Researcher: Ed Boyden

High-resolution imaging with conventional microscopes

Anne Trafton |

MIT researchers have developed a way to make extremely high-resolution images of tissue samples, at a fraction of the cost of other techniques that offer similar resolution.

The new technique relies on expanding tissue before imaging it with a conventional light microscope. Two years ago, the MIT team showed that it was possible to expand tissue volumes 100-fold, resulting in an image resolution of about 60 nanometers. Now, the researchers have shown that expanding the tissue a second time before imaging can boost the resolution to about 25 nanometers.

This level of resolution allows scientists to see, for example, the proteins that cluster together in complex patterns at brain synapses, helping neurons to communicate with each other. It could also help researchers to map neural circuits, says Ed Boyden, an associate professor of biological engineering and brain and cognitive sciences at MIT.

“We want to be able to trace the wiring of complete brain circuits,” says Boyden, the study’s senior author. “If you could reconstruct a complete brain circuit, maybe you could make a computational model of how it generates complex phenomena like decisions and emotions. Since you can map out the biomolecules that generate electrical pulses within cells and that exchange chemicals between cells, you could potentially model the dynamics of the brain.”

This approach could also be used to image other phenomena such as the interactions between cancer cells and immune cells, to detect pathogens without expensive equipment, and to map the cell types of the body.

Former MIT postdoc Jae-Byum Chang is the first author of the paper, which appears in the April 17 issue of Nature Methods.

Double expansion

To expand tissue samples, the researchers embed them in a dense, evenly generated gel made of polyacrylate, a very absorbent material that’s also used in diapers. Before the gel is formed, the researchers label the cell proteins they want to image, using antibodies that bind to specific targets. These antibodies bear “barcodes” made of DNA, which in turn are attached to cross-linking molecules that bind to the polymers that make up the expandable gel. The researchers then break down the proteins that normally hold the tissue together, allowing the DNA barcodes to expand away from each other as the gel swells.

These enlarged samples can then be labeled with fluorescent probes that bind the DNA barcodes, and imaged with commercially available confocal microscopes, whose resolution is usually limited to hundreds of nanometers.

Using that approach, the researchers were previously able to achieve a resolution of about 60 nanometers. However, “individual biomolecules are much smaller than that, say 5 nanometers or even smaller,” Boyden says. “The original versions of expansion microscopy were useful for many scientific questions but couldn’t equal the performance of the highest-resolution imaging methods such as electron microscopy.”

In their original expansion microscopy study, the researchers found that they could expand the tissue more than 100-fold in volume by reducing the number of cross-linking molecules that hold the polymer in an orderly pattern. However, this made the tissue unstable.

“If you reduce the cross-linker density, the polymers no longer retain their organization during the expansion process,” says Boyden, who is a member of MIT’s Media Lab and McGovern Institute for Brain Research. “You lose the information.”

Instead, in their latest study, the researchers modified their technique so that after the first tissue expansion, they can create a new gel that swells the tissue a second time — an approach they call “iterative expansion.”

Mapping circuits

Using iterative expansion, the researchers were able to image tissues with a resolution of about 25 nanometers, which is similar to that achieved by high-resolution techniques such as stochastic optical reconstruction microscopy (STORM). However, expansion microscopy is much cheaper and simpler to perform because no specialized equipment or chemicals are required, Boyden says. The method is also much faster and thus compatible with large-scale, 3-D imaging.

The resolution of expansion microscopy does not yet match that of scanning electron microscopy (about 5 nanometers) or transmission electron microscopy (about 1 nanometer). However, electron microscopes are very expensive and not widely available, and with those microscopes, it is difficult for researchers to label specific proteins.

In the Nature Methods paper, the MIT team used iterative expansion to image synapses — the connections between neurons that allow them to communicate with each other. In their original expansion microscopy study, the researchers were able to image scaffolding proteins, which help to organize the hundreds of other proteins found in synapses. With the new, enhanced resolution, the researchers were also able to see finer-scale structures, such as the location of neurotransmitter receptors located on the surfaces of the “postsynaptic” cells on the receiving side of the synapse.

“My hope is that we can, in the coming years, really start to map out the organization of these scaffolding and signaling proteins at the synapse,” Boyden says.

Combining expansion microscopy with a new tool called temporal multiplexing should help to achieve that, he believes. Currently, only a limited number of colored probes can be used to image different molecules in a tissue sample. With temporal multiplexing, researchers can label one molecule with a fluorescent probe, take an image, and then wash the probe away. This can then be repeated many times, each time using the same colors to label different molecules.

“By combining iterative expansion with temporal multiplexing, we could in principle have essentially infinite-color, nanoscale-resolution imaging over large 3-D volumes,” Boyden says. “Things are getting really exciting now that these different technologies may soon connect with each other.”

The researchers also hope to achieve a third round of expansion, which they believe could, in principle, enable resolution of about 5 nanometers. However, right now the resolution is limited by the size of the antibodies used to label molecules in the cell. These antibodies are about 10 to 20 nanometers long, so to get resolution below that, researchers would need to create smaller tags or expand the proteins away from each other first and then deliver the antibodies after expansion.

This study was funded by the National Institutes of Health Director’s Pioneer Award, the New York Stem Cell Foundation Robertson Award, the HHMI-Simons Faculty Scholars Award, and the Open Philanthropy Project.

Sensor traces dopamine released by single cells

Anne Trafton |

MIT chemical engineers have developed an extremely sensitive detector that can track single cells’ secretion of dopamine, a brain chemical responsible for carrying messages involved in reward-motivated behavior, learning, and memory.

Using arrays of up to 20,000 tiny sensors, the researchers can monitor dopamine secretion of single neurons, allowing them to explore critical questions about dopamine dynamics. Until now, that has been very difficult to do.

“Now, in real-time, and with good spatial resolution, we can see exactly where dopamine is being released,” says Michael Strano, the Carbon P. Dubbs Professor of Chemical Engineering and the senior author of a paper describing the research, which appears in the Proceedings of the National Academy of Sciences the week of Feb. 6.

