Nearly 150 years ago, scientists began to imagine how information might flow through the brain based on the shapes of neurons they had seen under the microscopes of the time. With today’s imaging technologies, scientists can zoom in much further, seeing the tiny synapses through which neurons communicate with one another, and even the molecules the cells use to relay their messages. These inside views can spark new ideas about how healthy brains work and reveal important changes that contribute to disease.
This sharper view of biology is not just about the advances that have made microscopes more powerful than ever before. Using methodology developed in the lab of MIT McGovern Institute for Brain Research investigator Edward Boyden, researchers around the world are imaging samples that have been swollen to as much as 20 times their original size so their finest features can be seen more clearly.
“It’s a very different way to do microscopy,” says Boyden, who is also a Howard Hughes Medical Institute (HHMI) investigator, a professor of brain and cognitive sciences and biological engineering, and a member of the Yang Tan Collective at MIT. “In contrast to the last 300 years of bioimaging, where you use a lens to magnify an image of light from an object, we physically magnify objects themselves.” Once a tissue is expanded, Boyden says, researchers can see more even with widely available, conventional microscopy hardware.
Boyden’s team introduced this approach, which they named expansion microscopy (ExM), in 2015. Since then, they have been refining the method and adding to its capabilities, while researchers at MIT and beyond deploy it to learn about life on the smallest of scales.
it’s advancing quite quickly across the fields of biology and medicine,” states boyden. “it’s being utilized for conditions like kidney disease, the neural circuits of fruit flies, seed genetics, microbial communities, alzheimer’s disease, viral studies, and much more.
Origins of ExM
To create expansion microscopy, Boyden and his team looked into using hydrogels—materials known for their exceptional ability to absorb water—which were previously utilized in everyday products like disposable diapers to maintain baby’s comfort. The laboratory led by Boyden proposed that these hydrogels might preserve their structural integrity even when absorbing many times their initial volume in water, causing an increase in spacing among their molecular constituents as they expand.
Following several trials, Boyden’s group identified four crucial stages for expanding tissue specimens to enhance visualization. Initially, the tissue needs to be saturated with a hydrogel. The elements within the tissue, specifically biomolecules, get fastened onto the interconnected network of the gel, attaching them firmly to the gel’s molecular structure. Next, the tissue undergoes chemical treatment to become more pliable before being immersed in water. Upon absorbing this moisture, the hydrogel increases in size causing the entire tissue sample to expand uniformly, maintaining the original spatial relationships among its parts intact.
Boyden along with his graduate students Fei Chen and Paul Tillberg
initial study on expansion microscopy
appeared in the journal
Science
In 2015, the team showed that when they spread out molecules previously packed within cells, details that appeared blurry through a conventional light microscope became clear and discrete. Standard light microscopes typically struggle to distinguish objects closer than approximately 300 nanometers due to physical limitations. However, with expansion microscopy, as stated by Boyden’s research group, they achieved an effective resolution of around 70 nanometers after expanding the sample four times.
Boyden states that this degree of precision is essential for biologists. He explains, “Ultimately, biology is essentially a discipline rooted in nanoscopic phenomena. Biomolecules operate at a nano scale, and their interactions occur across these minuscule distances. A significant number of critical issues in both biology and medicine revolve around understanding these nanoscale dynamics.”
Various advanced microscopes, each possessing distinct pros and cons, can reveal such detailed information. However, these techniques tend to be expensive and necessitate specific expertise, rendering them out of reach for many scientists. Expansion microscopy makes nanoscale imaging more accessible, according to Boyden. He explains, “This approach allows everyone to examine the fundamental components of life and understand their interactions.”
Empowering scientists
After Boyden’s group unveiled expansion microscopy in 2015, numerous scientific teams worldwide have produced countless studies detailing their findings achieved through this method. This approach has been particularly illuminating for neuroscientists as it has clarified complex neuronal circuitry, revealed the arrangement of specific proteins within and across synaptic junctions which aid neuron-to-neuron signaling, and identified alterations linked to both aging processes and diseases.
This technique has also proven highly beneficial for research outside the realm of neurology. Each week, Sabrina Axellon employs expansion microscopy in her laboratory at the Indiana University School of Medicine to examine the malaria parasite—a unicellular microbe brimming with intricate components necessary for invading and surviving within host organisms. Due to its minuscule size, many of these crucial features remain invisible under conventional optical microscopy techniques.
As a cell biologist, I’m finding myself without one of the most crucial tools for deducing protein functions, understanding organelle structures, shapes, and their roles, which essentially boils down to visual assessment,” she explains. Through this expansion technique, not only will she be able to observe the internal organelles within a malaria parasite, but also track how they form and monitor changes during division stages. Grasping these mechanisms, according to her, might guide pharmaceutical researchers toward discovering novel methods to disrupt the parasite’s lifecycle.
