Tag Archives: Edward S. Boyden

Viewing RNA (ribonucleic acid) more closely at the nanoscale with expansion microscopy (EXM) and off-the-shelf parts

A close cousin to DNA (deoxyribonucleic acid), RNA (ribonucleic acid) is a communicator according to a July 4, 2016 news item on ScienceDaily describing how a team at the Massachusetts Institute of Technology (MIT) managed to image RNA more precisely,

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, 2016 issue of Nature Methods.

A July 4, 2016 MIT news release (also on EurekAlert), which originated the news item, explains why scientists want a better look at RNA and how the MIT team accomplished the task,

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.

There’s a brief (30 secs.), silent video illustrating the work (something about a ‘Brainbow Hippocampus’) made available by MIT,


Here’s a link to and a citation for the paper,

Nanoscale imaging of RNA with expansion microscopy by Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj, & Edward S Boyden.     Nature Methods (2016)  doi:10.1038/nmeth.3899 Published online 04 July 2016

This paper is behind a paywall.

Nanotechnology and the US mega science project: BAM (Brain Activity Map) and more

The Brain Activity Map (BAM) project received budgetary approval as of this morning, Apr. 2, 2013 (I first mentioned BAM in my Mar. 4, 2013 posting when approval seemed imminent). From the news item, Obama Announces Huge Brain-Mapping Project, written by Stephanie Pappas for Yahoo News (Note: Links have been removed),

 President Barack Obama announced a new research initiative this morning (April 2) to map the human brain, a project that will launch with $100 million in funding in 2014.

The Brain Activity Map (BAM) project, as it is called, has been in the planning stages for some time. In the June 2012 issue of the journal Neuron, six scientists outlined broad proposals for developing non-invasive sensors and methods to experiment on single cells in neural networks. This February, President Obama made a vague reference to the project in his State of the Union address, mentioning that it could “unlock the answers to Alzheimer’s.”

In March, the project’s visionaries outlined their final goals in the journal Science. They call for an extended effort, lasting several years, to develop tools for monitoring up to a million neurons at a time. The end goal is to understand how brain networks function.

“It could enable neuroscience to really get to the nitty-gritty of brain circuits, which is the piece that’s been missing from the puzzle,” Rafael Yuste, the co-director of the Kavli Institute for Brain Circuits at Columbia University, who is part of the group spearheading the project, told LiveScience in March. “The reason it’s been missing is because we haven’t had the techniques, the tools.” [Inside the Brain: A Journey Through Time]

Not all neuroscientists support the project, however, with some arguing that it lacks clear goals and may cannibalize funds for other brain research.

….

I believe the $100M mentioned for 2014 would one installment in a series totaling up to $1B or more. In any event, it seems like a timely moment to comment on the communications campaign that has been waged on behalf of the BAM. It reminds me a little of the campaign for graphene, which was waged in the build up to the decision as to which two projects (in a field of six semi-finalists, then narrowed to a field of four finalists) should receive a FET (European Union’s Future and Emerging Technology) 1 billion euro research prize each. It seemed to me even a year or so before the decision that graphene’s win was a foregone conclusion but the organizers left nothing to chance and were relentless in their pursuit of attention and media coverage in the buildup to the final decision.

The most recent salvo in the BAM campaign was an attempt to link it with nanotechnology. A shrewd move given that the US has spent well over $1B since the US National Nanotechnology Initiative (NNI) was first approved in 2000. Linking the two projects means the NNI can lend a little authority to the new project (subtext: we’ve supported a mega-project before and that was successful) while the new project BAM can imbue the ageing NNI with some excitement.

Here’s more about nanotechnology and BAM from a Mar. 27, 2013 Spotlight article by Michael Berger on Nanowerk,

A comprehensive understanding of the brain remains an elusive, distant frontier. To arrive at a general theory of brain function would be an historic event, comparable to inferring quantum theory from huge sets of complex spectra and inferring evolutionary theory from vast biological field work. You might have heard about the proposed Brain Activity Map – a project that, like the Human Genome Project, will tap the hive mind of experts to make headway in the understanding of the field. Engineers and nanotechnologists will be needed to help build ever smaller devices for measuring the activity of individual neurons and, later, to control how those neurons function. Computer scientists will be called upon to develop methods for storing and analyzing the vast quantities of imaging and physiological data, and for creating virtual models for studying brain function. Neuroscientists will provide critical biological expertise to guide the research and interpret the results.

Berger goes on to highlight some of the ways nanotechnology-enabled devices could contribute to the effort. He draws heavily on a study published Mar. 20, 2013 online in ACS (American Chemical Society)Nano. Shockingly, the article is open access. Given that this is the first time I’ve come across an open access article in any of the American Chemical Society’s journals, I suspect that there was payment of some kind involved to make this information freely available. (The practice of allowing researchers to pay more in order to guarantee open access to their research in journals that also have articles behind paywalls seems to be in the process of becoming more common.)

Here’s a citation and a link to the article about nanotechnology and BAM,

Nanotools for Neuroscience and Brain Activity Mapping by A. Paul Alivisatos, Anne M. Andrews, Edward S. Boyden, Miyoung Chun, George M. Church, Karl Deisseroth, John P. Donoghue, Scott E. Fraser, Jennifer Lippincott-Schwartz, Loren L. Looger, Sotiris Masmanidis, Paul L. McEuen, Arto V. Nurmikko, Hongkun Park, Darcy S. Peterka, Clay Reid, Michael L. Roukes, Axel Scherer, Mark Schnitzer, Terrence J. Sejnowski, Kenneth L. Shepard, Doris Tsao, Gina Turrigiano, Paul S. Weiss, Chris Xu, Rafael Yuste, and Xiaowei Zhuang. ACS Nano, 2013, 7 (3), pp 1850–1866 DOI: 10.1021/nn4012847 Publication Date (Web): March 20, 2013
Copyright © 2013 American Chemical Society

As these things go, it’s a readable article for people without a neuroscience education provided they don’t mind feeling a little confused from time to time. From Nanotools for Neuroscience and Brain Activity Mapping (Note: Footnotes and links removed),

The Brain Activity Mapping (BAM) Project (…) has three goals in terms of building tools for neuroscience capable of (…) measuring the activity of large sets of neurons in complex brain circuits, (…) computationally analyzing and modeling these brain circuits, and (…) testing these models by manipulating the activities of chosen sets of neurons in these brain circuits.