Strano and his colleagues have already demonstrated that dopamine release occurs differently than scientists expected in a type of neural progenitor cell, helping to shed light on how dopamine may exert its effects in the brain.

The paper’s lead author is Sebastian Kruss, a former MIT postdoc who is now at Göttingen University, in Germany. Other authors are Daniel Salem and Barbara Lima, both MIT graduate students; Edward Boyden, an associate professor of biological engineering and brain and cognitive sciences, as well as a member of the MIT Media Lab and the McGovern Institute for Brain Research; Lela Vukovic, an assistant professor of chemistry at the University of Texas at El Paso; and Emma Vander Ende, a graduate student at Northwestern University.

“A global effect”

Dopamine is a neurotransmitter that plays important roles in learning, memory, and feelings of reward, which reinforce positive experiences.

Neurotransmitters allow neurons to relay messages to nearby neurons through connections known as synapses. However, unlike most other neurotransmitters, dopamine can exert its effects beyond the synapse: Not all dopamine released into a synapse is taken up by the target cell, allowing some of the chemical to diffuse away and affect other nearby cells.

“It has a local effect, which controls the signaling through the neurons, but also it has a global effect,” Strano says. “If dopamine is in the region, it influences all the neurons nearby.”

Tracking this dopamine diffusion in the brain has proven difficult. Neuroscientists have tried using electrodes that are specialized to detect dopamine, but even using the smallest electrodes available, they can place only about 20 near any given cell.

“We’re at the infancy of really understanding how these packets of chemicals move and their directionality,” says Strano, who decided to take a different approach.

Strano’s lab has previously developed sensors made from arrays of carbon nanotubes — hollow, nanometer-thick cylinders made of carbon, which naturally fluoresce when exposed to laser light. By wrapping these tubes in different proteins or DNA strands, scientists can customize them to bind to different types of molecules.

The carbon nanotube sensors used in this study are coated with a DNA sequence that makes the sensors interact with dopamine. When dopamine binds to the carbon nanotubes, they fluoresce more brightly, allowing the researchers to see exactly where the dopamine was released. The researchers deposited more than 20,000 of these nanotubes on a glass slide, creating an array that detects any dopamine secreted by a cell placed on the slide.

Dopamine diffusion

In the new PNAS study, the researchers used these dopamine sensors to explore a longstanding question about dopamine release in the brain: From which part of the cell is dopamine secreted?

To help answer that question, the researchers placed individual neural progenitor cells known as PC-12 cells onto the sensor arrays. PC-12 cells, which develop into neuron-like cells under the right conditions, have a starfish-like shape with several protrusions that resemble axons, which form synapses with other cells.

After stimulating the cells to release dopamine, the researchers found that certain dopamine sensors near the cells lit up immediately, while those farther away turned on later as the dopamine diffused away. Tracking those patterns over many seconds allowed the researchers to trace how dopamine spreads away from the cells.

Strano says one might expect to see that most of the dopamine would be released from the tips of the arms extending out from the cells. However, the researchers found that in fact more dopamine came from the sides of the arms.

“We have falsified the notion that dopamine should only be released at these regions that will eventually become the synapses,” Strano says. “This observation is counterintuitive, and it’s a new piece of information you can only obtain with a nanosensor array like this one.”

The team also showed that most of the dopamine traveled away from the cell, through protrusions extending in opposite directions. “Even though dopamine is not necessarily being released only at the tip of these protrusions, the direction of release is associated with them,” Salem says.

Other questions that could be explored using these sensors include how dopamine release is affected by the direction of input to the cell, and how the presence of nearby cells influences each cell’s dopamine release.

The research was funded by the National Science Foundation, the National Institutes of Health, a University of Illinois Center for the Physics of Living Cells Postdoctoral Fellowship, the German Research Foundation, and a Liebig Fellowship.

Researchers create synthetic cells to isolate genetic circuits

Anne Trafton |

Synthetic biology allows scientists to design genetic circuits that can be placed in cells, giving them new functions such as producing drugs or other useful molecules. However, as these circuits become more complex, the genetic components can interfere with each other, making it difficult to achieve more complicated functions.

MIT researchers have now demonstrated that these circuits can be isolated within individual synthetic “cells,” preventing them from disrupting each other. The researchers can also control communication between these cells, allowing for circuits or their products to be combined at specific times.

“It’s a way of having the power of multicomponent genetic cascades, along with the ability to build walls between them so they won’t have cross-talk. They won’t interfere with each other in the way they would if they were all put into a single cell or into a beaker,” says Edward Boyden, an associate professor of biological engineering and brain and cognitive sciences at MIT. Boyden is also a member of MIT’s Media Lab and McGovern Institute for Brain Research, and an HHMI-Simons Faculty Scholar.

This approach could allow researchers to design circuits that manufacture complex products or act as sensors that respond to changes in their environment, among other applications.

Boyden is the senior author of a paper describing this technique in the Nov. 14 issue of Nature Chemistry. The paper’s lead authors are former MIT postdoc Kate Adamala, who is now an assistant professor at the University of Minnesota, and former MIT grad student Daniel Martin-Alarcon. Katriona Guthrie-Honea, a former MIT research assistant, is also an author of the paper.

Circuit control

The MIT team encapsulated their genetic circuits in droplets known as liposomes, which have a fatty membrane similar to cell membranes. These synthetic cells are not alive but are equipped with much of the cellular machinery necessary to read DNA and manufacture proteins.

By segregating circuits within their own liposomes, the researchers are able to create separate circuit subroutines that could not run in the same container at the same time, but can run in parallel to each other, communicating in controlled ways. This approach also allows scientists to repurpose the same genetic tools, including genes and transcription factors (proteins that turn genes on or off), to do different tasks within a network.

“If you separate circuits into two different liposomes, you could have one tool doing one job in one liposome, and the same tool doing a different job in the other liposome,” Martin-Alarcon says. “It expands the number of things that you can do with the same building blocks.”