Absalon emphasizes that the accessibility of expansion microscopy holds significant importance in the realm of parasitology, especially since much research occurs in regions with constrained resources such as Africa, South America, and Asia. These areas have seen workshops and training initiatives aimed at disseminating this technology to researchers who are closely affected by diseases like malaria and various parasites. As Absalon points out, these scientists now possess the capability for super-resolution imaging without requiring highly sophisticated equipment.
Always improving
Starting in 2015, Boyden’s multidisciplinary team has explored numerous innovative methods to enhance expansion microscopy and apply it in novel applications. The techniques they employ nowadays offer superior tagging capabilities, increased magnification ratios, and enhanced image clarity. Features as close as 20 nanometers apart can now be distinguished individually using a light microscope thanks to these advancements.
They have adjusted their procedures to accommodate various crucial specimen types, including whole nematodes (a favorite for neuroscientists, developmental biologists, and others), as well as clinical specimens. Regarding the latter, they demonstrated that expansion techniques can uncover minor indicators of illness, potentially leading to earlier or more economical diagnostic methods.
Initially, the team fine-tuned its technique to visualize proteins within cells by tagging targeted proteins and embedding them into a hydrogel before expanding it. Now, through an improved method of handling specimens, investigators have the ability to re-stain expanded samples repeatedly with various markers for numerous rounds of imaging. This allows scientists to determine the locations of many distinct proteins in identical tissues. Consequently, this enables researchers to observe the spatial arrangement of these molecules relative to each other and potential interactions between them, as well as analyze extensive arrays of proteins to detect alterations caused by diseases.
However, improved protein visualization was merely the starting point for expansion microscopy. As Boyden states, “Our aim is to observe everything.” The team aspires to visualize all types of biomolecules with an unprecedented level of accuracy reaching up to the atomic scale. Although they haven’t achieved this goal completely yet, using advanced probes and refined techniques has made it feasible to examine not only proteins but also RNA and lipids within enlarged tissue specimens.
By tagging lipids, which make up the membranes enveloping cells, scientists can now clearly delineate cell structures within enlarged tissue samples. This increased clarity provided by expansion allows for detailed tracing of the thin extensions of neurons throughout an image.
Typically, researchers have relied on electron microscopy, which generates exquisitely detailed pictures but requires expensive equipment, to map the brain’s circuitry. “Now, you can get images that look a lot like electron microscopy images, but on regular old light microscopes—the kind that everybody has access to,” Boyden says.
Boyden says expansion can be powerful in combination with other cutting-edge tools. When expanded samples are used with an ultra-fast imaging method developed by Eric Betzig, an HHMI investigator at the University of California at Berkeley, called lattice light-sheet microscopy, the entire brain of a fruit fly can be imaged at high resolution in just a few days.
When RNA molecules are fixed within a hydrogel matrix and subsequently sequenced without being moved, researchers gain precise insight into their location within cells—their placement relative to protein construction sites—demonstrated through a partnership between Boyden’s group and Harvard genetics researcher George Church along with former MIT professor Aviv RegeV. “Expansion essentially enhances the resolution of numerous technological methods,” explains Boyden. “Whether you’re conducting mass spectrometry imaging, X-ray imaging, or Raman spectroscopy, expansion refines your equipment.”
Expanding possibilities
A decade has passed since the initial showcase of expansion microscopy’s capabilities, and Boyden along with his team remains dedicated to enhancing this technology further. As stated by him, “Our aim is to refine it for various types of challenges, and ensuring these advancements are quicker, superior, and less expensive stays crucial.” However, the progression of expansion microscopy won’t solely depend on innovations from the Boyden laboratory. He elaborates, saying, “The process is straightforward enough both to execute and adapt; hence numerous others are advancing expansion techniques either through our partnership or independently.”
Boyden highlights a team spearheaded by Silvio Rizzoli from the University Medical Center Göttingen in Germany. This team, working alongside Boyden, has refined the expansion technique to better visualize protein structures. Meanwhile, researchers under Jae-Byum Chang at the Korea Advanced Institute of Science and Technology—a previous postdoctoral researcher in Boyden’s lab—have developed methods to enlarge whole specimens such as mouse embryos and young zebrafish. Collaborating closely with Boyden, they aim to offer unprecedented insights into developmental mechanisms and extensive neural pathways.
Mapping the intricate networks inside the brain’s complex neural pathways might be simplified using expansion microscopy-based connectomics, a technique created by Johann Danzl and his team at the Institute of Science and Technology in Austria. This method leverages the high-resolution imaging and detailed molecular insights provided by expansion microscopy.
“Expansion allows us to observe a biological system at its most fundamental level, which is truly beautiful,” according to Boyden.
His group is determined to test the boundaries of this technique from a physical standpoint and expects to uncover fresh possibilities for exploration along the way. He explains, “By mapping the brain or any biological system down to the scale of individual molecules, we could potentially understand how these components function collectively as a network—the true essence of how life functions.”
This story is republished courtesy of MIT News.
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