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(Image caption: A new technique called magnified analysis of proteome (MAP), developed at MIT, allows researchers to peer at molecules within cells or take a wider view of the long-range connections between neurons. Credit: Courtesy of the researchers)

Imaging the brain at multiple size scales

MIT researchers have developed a new technique for imaging brain tissue at multiple scales, allowing them to peer at molecules within cells or take a wider view of the long-range connections between neurons.

This technique, known as magnified analysis of proteome (MAP), should help scientists in their ongoing efforts to chart the connectivity and functions of neurons in the human brain, says Kwanghun Chung, the Samuel A. Goldblith Assistant Professor in the Departments of Chemical Engineering and Brain and Cognitive Sciences, and a member of MIT’s Institute for Medical Engineering and Science (IMES) and Picower Institute for Learning and Memory.

“We use a chemical process to make the whole brain size-adjustable, while preserving pretty much everything. We preserve the proteome (the collection of proteins found in a biological sample), we preserve nanoscopic details, and we also preserve brain-wide connectivity,” says Chung, the senior author of a paper describing the method in the July 25 issue of Nature Biotechnology.

The researchers also showed that the technique is applicable to other organs such as the heart, lungs, liver, and kidneys.

The paper’s lead authors are postdoc Taeyun Ku, graduate student Justin Swaney, and visiting scholar Jeong-Yoon Park.

Multiscale imaging

The new MAP technique builds on a tissue transformation method known as CLARITY, which Chung developed as a postdoc at Stanford University. CLARITY preserves cells and molecules in brain tissue and makes them transparent so the molecules inside the cell can be imaged in 3-D. In the new study, Chung sought a way to image the brain at multiple scales, within the same tissue sample.

“There is no effective technology that allows you to obtain this multilevel detail, from brain region connectivity all the way down to subcellular details, plus molecular information,” he says.

To achieve that, the researchers developed a method to reversibly expand tissue samples in a way that preserves nearly all of the proteins within the cells. Those proteins can then be labeled with fluorescent molecules and imaged.

The technique relies on flooding the brain tissue with acrylamide polymers, which can form a dense gel. In this case, the gel is 10 times denser than the one used for the CLARITY technique, which gives the sample much more stability. This stability allows the researchers to denature and dissociate the proteins inside the cells without destroying the structural integrity of the tissue sample.

Before denaturing the proteins, the researchers attach them to the gel using formaldehyde, as Chung did in the CLARITY method. Once the proteins are attached and denatured, the gel expands the tissue sample to four or five times its original size.

“It is reversible and you can do it many times,” Chung says. “You can then use off-the-shelf molecular markers like antibodies to label and visualize the distribution of all these preserved biomolecules.”

There are hundreds of thousands of commercially available antibodies that can be used to fluorescently tag specific proteins. In this study, the researchers imaged neuronal structures such as axons and synapses by labeling proteins found in those structures, and they also labeled proteins that allow them to distinguish neurons from glial cells.

“We can use these antibodies to visualize any target structures or molecules,” Chung says. “We can visualize different neuron types and their projections to see their connectivity. We can also visualize signaling molecules or functionally important proteins.”

High resolution

Once the tissue is expanded, the researchers can use any of several common microscopes to obtain images with a resolution as high as 60 nanometers — much better than the usual 200 to 250-nanometer limit of light microscopes, which are constrained by the wavelength of visible light. The researchers also demonstrated that this approach works with relatively large tissue samples, up to 2 millimeters thick.

“This is, as far as I know, the first demonstration of super-resolution proteomic imaging of millimeter-scale samples,” Chung says.

“This is an exciting advance for brain mapping, a technique that reveals the molecular and connectional architecture of the brain with unprecedented detail,” says Sebastian Seung, a professor of computer science at the Princeton Neuroscience Institute, who was not involved in the research.

Currently, efforts to map the connections of the human brain rely on electron microscopy, but Chung and colleagues demonstrated that the higher-resolution MAP imaging technique can trace those connections more accurately.

Chung’s lab is now working on speeding up the imaging and the image processing, which is challenging because there is so much data generated from imaging the expanded tissue samples.

“It’s already easier than other techniques because the process is really simple and you can use off-the-shelf molecular markers, but we are trying to make it even simpler,” Chung says.