This approach also enables communication between circuits from different types of organisms, such as bacteria and mammals.

As a demonstration, the researchers created a circuit that uses bacterial genetic parts to respond to a molecule known as theophylline, a drug similar to caffeine. When this molecule is present, it triggers another molecule known as doxycycline to leave the liposome and enter another set of liposomes containing a mammalian genetic circuit. In those liposomes, doxycycline activates a genetic cascade that produces luciferase, a protein that generates light.

Using a modified version of this approach, scientists could create circuits that work together to produce biological therapeutics such as antibodies, after sensing a particular molecule emitted by a brain cell or other cell.

“If you think of the bacterial circuit as encoding a computer program, and the mammalian circuit is encoding the factory, you could combine the computer code of the bacterial circuit and the factory of the mammalian circuit into a unique hybrid system,” Boyden says.

The researchers also designed liposomes that can fuse with each other in a controlled way. To do that, they programmed the cells with proteins called SNAREs, which insert themselves into the cell membrane. There, they bind to corresponding SNAREs found on surfaces of other liposomes, causing the synthetic cells to fuse. The timing of this fusion can be controlled to bring together liposomes that produce different molecules. When the cells fuse, these molecules are combined to generate a final product.

More modularity

The researchers believe this approach could be used for nearly any application that synthetic biologists are already working on. It could also allow scientists to pursue potentially useful applications that have been tried before but abandoned because the genetic circuits interfered with each other too much.

“The way that we wrote this paper was not oriented toward just one application,” Boyden says. “The basic question is: Can you make these circuits more modular? If you have everything mishmashed together in the cell, but you find out that the circuits are incompatible or toxic, then putting walls between those reactions and giving them the ability to communicate with each other could be very useful.”

Vincent Noireaux, an associate professor of physics at the University of Minnesota, described the MIT approach as “a rather novel method to learn how biological systems work.”

“Using cell-free expression has several advantages: Technically the work is reduced to cloning (nowadays fast and easy), we can link information processing to biological function like living cells do, and we work in isolation with no other gene expression occurring in the background,” says Noireaux, who was not involved in the research.

Another possible application for this approach is to help scientists explore how the earliest cells may have evolved billions of years ago. By engineering simple circuits into liposomes, researchers could study how cells might have evolved the ability to sense their environment, respond to stimuli, and reproduce.

“This system can be used to model the behavior and properties of the earliest organisms on Earth, as well as help establish the physical boundaries of Earth-type life for the search of life elsewhere in the solar system and beyond,” Adamala says.

Newly discovered neural connections may be linked to emotional decision-making

Anne Trafton |

MIT neuroscientists have discovered connections deep within the brain that appear to form a communication pathway between areas that control emotion, decision-making, and movement. The researchers suspect that these connections, which they call striosome-dendron bouquets, may be involved in controlling how the brain makes decisions that are influenced by emotion or anxiety.

This circuit may also be one of the targets of the neural degeneration seen in Parkinson’s disease, says Ann Graybiel, an Institute Professor at MIT, member of the McGovern Institute for Brain Research, and the senior author of the study.

Graybiel and her colleagues were able to find these connections using a technique developed at MIT known as expansion microscopy, which enables scientists to expand brain tissue before imaging it. This produces much higher-resolution images than would otherwise be possible with conventional microscopes.

That technique was developed in the lab of Edward Boyden, an associate professor of biological engineering and brain and cognitive sciences at the MIT Media Lab, who is also an author of this study. Jill Crittenden, a research scientist at the McGovern Institute, is the lead author of the paper, which appears in the Proceedings of the National Academy of Sciences the week of Sept. 19.

Tracing a circuit

In this study, the researchers focused on a small region of the brain known as the striatum, which is part of the basal ganglia — a cluster of brain centers associated with habit formation, control of voluntary movement, emotion, and addiction. Malfunctions of the basal ganglia have been associated with Parkinson’s and Huntington’s diseases, as well as autism, obsessive-compulsive disorder, and Tourette’s syndrome.

Much of the striatum is uncharted territory, but Graybiel’s lab has previously identified clusters of cells there known as striosomes. She also found that these clusters receive very specific input from parts of the brain’s prefrontal cortex involved in processing emotions, and showed that this communication pathway is necessary for making decisions that require an anxiety-provoking cost-benefit analysis, such as choosing whether to take a job that pays more but forces a move away from family and friends.

Her studies also suggested that striosomes relay information to cells within a region called the substantia nigra, one of the brain’s main dopamine-producing centers. Dopamine has many functions in the brain, including roles in initiating movement and regulating mood.

To figure out how these regions might be communicating, Graybiel, Crittenden, and their colleagues used expansion microscopy to image the striosomes and discovered extensive connections between those clusters of cells and dopamine-producing cells of the substantia nigra. The dopamine-producing cells send down many tiny extensions known as dendrites that become entwined with axons that come up to meet them from the striosomes, forming a bouquet-like structure.

“With expansion microscopy, we could finally see direct connections between these cells by unraveling their unusual rope-like bundles of axons and dendrites,” Crittenden says. “What’s really exciting to us is we can see that it’s small discrete clusters of dopamine cells with bundles that are being targeted.”

Hard decisions

This finding expands the known decision-making circuit so that it encompasses the prefrontal cortex, striosomes, and a subset of dopamine-producing cells. Together, the striosomes may be acting as a gatekeeper that absorbs sensory and emotional information coming from the cortex and integrates it to produce a decision on how to react, which is initiated by the dopamine-producing cells, the researchers say.

To explore that possibility, the researchers plan to study mice in which they can selectively activate or shut down the striosome-dendron bouquet as the mice are prompted to make decisions requiring a cost-benefit analysis.

The researchers also plan to investigate whether these connections are disrupted in mouse models of Parkinson’s disease. MRI studies and postmortem analysis of brains of Parkinson’s patients have shown that death of dopamine cells in the substantia nigra is strongly correlated with the disease, but more work is needed to determine if this subset overlaps with the dopamine cells that form the striosome-dendron bouquets.

