elements of the mouse brain circuitry, including synapses (areas where two neurons connect), dendrites (nerve cell projections), and many axons (wires carrying electrical information).
For Andreas, it’s the ability to image intact brains that’s most exciting. “Volume EM has been revolutionary for seeing things inside the cell in 3D, but it comes with limitations for mapping neuron connections inside mammalian brains, which are too large to be reliably sliced into tiny sections.
“We’re excited that our protocol and use of powerful radiation-resistant material have allowed brain tissue to be imaged at extraordinary resolution. Refining this technique further could bring us a small step closer to a future goal in the field: mapping the mouse brain connectome, which is tens of thousands of times bigger than the fruit fly.”
“Now we’ve shown that X-ray imaging is suitable for mapping the fine detail of delicate biological tissue samples in 3D, we’re continuing to make the methods better and better to improve the field of view and the resolution,” adds Carles.
“How large are the samples we can image? Can we obtain finer detail? With new technological advances, we’re lifting the limitations on our understanding of the function of biological tissues such as the brain.”
