Mapping all the connections among the brain’s 100 billion neurons is one of the great scientific and engineering challenges of our time. Scientists, including those at the University of Chicago, began this work in earnest about a decade ago in a quest to build a complete wiring diagram of the brain. This diagram – known as the connectome – will help researchers reverse engineer the brain and unlock secrets of how it works at the most fundamental level.
So far, they’ve made tremendous progress on some of the initial technical challenges. The processes for preparing brain samples to take images under a microscope efficiently have vastly improved. Artificial intelligence, machine learning models, and raw computing horsepower are now up to the task of organizing and analyzing the vast amounts of data contained in these images. But one bottleneck remains: it still takes too long to capture images of each brain sample.
“We are kind of stuck at some hard limits on how quickly we can image the data, and there's not an obvious path how to get past those with the kinds of microscopes we’ve been using,” said Gregg Wildenberg, a staff scientist in the Department of Neurobiology at UChicago.
Wildenberg works with Narayanan “Bobby” Kasthuri, Assistant Professor of Neurobiology at UChicago and neuroscience researcher at Argonne National Laboratory, who has been pioneering connectome research over the past decade. Now, they are joining a team of scientists from UChicago, Chicago State University (CSU), and the University of Illinois Chicago (UIC) to adapt a different imaging technology, called photoemission electron microscopy (PEEM), for connectomics and drastically increase the speed of capturing brain images. The group recently received a $4.8 million, three-year grant from the Brain Research Through Advancing Innovative Neurotechnologies (BRAIN) Initiative, part of the National Institutes of Health (NIH).
Brains on a deli slicer
Until now, connectome researchers have been using two kinds of microscopes: scanning electron microscopy (SEM) and transmission electron microscopy (TEM), each with their own advantages and disadvantages. SEM is fairly forgiving with samples, which can be easily deposited on standard silicon wafers, and it works with samples with a wide range of thicknesses. But SEM also scans across the sample pixel by pixel in a grid pattern, which can be very slow. TEM solves this problem by capturing large regions of an image simultaneously, but the samples must be extremely thin and mounted on fragile grids, which are difficult to work with, especially in large volumes.
PEEM isn’t new; it’s been used since the 1980s to scan two dimensional hard surfaces and materials. It combines elements of SEM and TEM, using photons to capture the whole image simultaneously but with sturdier equipment for mounting the samples inside a vacuum chamber. Sarah King, Assistant Professor of Chemistry at UChicago and a co-investigator on the project, specializes in using PEEM to look at thin, flat sections of two-dimensional materials. She has considered using PEEM for biological molecules, but not whole tissues and organs because she assumed the high vacuum pressures would damage the samples--until one day she received an email from Wildenberg and Kasthuri asking if they could use it to scan slices of mouse brains, offering her a free coffee and a pastry as a bribe.
“It was one of the weirder emails I’ve received in my life,” she said. “The idea of putting a whole mouse brain in a microscope with more vacuum than outer space seemed like it would be in vain, but for a free coffee and a pastry, sure, I’ll listen to your pitch.”
As it turned out, the sample preparation techniques Kasthuri and Wildenberg have perfected over the years lend themselves well to PEEM. Viewing synapses, the connections between brain cells, requires imaging a slice of brain thousands of times thinner than a sheet of paper. Mapping an entire mouse brain would require hundreds of thousands of these slices, so they industrialized the process of creating samples. “We try to turn brains into as close of a material science problem as possible,” Wildenberg said.
First, the brains are dehydrated and stained with dyes, then fixed in a hard resin. Next, they’re placed on what everyone on the team calls “the deli slicer,” which shaves off ultrathin slice after slice to float away on water to a conveyer belt that places them sequentially on a substrate for imaging.
The first few images they scanned in King’s lab turned out surprisingly well. "It took significant pioneering spirit to introduce biological samples into an environment usually only operated for surface science in ultra-high-vacuum conditions,” said Kevin Boergens, Research Assistant Professor of Physics at UIC and another co-investigator on the project. “The images look great, it’s super promising. And the cool thing is, because you use UV light to make the image, the promise is that you can scale it quite easily.”
Democratizing neuroscience
The team will begin by retrofitting a standard PEEM microscope to handle the large quantity of deli-thin brain slices. For all the high-tech, expensive equipment needed to capture brain images, however, the rest of the field of connectomics is surprisingly egalitarian. Once researchers publish their data in online repositories, anyone can download it and do their own analyses, asking new questions or revisiting previous studies. And since today’s high-resolution microscopes capture the entire anatomy of the brain and its cells, that secondary research doesn’t have to be only about how neurons connect. Students can study other structures of the brain or investigate specific structures of cells.
The new grant helps democratize brain research further through a collaboration with Andrew Maselli, Professor of Biology at CSU. Maselli has connected students to work in the lab with Wildenberg and Kasthuri, learning the ins and outs of preparing brain samples, working with microscopes, and analyzing the imaging data. The new project will introduce CSU students to working with PEEM and open avenues for them to conduct their own secondary research using the data the team generates.
"At Chicago State, we are proud to provide undergraduate students the chance to engage in groundbreaking biological research, collaborating with leading institutions across Chicago. This project brings together diverse teams to solve complex scientific questions and transforms students' academic journeys—allowing them to directly contribute to mapping the brain using cutting-edge technology,” Maselli said. "For undergraduate students, participating in research of this magnitude will empower them to ask bold questions and be part of scientific breakthroughs that they will remember throughout their careers."
King says the project shows what kind of opportunities can come from taking a chance and answering an email out of the blue (not to mention the free coffee).
“There's still a lot of engineering and programming to figure out, but it's really this beautiful marriage of material science, chemistry, physics and neurobiology,” King said. “And it's only possible because of the close collaboration between the Biological Sciences Division and Physical Sciences Division at UChicago and our amazing partners at UIC and Chicago State. It’s bringing diverse perspectives together on the science and opening the doors for students to learn all these tools that are incredibly important in terms of being part of a 21st century workforce.”
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