Seeing RNA at the nanoscale

Anne Trafton |

Cells contain thousands of messenger RNA molecules, which carry copies of DNA’s genetic instructions to the rest of the cell. MIT engineers have now developed a way to visualize these molecules in higher resolution than previously possible in intact tissues, allowing researchers to precisely map the location of RNA throughout cells.

Key to the new technique is expanding the tissue before imaging it. By making the sample physically larger, it can be imaged with very high resolution using ordinary microscopes commonly found in research labs.

“Now we can image RNA with great spatial precision, thanks to the expansion process, and we also can do it more easily in large intact tissues,” says Ed Boyden, an associate professor of biological engineering and brain and cognitive sciences at MIT, a member of MIT’s Media Lab and McGovern Institute for Brain Research, and the senior author of a paper describing the technique in the July 4 issue of Nature Methods.

Studying the distribution of RNA inside cells could help scientists learn more about how cells control their gene expression and could also allow them to investigate diseases thought to be caused by failure of RNA to move to the correct location.

Boyden and colleagues first described the underlying technique, known as expansion microscopy (ExM), last year, when they used it to image proteins inside large samples of brain tissue. In a paper appearing in Nature Biotechnology on July 4, the MIT team has now presented a new version of the technology that employs off-the-shelf chemicals, making it easier for researchers to use.

MIT graduate students Fei Chen and Asmamaw Wassie are the lead authors of the Nature Methods paper, and Chen and graduate student Paul Tillberg are the lead authors of the Nature Biotechnology paper.

A simpler process

The original expansion microscopy technique is based on embedding tissue samples in a polymer that swells when water is added. This tissue enlargement allows researchers to obtain images with a resolution of around 70 nanometers, which was previously possible only with very specialized and expensive microscopes.

However, that method posed some challenges because it requires generating a complicated chemical tag consisting of an antibody that targets a specific protein, linked to both a fluorescent dye and a chemical anchor that attaches the whole complex to a highly absorbent polymer known as polyacrylate. Once the targets are labeled, the researchers break down the proteins that hold the tissue sample together, allowing it to expand uniformly as the polyacrylate gel swells.

In their new studies, to eliminate the need for custom-designed labels, the researchers used a different molecule to anchor the targets to the gel before digestion. This molecule, which the researchers dubbed AcX, is commercially available and therefore makes the process much simpler.

AcX can be modified to anchor either proteins or RNA to the gel. In the Nature Biotechnology study, the researchers used it to anchor proteins, and they also showed that the technique works on tissue that has been previously labeled with either fluorescent antibodies or proteins such as green fluorescent protein (GFP).

“This lets you use completely off-the-shelf parts, which means that it can integrate very easily into existing workflows,” Tillberg says. “We think that it’s going to lower the barrier significantly for people to use the technique compared to the original ExM.”

Using this approach, it takes about an hour to scan a piece of tissue 500 by 500 by 200 microns, using a light sheet fluorescence microscope. The researchers showed that this technique works for many types of tissues, including brain, pancreas, lung, and spleen.

Imaging RNA

In the Nature Methods paper, the researchers used the same kind of anchoring molecule but modified it to target RNA instead. All of the RNAs in the sample are anchored to the gel, so they stay in their original locations throughout the digestion and expansion process.

After the tissue is expanded, the researchers label specific RNA molecules using a process known as fluorescence in situ hybridization (FISH), which was originally developed in the early 1980s and is widely used. This allows researchers to visualize the location of specific RNA molecules at high resolution, in three dimensions, in large tissue samples.

This enhanced spatial precision could allow scientists to explore many questions about how RNA contributes to cellular function. For example, a longstanding question in neuroscience is how neurons rapidly change the strength of their connections to store new memories or skills. One hypothesis is that RNA molecules encoding proteins necessary for plasticity are stored in cell compartments close to the synapses, poised to be translated into proteins when needed.

With the new system, it should be possible to determine exactly which RNA molecules are located near the synapses, waiting to be translated.
“People have found hundreds of these locally translated RNAs, but it’s hard to know where exactly they are and what they’re doing,” Chen says. “This technique would be useful to study that.”

Boyden’s lab is also interested in using this technology to trace the connections between neurons and to classify different subtypes of neurons based on which genes they are expressing.

The research was funded by the Open Philanthropy Project, the New York Stem Cell Foundation Robertson Award, the National Institutes of Health, the National Science Foundation, and Jeremy and Joyce Wertheimer.

Controlling RNA in living cells

Anne Trafton |

MIT researchers have devised a new set of proteins that can be customized to bind arbitrary RNA sequences, making it possible to image RNA inside living cells, monitor what a particular RNA strand is doing, and even control RNA activity.

The new strategy is based on human RNA-binding proteins that normally help guide embryonic development. The research team adapted the proteins so that they can be easily targeted to desired RNA sequences.

“You could use these proteins to do measurements of RNA generation, for example, or of the translation of RNA to proteins,” says Edward Boyden, an associate professor of biological engineering and brain and cognitive sciences at the MIT Media Lab. “This could have broad utility throughout biology and bioengineering.”

Unlike previous efforts to control RNA with proteins, the new MIT system consists of modular components, which the researchers believe will make it easier to perform a wide variety of RNA manipulations.

“Modularity is one of the core design principles of engineering. If you can make things out of repeatable parts, you don’t have to agonize over the design. You simply build things out of predictable, linkable units,” says Boyden, who is also a member of MIT’s McGovern Institute for Brain Research.

Boyden is the senior author of a paper describing the new system in the Proceedings of the National Academy of Sciences. The paper’s lead authors are postdoc Katarzyna Adamala and grad student Daniel Martin-Alarcon.