The Art of Lying

How to Make a Motor Neuron

A team of scientists has uncovered details of the cellular mechanisms that control the direct programming of stem cells into motor neurons. The scientists analyzed changes that occur in the cells over the course of the reprogramming process. They discovered a dynamic, multi-step process in which multiple independent changes eventually converge to change the stem cells into motor neurons.

“There is a lot of interest in generating motor neurons to study basic developmental processes as well as human diseases like ALS and spinal muscular atrophy,” said Shaun Mahony, assistant professor of biochemistry and molecular biology at Penn State and one of the lead authors of the paper. “By detailing the mechanisms underlying the direct programing of motor neurons from stem cells, our study not only informs the study of motor neuron development and its associated diseases, but also informs our understanding of the direct programming process and may help with the development of techniques to generate other cell types.”

The direct programming technique could eventually be used to regenerate missing or damaged cells by converting other cell types into the missing one. The research findings, which appear online in the journal Cell Stem Cell on December 8, 2016, show the challenges facing current cell-replacement technology, but they also outline a potential pathway to the creation of more viable methods.

“Despite having a great therapeutic potential, direct programming is generally inefficient and doesn’t fully take into account molecular complexity,” said Esteban Mazzoni, an assistant professor in New York University’s Department of Biology and one of the lead authors of the study. “However, our findings point to possible new avenues for enhanced gene-therapy methods.”

The researchers had shown previously that they can transform mouse embryonic stem cells into motor neurons by expressing three transcription factors – genes that control the expression of other genes – in the stem cells. The transformation takes about two days. In order to better understand the cellular and genetic mechanisms responsible for the transformation, the researchers analyzed how the transcription factors bound to the genome, changes in gene expression, and modifications to chromatin at 6-hour intervals during the transformation.

“We have a very efficient system in which we can transform stem cells into motor neurons with something like a 90 to 95 percent success rate by adding the cocktail of transcription factors,” said Mahony. “Because of that efficiency, we were able to use our system to tease out the details of what actually happens in the cell during this transformation.”

“A cell in an embryo develops by passing through several intermediate stages,” noted Uwe Ohler, senior researcher at the Max Delbrück Center for Molecular Medicine (MDC) in Berlin and one of the lead authors of the work. “But in direct programming we don’t have that: we replace the gene transcription network of the cell with a completely new one at once, without the progression through intermediate stages. We asked, what are the timing and kinetics of chromatin changes and transcription events that directly lead to the final cell fate?“

The research team found surprising complexity – programming of these stem cells into neurons is the result of two independent transcriptional processes that eventually converge. Early on in the process, two of the transcription factors – Isl1 and Lhx3 – work in tandem, binding to the genome and beginning a cascade of events including changes to chromatin structure and gene expression in the cells. The third transcription factor, Ngn2, acts independently making additional changes to gene expression. Later in the transformation process, Isl1 and Lhx3 rely on changes in the cell initiated by Ngn2 to help complete the transformation. In order for direct programming to successfully achieve cellular conversion, it must coordinate the activity of the two processes.

“Many have found direct programming to be a potentially attractive method as it can be performed either in vitro – outside of a living organism – or in vivo – inside the body and, importantly, at the site of cellular damage,” said Mazzoni. “However, questions remain about its viability to repair cells – especially given the complex nature of the biological process. Looking ahead, we think it’s reasonable to use this newly gained knowledge to, for instance, manipulate cells in the spinal cord to replace the neurons required for voluntary movement that are destroyed by afflictions such as ALS.”

winter sunrise reflections

by Denny Bitte

Engineers build world’s lightest mechanical watch thanks to graphene

An ultralight high-performance mechanical watch made with graphene is unveiled today in Geneva at the Salon International De La Haute Horlogerie thanks to a unique collaboration.

The University of Manchester has collaborated with watchmaking brand Richard Mille and McLaren F1 to create world’s lightest mechanical chronograph by pairing leading graphene research with precision engineering.

The RM 50-03 watch was made using a unique composite incorporating graphene to manufacture a strong but lightweight new case to house the delicate watch mechanism. The graphene composite known as Graph TPT weighs less than previous similar materials used in watchmaking.

Graphene is the world’s first two-dimensional material at just one-atom thick. It was first isolated at The University of Manchester in 2004 and has the potential to revolutionise a large number of applications including, high-performance composites for the automotive and aerospace industries, as well as flexible, bendable mobile phones and tablets and next-generation energy storage.

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