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Multiplex Rainbow Technology Offers New View of the Brain

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Proteins imaged with this new approach
Caption: Confocal LNA-PRISM imaging of neuronal synapses. Conventional images of cell nuclei and two proteins (top row, three images on the left), along with 11 PRISM images of proteins and one composite, multiplexed image (bottom row, right). Credit: Adapted from Guo SM, Nature Communications, 2019

The NIH-led Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative is revolutionizing our understanding of how the brain works through its creation of new imaging tools. One of the latest advances—used to produce this rainbow of images—makes it possible to view dozens of proteins in rapid succession in a single tissue sample containing thousands of neural connections, or synapses.

Apart from their colors, most of these images look nearly identical at first glance. But, upon closer inspection, you’ll see some subtle differences among them in both intensity and pattern. That’s because the images capture different proteins within the complex network of synapses—and those proteins may be present in that network in different amounts and locations. Such findings may shed light on key differences among synapses, as well as provide new clues into the roles that synaptic proteins may play in schizophrenia and various other neurological disorders.

Synapses contain hundreds of proteins that regulate the release of chemicals called neurotransmitters, which allow neurons to communicate. Each synaptic protein has its own specific job in the process. But there have been longstanding technical difficulties in observing synaptic proteins at work. Conventional fluorescence microscopy can visualize at most four proteins in a synapse.

As described in Nature Communications [1], researchers led by Mark Bathe, Massachusetts Institute of Technology (MIT), Cambridge, and Jeffrey Cottrell, Broad Institute of MIT and Harvard, Cambridge, have just upped this number considerably while delivering high quality images. They did it by adapting an existing imaging method called DNA PAINT [2]. The researchers call their adapted method PRISM. It is short for: Probe-based Imaging for Sequential Multiplexing.

Here’s how it works: First, researchers label proteins or other molecules of interest using antibodies that recognize those proteins. Those antibodies include a unique DNA probe that helps with the next important step: making the proteins visible under a microscope.

To do it, they deliver short snippets of complementary fluorescent DNA, which bind the DNA-antibody probes. While each protein of interest is imaged separately, researchers can easily wash the probes from a sample to allow a series of images to be generated, each capturing a different protein of interest.

In the original DNA PAINT, the DNA strands bind and unbind periodically to create a blinking fluorescence that can be captured using super-resolution microscopy. But that makes the process slow, requiring about half an hour for each protein.

To speed things up with PRISM, Bathe and his colleagues altered the fluorescent DNA probes. They used synthetic DNA that’s specially designed to bind more tightly or “lock” to the DNA-antibody. This gives a much brighter signal without the blinking effect. As a result, the imaging can be done faster, though at slightly lower resolution.

Though the team now captures images of 12 proteins within a sample in about an hour, this is just a start. As more DNA-antibody probes are developed for synaptic proteins, the team can readily ramp up this number to 30 protein targets.

Thanks to the BRAIN Initiative, researchers now possess a powerful new tool to study neurons. PRISM will help them learn more mechanistically about the inner workings of synapses and how they contribute to a range of neurological conditions.


[1] Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes. Guo SM, Veneziano R, Gordonov S, Li L, Danielson E, Perez de Arce K, Park D, Kulesa AB, Wamhoff EC, Blainey PC, Boyden ES, Cottrell JR, Bathe M. Nat Commun. 2019 Sep 26;10(1):4377.

[2] Super-resolution microscopy with DNA-PAINT. Schnitzbauer J, Strauss MT, Schlichthaerle T, Schueder F, Jungmann R. Nat Protoc. 2017 Jun;12(6):1198-1228.


Schizophrenia (National Institute of Mental Health)

Mark Bathe (Massachusetts Institute of Technology, Cambridge)

Jeffrey Cottrell (Broad Institute of MIT and Harvard, Cambridge)

Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative (NIH)

NIH Support: National Institute of Mental Health; National Human Genome Research Institute; National Institute of Neurological Disorders and Stroke; National Institute of Environmental Health Sciences

Big Data and Imaging Analysis Yields High-Res Brain Map

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The HCP’s multi-modal cortical parcellation

Caption: Map of 180 areas in the left and right hemispheres of the cerebral cortex.
Credit: Matthew F. Glasser, David C. Van Essen, Washington University Medical School, Saint Louis, Missouri

Neuroscientists have been working for a long time to figure out how the human brain works, and that has led many through the years to attempt to map its various regions and create a detailed atlas of their complex geography and functions. While great progress has been made in recent years, existing brain maps have remained relatively blurry and incomplete, reflecting only limited aspects of brain structure or function and typically in just a few people.

In a study reported recently in the journal Nature, an NIH-funded team of researchers has begun to bring this map of the human brain into much sharper focus [1]. By combining multiple types of cutting-edge brain imaging data from more than 200 healthy young men and women, the researchers were able to subdivide the cerebral cortex, the brain’s outer layer, into 180 specific areas in each hemisphere. Remarkably, almost 100 of those areas had never before been described. This new high-resolution brain map will advance fundamental understanding of the human brain and will help to bring greater precision to the diagnosis and treatment of many brain disorders.

Snapshots of Life: Behold the Beauty of the Eye

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Colorized cross section of a mouse eye

Credit: Bryan William Jones and Robert E. Marc, University of Utah

The eye is a complex marvel of nature. In fact, there are some 70 to 80 kinds of cells in the mammalian retina. This image beautifully illuminates the eye’s complexity, on a cellular level—showing how these cells are arranged and wired together to facilitate sight.

“Reading” the image from left to right, we first find the muscle cells, in peach, that move the eye in its socket. The green layer, next, is the sclera—the white part of the eye. The spongy-looking layers that follow provide blood to the retina. The thin layer of yellow is the retinal pigment epithelium. The photoreceptors, in shades of pink, detect photons and transmit the information to the next layer down: the bipolar and horizontal cells (purple). From the bipolar cells, information flows to the amacrine and ganglion cells (blue, green, and turquoise) and then out of the retina via the optic nerve (the white plume that seems to billow out across the upper-right side of the eye), which transmits data to the brain for processing.

The Symphony Inside Your Brain

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Ever wonder what is it that makes you, you? Depending on whom you ask, there are a lot of different answers, but these days some of the world’s top neuroscientists might say: “You are your connectome.”

Grid of major pathways in human brain’s left hemisphere. Using diffusion spectrum imaging, which tracks movement of water through nerve fibers, researchers can trace groups of neurons as they cross from one region of the brain to another in living individuals. Credit: Van Wedeen, Massachusetts General/Harvard Medical School

The connectome refers to the exquisitely interconnected network of neurons (nerve cells) in your brain. Like the genome, the microbiome, and other exciting “ome” fields, the effort to map the connectome and decipher the electrical signals that zap through it to generate your thoughts, feelings, and behaviors has become possible through development of powerful new tools and technologies.

For some time, neuroscientists have been able to infer loosely the main functions of certain brain regions by studying patients with head injuries, brain tumors, and neurological diseases—or by measuring levels of oxygen or glucose consumption in healthy people’s brains during particular activities. But all along it’s been rather clear that these inferences were overly simplistic.  Now, new advances in computer science, math, and imaging and data visualization are empowering us to study the human brain as an entire organ, and at a level of detail not previously imagined possible in a living person.