Modular code

Living cells contain many types of RNA that perform different roles. One of the best known varieties is messenger RNA (mRNA), which is copied from DNA and carries protein-coding information to cell structures called ribosomes, where mRNA directs protein assembly in a process called translation. Monitoring mRNA could tell scientists a great deal about which genes are being expressed in a cell, and tweaking the translation of mRNA would allow them to alter gene expression without having to modify the cell’s DNA.

To achieve this, the MIT team set out to adapt naturally occurring proteins called Pumilio homology domains. These RNA-binding proteins include sequences of amino acids that bind to one of the ribonucleotide bases or “letters” that make up RNA sequences — adenine (A), thymine (T), uracil (U), and guanine (G).

In recent years, scientists have been working on developing these proteins for experimental use, but until now it was more of a trial-and-error process to create proteins that would bind to a particular RNA sequence.

“It was not a truly modular code,” Boyden says, referring to the protein’s amino acid sequences. “You still had to tweak it on a case-by-case basis. Whereas now, given an RNA sequence, you can specify on paper a protein to target it.”

To create their code, the researchers tested out many amino acid combinations and found a particular set of amino acids that will bind each of the four bases at any position in the target sequence. Using this system, which they call Pumby (for Pumilio-based assembly), the researchers effectively targeted RNA sequences varying in length from six to 18 bases.

“I think it’s a breakthrough technology that they’ve developed here,” says Robert Singer, a professor of anatomy and structural biology, cell biology, and neuroscience at Albert Einstein College of Medicine, who was not involved in the research. “Everything that’s been done to target RNA so far requires modifying the RNA you want to target by attaching a sequence that binds to a specific protein. With this technique you just design the protein alone, so there’s no need to modify the RNA, which means you could target any RNA in any cell.”

RNA manipulation

In experiments in human cells grown in a lab dish, the researchers showed that they could accurately label mRNA molecules and determine how frequently they are being translated. First, they designed two Pumby proteins that would bind to adjacent RNA sequences. Each protein is also attached to half of a green fluorescent protein (GFP) molecule. When both proteins find their target sequence, the GFP molecules join and become fluorescent — a signal to the researchers that the target RNA is present.

Furthermore, the team discovered that each time an mRNA molecule is translated, the GFP gets knocked off, and when translation is finished, another GFP binds to it, enhancing the overall fluorescent signal. This allows the researchers to calculate how often the mRNA is being read.

This system can also be used to stimulate translation of a target mRNA. To achieve that, the researchers attached a protein called a translation initiator to the Pumby protein. This allowed them to dramatically increase translation of an mRNA molecule that normally wouldn’t be read frequently.

“We can turn up the translation of arbitrary genes in the cell without having to modify the genome at all,” Martin-Alarcon says.

The researchers are now working toward using this system to label different mRNA molecules inside neurons, allowing them to test the idea that mRNAs for different genes are stored in different parts of the neuron, helping the cell to remain poised to perform functions such as storing new memories. “Until now it’s been very difficult to watch what’s happening with those mRNAs, or to control them,” Boyden says.

These RNA-binding proteins could also be used to build molecular assembly lines that would bring together enzymes needed to perform a series of reactions that produce a drug or another molecule of interest.

Edward Boyden wins BBVA Foundation Frontiers of Knowledge Award

Anne Trafton |

Edward S. Boyden, a professor of media arts and sciences, biological engineering, and brain and cognitive sciences at MIT, has won the BBVA Foundation Frontiers of Knowledge Award in Biomedicine for his role in the development of optogenetics, a technique for controlling brain activity with light. Gero Miesenböck of the University of Oxford and Karl Deisseroth of Stanford University were also honored with the prize for their role in developing and refining the technique.

The BBVA Foundation Frontiers of Knowledge Awards are given annually for “outstanding contributions and radical advances in a broad range of scientific, technological and artistic areas.” The €400.000 prize in the category of biomedicine will be shared among the three neuroscientists.

“If we imagine the brain as a computer, optogenetics is a keyboard that allows us to send extremely precise commands,” says Boyden, a a faculty member at the MIT Media Lab with a joint appointment at MIT’s McGovern Institute for Brain Research. “It is a tool whereby we can control the brain with exquisite precision.”

Boyden joins an illustrious list of prize laureates including physicist Stephen Hawking and artificial intelligence pioneer Marvin Minsky of MIT, who died on January 24.

The BBVA Foundation will host the winners at an awards ceremony on June 21, 2016 at the foundation’s headquarters in Madrid, Spain.

About the BBVA Foundation Frontiers of Knowledge Awards

The BBVA Foundation promotes, funds and disseminates world-class scientific research and artistic creation, in the conviction that science, culture and knowledge hold the key to better opportunities for all world citizens. The Foundation designs and implements its programs in partnership with some of the leading scientific and cultural organizations in Spain and abroad, striving to identify and prioritize those projects with the power to significantly advance the frontiers of the known world.

The juries in each of eight categories are made up of leading international experts in their respective fields, who arrive at their decisions in a wholly independent manner, applying internationally recognized metrics of excellence. The BBVA Foundation is aided in the organization of the awards by the Spanish National Research Council (CSIC).

Ed Boyden wins 2016 Breakthrough Prize in Life Sciences

Anne Trafton |

MIT researchers took home several awards last night at the 2016 Breakthrough Prize ceremony at NASA’s Ames Research Center in Mountain View, California.

Edward Boyden, an associate professor of media arts and sciences, biological engineering, and brain and cognitive sciences, was one of five scientists honored with the Breakthrough Prize in Life Sciences, given for “transformative advances toward understanding living systems and extending human life.” He will receive $3 million for the award.

MIT physicists also contributed to a project that won the Breakthrough Prize in Fundamental Physics. That prize went to five experiments investigating the oscillation of subatomic particles known as neutrinos. More than 1,300 contributing physicists will share in the recognition for their work, according to the award announcement. Those physicists include MIT associate professor of physics Joseph Formaggio and his team, as well as MIT assistant professor of physics Lindley Winslow.