As described below, many different approaches can, and likely will, be taken to achieve these goals as neural circuits of increasing size and complexity are studied and probed.

The BAM project will focus both on dynamic voltage activity and on chemical neurotransmission. With an estimated 85 billion neurons, 100 trillion synapses, and 100 chemical neurotransmitters in the human brain,(…) this is a daunting task. Thus, the BAM project will start with model organisms, neural circuits (vide infra), and small subsets of specific neural circuits in humans.

Among the approaches that show promise for the required dynamic, parallel measurements are optical and electro-optical methods that can be used to sense neural cell activity such as Ca2+,(7) voltage,(…) and (already some) neurotransmitters;(…) electrophysiological approaches that sense voltages and some electrochemically active neurotransmitters;(…) next-generation photonics-based probes with multifunctional capabilities;(18) synthetic biology approaches for recording histories of function;(…) and nanoelectronic measurements of voltage and local brain chemistry.(…) We anticipate that tools developed will also be applied to glia and more broadly to nanoscale and microscale monitoring of metabolic processes.

Entirely new tools will ultimately be required both to study neurons and neural circuits with minimal perturbation and to study the human brain. These tools might include “smart”, active nanoscale devices embedded within the brain that report on neural circuit activity wirelessly and/or entirely new modalities of remote sensing of neural circuit dynamics from outside the body. Remarkable advances in nanoscience and nanotechnology thus have key roles to play in transduction, reporting, power, and communications.

One of the ultimate goals of the BAM project is that the knowledge acquired and tools developed will prove useful in the intervention and treatment of a wide variety of diseases of the brain, including depression, epilepsy, Parkinson’s, schizophrenia, and others. We note that tens of thousands of patients have already been treated with invasive (i.e., through the skull) treatments. [emphases mine] While we hope to reduce the need for such measures, greatly improved and more robust interfaces to the brain would impact effectiveness and longevity where such treatments remain necessary.

Perhaps not so coincidentally, there was this Mar. 29, 2013 news item on Nanowerk,

Some human cells forget to empty their trash bins, and when the garbage piles up, it can lead to Parkinson’s disease and other genetic and age-related disorders. Scientists don’t yet understand why this happens, and Rice University engineering researcher Laura Segatori is hoping to change that, thanks to a prestigious five-year CAREER Award from the National Science Foundation (NSF).

Segatori, Rice’s T.N. Law Assistant Professor of Chemical and Biomolecular Engineering and assistant professor of bioengineering and of biochemistry and cell biology, will use her CAREER grant to create a toolkit for probing the workings of the cellular processes that lead to accumulation of waste material and development of diseases, such as Parkinson’s and lysosomal storage disorders. Each tool in the kit will be a nanoparticle — a speck of matter about the size of a virus — with a specific shape, size and charge.  [emphases mine] By tailoring each of these properties, Segatori’s team will create a series of specialized probes that can undercover the workings of a cellular process called autophagy.

“Eventually, once we understand how to design a nanoparticle to activate autophagy, we will use it as a tool to learn more about the autophagic process itself because there are still many question marks in biology regarding how this pathway works,” Segatori said. “It’s not completely clear how it is regulated. It seems that excessive autophagy may activate cell death, but it’s not yet clear. In short, we are looking for more than therapeutic applications. We are also hoping to use these nanoparticles as tools to study the basic science of autophagy.”

There is no direct reference to BAM but there are some intriguing correspondences.

Finally, there is no mention of nanotechnology in this radio broadcast/podcast and transcript but it does provide more information about BAM (for many folks this was first time they’d heard about the project) and the hopes and concerns this project raises while linking it to the Human Genome Project. From the Mar. 31, 2013 posting of a transcript and radio (Kera News; a National Public Radio station) podcast titled, Somewhere Over the Rainbow: The Journey to Map the Human Brain,

During the State of the Union, President Obama said the nation is about to embark on an ambitious project: to examine the human brain and create a road map to the trillions of connections that make it work.

“Every dollar we invested to map the human genome returned $140 to our economy — every dollar,” the president said. “Today, our scientists are mapping the human brain to unlock the answers to Alzheimer’s.”

Details of the project have slowly been leaking out: $3 billion, 10 years of research and hundreds of scientists. The National Institutes of Health is calling it the Brain Activity Map.

Obama isn’t the first to tout the benefits of a huge government science project. But can these projects really deliver? And what is mapping the human brain really going to get us?

Whether one wants to call it a public relations campaign or a marketing campaign is irrelevant. Science does not take place in an environment where data and projects are considered dispassionately. Enormous amounts of money are spent to sway public opinion and policymakers’ decisions.

ETA Ap. 3, 2013: Here are more stories about BAM and the announcement:

BRAIN Initiative Launched to Unlock Mysteries of Human Mind

Obama’s BRAIN Only 1/13 The Size Of Europe’s

BRAIN Initiative Builds on Efforts of Leading Neuroscientists and Nanotechnologists