Larry Guth, an MIT professor of mathematics, was honored with the New Horizons in Mathematics Prize, which is given to promising junior researchers who have already produced important work in mathematics. Liang Fu, an assistant professor of physics, was honored with the New Horizons in Physics Prize, which is awarded to promising junior researchers who have already produced important work in fundamental physics.

“By challenging conventional thinking and expanding knowledge over the long term, scientists can solve the biggest problems of our time,” said Mark Zuckerberg, chairman and CEO of Facebook, and one of the prizes’ founders. “The Breakthrough Prize honors achievements in science and math so we can encourage more pioneering research and celebrate scientists as the heroes they truly are.”

Optogenetics

Boyden was honored for the development and implementation of optogenetics, a technique in which scientists can control neurons by shining light on them. Karl Deisseroth, a Stanford University professor who worked with Boyden to pioneer the technique, was also honored with one of the life sciences prizes.

Optogenetics relies on light-sensitive proteins, originally isolated from bacteria and algae. About 10 years ago, Boyden and Deisseroth began engineering neurons to express these proteins, allowing them to selectively stimulate or silence them with pulses of light. More recently, Boyden has developed additional proteins that are even more sensitive to light and can respond to different colors.

Scientists around the world have used optogenetics to reveal the brain circuitry underlying normal neural function as well as neurological disorders such as autism, obsessive-compulsive disorder, and depression.

Boyden is a member of the MIT Media Lab and MIT’s McGovern Institute for Brain Research.

Neutrino oscillations

The Breakthrough Prize in Fundamental Physics was awarded to five research projects investigating the nature of neutrinos: Daya Bay (China); KamLAND (Japan); K2K/T2K (Japan); Sudbury Neutrino Observatory (Canada); and Super-Kamiokande (Japan). Researchers with these experiments were recognized “for the fundamental discovery of neutrino oscillations, revealing a new frontier beyond, and possibly far beyond, the standard model of particle physics.”

Formaggio and his team at MIT have been collaborating on the Sudbury Neutrino Observatory (SNO) project since 2005. Research at the observatory, 2 kilometers underground in a mine near Sudbury, Ontario, demonstrated that neutrinos change their type — or “flavor” — on their way to Earth from the sun.

Winslow has been a collaborator on KamLAND, located in a mine in Japan, since 2001. Using antineutrinos from nuclear reactors, this experiment demonstrated that the change in flavor was energy-dependent. The combination of these results solved the solar neutrino puzzle and proved that neutrinos have mass.

The MIT SNO group has participated heavily on the analysis of neutrino data, particularly during that experiment’s final measurement phase. The MIT KamLAND group is involved with the next phase, KamLAND-Zen, which is searching for a rare nuclear process that if observed, would make neutrinos their own antiparticles.

Reaching new horizons

Guth, who will receive a $100,000 prize, was honored for his “ingenious and surprising solutions to long standing open problems in symplectic geometry, Riemannian geometry, harmonic analysis, and combinatorial geometry.”

Guth’s work at MIT focuses on combinatorics, or the study of discrete structures, and how sets of lines intersect each other in space. He also works in the area of harmonic analysis, studying how sound waves interact with each other.

Guth’s father, MIT physicist Alan Guth, won the inaugural Breakthrough Prize in Fundamental Physics in 2015.

Fu will share a New Horizons in Physics Prize with two other researchers: B. Andrei Bernevig of Princeton University and Xiao-Liang Qi of Stanford University. The physicists were honored for their “outstanding contributions to condensed matter physics, especially involving the use of topology to understand new states of matter.”

Fu works on theories of topological insulators — a new class of materials whose surfaces can freely conduct electrons even though their interiors are electrical insulators — and topological superconductors. Such materials may provide insight into quantum physics and have possible applications in creating transistors based on the spin of particles rather than their charge.

Yesterday’s prize ceremony was hosted by producer/actor/director Seth MacFarlane; awards were presented by the prize sponsors and by celebrities including actors Russell Crowe, Hilary Swank, and Lily Collins. The Breakthrough Prizes were founded by Sergey Brin and Anne Wojcicki, Jack Ma and Cathy Zhang, Yuri and Julia Milner, and Mark Zuckerberg and Priscilla Chan.

“Breakthrough Prize laureates are making fundamental discoveries about the universe, life, and the mind,” Yuri Milner said. “These fields of investigation are advancing at an exponential pace, yet the biggest questions remain to be answered.”

Bold new microscopies for the brain

by Elizabeth Doherty |

McGovern researchers create unexpected new approaches to microscopy that are changing the way scientists look at the brain.

Ask McGovern Investigator Ed Boyden about his ten-year plan and you’ll get an immediate and straight-faced answer: “We would like to understand the brain.”

He means it. Boyden intends to map all of the cells in a brain, all of their connections, and even all of the molecules that form those connections and determine their strengths. He also plans to study how information flows through the brain and to use this to generate a working model. “I’d love to be able to load a map of an entire brain into a computer and see if we can simulate the brain,” he says.

Boyden likens the process to reverse-engineering a computer by opening it up and looking inside. The analogy, though not perfect, provides a sense of the enormity of the task ahead. As complicated as computers are, brains are far more complex, and they are also much harder to visualize, given the need to see features at multiple scales. For example, signals travel from cell to cell through synaptic connections that are measured in nanometers, but the signals are then propagated along nerve fibers that may span several centimeters—a difference of more than a million-fold. Modern microscopes make it possible to study features at one scale or the other, but not both together. Similarly, there are methods for visualizing electrical activity in single neurons or in whole brains, but there is no way to see both at once. So Boyden is building his own tools, and in the process is pushing the limits of imagination. “Our group is often trying to do the opposite of what other people do,” Boyden says.

Boyden’s new methods are part of a broader push to understand the brain’s connectivity, an objective that gained impetus two years ago with the President’s BRAIN Initiative, and with allied efforts such as the NIH-funded Human Connectome Project. Hundreds of researchers have already downloaded Boyden’s recently published protocols, including colleagues at the McGovern Institute who are using them to advance their studies of brain function and disease.

Just add water

Under the microscope, the brain section prepared by Jill Crittenden looks like a tight bundle of threads. The nerve fibers are from a mouse brain, from a region known to degenerate in humans with Parkinson’s disease. The loss of the tiny synaptic connections between these fibers may be the earliest signs of degeneration, so Crittenden, a research scientist who has been studying this disease for several years in the lab of McGovern Investigator Ann Graybiel, wants to be able to see them.

But she can’t. They are far too small— smaller than a wavelength of light, meaning they are beyond the limit for optical microscopy. To bring these structures into view, one of Boyden’s technologies, called expansion microscopy (ExM), simply makes the specimen bigger, allowing it to be viewed on a conventional laboratory microscope.

The idea is at once obvious and fantastical. “Expansion microscopy is the kind of thing scientists daydream about,” says Paul Tillberg, a graduate student in Boyden’s lab. “You either shrink the scientist or expand the specimen.”

Leaving Crittenden’s sample in place, Tillberg adds water. Minutes later, the tissue has expanded and become transparent, a ghostly and larger version of its former self.

Crittenden takes another look through the scope. “It’s like someone has loosened up all the fibers. I can see each one independently, and see them interconnecting,” she says. “ExM will add a lot of power to the tools we’ve developed for visualizing the connections we think are degenerating.”

It took Tillberg and his fellow graduate student Fei Chen several months of brainstorming to find a plausible way to make ExM a reality. They had found inspiration in the work of MIT physicist Toyoichi Tanaka, who in the 1970s had studied smart gels, polymers that rapidly expand in response to a change in environment. One familiar example is the absorbent material in baby diapers, and Boyden’s team turned to this substance for the expansion technique.

The process they devised involves several steps. The tissue is first labeled using fluorescent antibodies that bind to molecules of interest, and then it is impregnated with the gel-forming material. Once the gel has set, the fluorescent markers are anchored to the gel, and the original tissue sample is digested, allowing the gel to stretch evenly in all directions.

When water is added, the gel expands and the fluorescent markers spread out like a picture on a balloon. Remarkably, the 3D shapes of even the finest structures are faithfully preserved during the expansion, making it possible to see them using a conventional microscope. By labeling molecules with different colors, the researchers can even distinguish pre-synaptic from post-synaptic structures. Boyden plans eventually to use hundreds, possibly thousands, of colors, and to increase the expansion factor to 10 times original size, equivalent to a 1000-fold increase in volume.

ExM is not the only way to see fine structures such as synapses; they can also be visualized by electron microcopy, or by recently-developed ‘super-resolution’ optical methods that garnered a 2014 Nobel Prize. These techniques, however, require expensive equipment, and the images are very time-consuming to produce.

“With ExM, because the sample is physically bigger, you can scan it very quickly using just a regular microscope,” says Boyden.

Boyden is already talking to other leading researchers in the field, including Kwanghun Chung at MIT and George Church at Harvard, about ways to further enhance the ExM method. Within the McGovern Institute, among those who expect to benefit from these advances is Guoping Feng, who is developing mouse models of autism, schizophrenia and other disorders by introducing some of the same genetic changes seen in humans with these disorders. Many of the genes associated with autism and schizophrenia play a role in the formation of synapses, but even with the mouse models at his disposal, Feng isn’t sure what goes wrong with them because they are so hard to see. “If we can make parts of the brain bigger, we might be able to see how the assembly of this synaptic machinery changes in different disorders,” he says.

3D Movies Without Special Glasses

Another challenge facing Feng and many other researchers is that many brain functions, and many brain diseases, are not confined to one area, but are widely distributed across the brain. Trying to understand these processes by looking through a small microscopic window has been compared to watching a soccer game by observing just a single square foot of the playing field.

No current technology can capture millisecond-by-millisecond electrical events across the entire living brain, so Boyden and collaborators in Vienna, Austria, decided to develop one. They turned to a method called light field microscopy (LFM) as a way to capture 3D movies of an animal’s thoughts as they flash through the entire nervous system.

The idea is mind-boggling to imagine, but the hardware is quite simple. The instrument records images in depth the same way humans do, using multiple ‘eyes’ to send slightly offset 2D images to a computer that can reconstruct a 3D image of the world. (The idea had been developed in the 1990s by Boyden’s MIT colleague Ted Adelson, and a similar method was used to create Google Street View.) Boyden and his collaborators started with a microscope of standard design, attached a video camera, and inserted between them a six-by-six array of miniature lenses, designed in Austria, that projects a grid of offset images into the camera and the computer.

The rest is math. “We take the multiple, superimposed flat images projected through the lens array and combine them into a volume,” says Young-Gyu Yoon, a graduate student in the Boyden lab who designed and wrote the software.

Another graduate student, Nikita Pak, used the new method to measure neural activity in C. elegans, a tiny worm whose entire nervous system consists of just 302 neurons. By using a worm that had been genetically engineered so that its neurons light up when they become electrically active, Pak was able to make 3D movies of the activity in the entire nervous system. “The setup is just so simple,” he says. “Every time I use it, I think it’s cool.”

The team then tested their method on a larger brain, that of the larval zebra fish. They presented the larvae with a noxious odor, and found that it triggered activity in around 5000 neurons, over a period of about three minutes. Even with this relatively simple example, activity is distributed widely throughout the brain, and would be difficult to detect with previous techniques. Boyden is now working towards recording activity over much longer timespans, and he also envisions scaling it up to image the much more complex brains of mammals.

He hopes to start with the smallest known mammal, the Etruscan shrew. This animal resembles a mouse, but it is ten times smaller, no bigger than a thimble. Its brain is also much smaller, with only a few million neurons, compared to 100 million in a mouse.

Whole brain imaging in this tiny creature could provide an unprecedented view of mammalian brain activity, including its disruption in disease states. Feng cites sensory overload in autism as an example. “If we can see how sensory activity spreads through the brain, we can start to understand how overload starts and how it spills over to other brain areas,” he says.

Visions of Convergence

While Boyden’s microscopy technologies are providing his colleagues with new ways to study brain disorders, Boyden himself hopes to use them to understand the brain as a whole. He plans to use ExM to map connections and identify which molecules are where; 3D whole-brain imaging to trace brain activity as it unfolds in real time, and optogenetics techniques to stimulate the brain and directly record the resulting activity. By combining all three tools together, he hopes to pin stimuli and activity to the molecules and connections on the map and then use that to build a computational model that simulates brain activity.

The plan is grandiose, and the tools aren’t all ready yet, but to make the scheme plausible in the proposed timeframe, Boyden is adhering to a few principles. His methods are fast, capturing information-dense images rapidly rather than scanning over days, and inclusive, imaging whole brains rather than chunks that need to be assembled. They are also accessible, so researchers don’t need to spend large sums to acquire specialized equipment or expertise in-house.

The challenges ahead might appear insurmountable at times, but Boyden is undeterred. He moves forward, his mind open to even the most far-fetched ideas, because they just might work.

MIT team enlarges brain samples, making them easier to image

Anne Trafton |

Beginning with the invention of the first microscope in the late 1500s, scientists have been trying to peer into preserved cells and tissues with ever-greater magnification. The latest generation of so-called “super-resolution” microscopes can see inside cells with resolution better than 250 nanometers.

A team of researchers from MIT has now taken a novel approach to gaining such high-resolution images: Instead of making their microscopes more powerful, they have discovered a method that enlarges tissue samples by embedding them in a polymer that swells when water is added. This allows specimens to be physically magnified, and then imaged at a much higher resolution.

This technique, which uses inexpensive, commercially available chemicals and microscopes commonly found in research labs, should give many more scientists access to super-resolution imaging, the researchers say.

“Instead of acquiring a new microscope to take images with nanoscale resolution, you can take the images on a regular microscope. You physically make the sample bigger, rather than trying to magnify the rays of light that are emitted by the sample,” says Ed Boyden, an associate professor of biological engineering and brain and cognitive sciences at MIT.

Boyden is the senior author of a paper describing the new method in the Jan. 15 online edition of Science. Lead authors of the paper are graduate students Fei Chen and Paul Tillberg.

Physical magnification

Most microscopes work by using lenses to focus light emitted from a sample into a magnified image. However, this approach has a fundamental limit known as the diffraction limit, which means that it can’t be used to visualize objects much smaller than the wavelength of the light being used. For example, if you are using blue-green light with a wavelength of 500 nanometers, you can’t see anything smaller than 250 nanometers.

“Unfortunately, in biology that’s right where things get interesting,” says Boyden, who is a member of MIT’s Media Lab and McGovern Institute for Brain Research. Protein complexes, molecules that transport payloads in and out of cells, and other cellular activities are all organized at the nanoscale.

Scientists have come up with some “really clever tricks” to overcome this limitation, Boyden says. However, these super-resolution techniques work best with small, thin samples, and take a long time to image large samples. “If you want to map the brain, or understand how cancer cells are organized in a metastasizing tumor, or how immune cells are configured in an autoimmune attack, you have to look at a large piece of tissue with nanoscale precision,” he says.

To achieve this, the MIT team focused its attention on the sample rather than the microscope. Their idea was to make specimens easier to image at high resolution by embedding them in an expandable polymer gel made of polyacrylate, a very absorbent material commonly found in diapers.

Before enlarging the tissue, the researchers first label the cell components or proteins that they want to examine, using an antibody that binds to the chosen targets. This antibody is linked to a fluorescent dye, as well as a chemical anchor that can attach the dye to the polyacrylate chain.

Once the tissue is labeled, the researchers add the precursor to the polyacrylate gel and heat it to form the gel. They then digest the proteins that hold the specimen together, allowing it to expand uniformly. The specimen is then washed in salt-free water to induce a 100-fold expansion in volume. Even though the proteins have been broken apart, the original location of each fluorescent label stays the same relative to the overall structure of the tissue because it is anchored to the polyacrylate gel.

“What you’re left with is a three-dimensional, fluorescent cast of the original material. And the cast itself is swollen, unimpeded by the original biological structure,” Tillberg says.

The MIT team imaged this “cast” with commercially available confocal microscopes, commonly used for fluorescent imaging but usually limited to a resolution of hundreds of nanometers. With their enlarged samples, the researchers achieved resolution down to 70 nanometers. “The expansion microscopy process … should be compatible with many existing microscope designs and systems already in laboratories,” Chen adds.

Large tissue samples

Using this technique, the MIT team was able to image a section of brain tissue 500 by 200 by 100 microns with a standard confocal microscope. Imaging such large samples would not be feasible with other super-resolution techniques, which require minutes to image a tissue slice only 1 micron thick and are limited in their ability to image large samples by optical scattering and other aberrations.

“The exciting part is that this approach can acquire data at the same high speed per pixel as conventional microscopy, contrary to most other methods that beat the diffraction limit for microscopy, which can be 1,000 times slower per pixel,” says George Church, a professor of genetics at Harvard Medical School who was not part of the research team.

“The other methods currently have better resolution, but are harder to use, or slower,” Tillberg says. “The benefits of our method are the ease of use and, more importantly, compatibility with large volumes, which is challenging with existing technologies.”

The researchers envision that this technology could be very useful to scientists trying to image brain cells and map how they connect to each other across large regions.

“There are lots of biological questions where you have to understand a large structure,” Boyden says. “Especially for the brain, you have to be able to image a large volume of tissue, but also to see where all the nanoscale components are.”

While Boyden’s team is focused on the brain, other possible applications for this technique include studying tumor metastasis and angiogenesis (growth of blood vessels to nourish a tumor), or visualizing how immune cells attack specific organs during autoimmune disease.

The research was funded by the National Institutes of Health, the New York Stem Cell Foundation, Jeremy and Joyce Wertheimer, the National Science Foundation, and the Fannie and John Hertz Foundation.