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Taking Brain Imaging Even Deeper

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Thanks to yet another amazing advance made possible by the NIH-led supported the Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative, I can now take you on a 3D fly-through of all six layers of the part of the mammalian brain that processes external signals into vision. This unprecedented view is made possible by three-photon microscopy, a low-energy imaging approach that is allowing researchers to peer deeply within the brains of living creatures without damaging or killing their brain cells.

The basic idea of multi-photon microscopy is this: for fluorescence microscopy to work, you want to deliver a specific energy level of photons (usually with a laser) to excite a fluorescent molecule, so that it will emit light at a slightly lower energy (longer wavelength) and be visualized as a burst of colored light in the microscope. That’s how fluorescence works. Green fluorescent protein (GFP) is one of many proteins that can be engineered into cells or mice to make that possible.

But for that version of the approach to work on tissue, the excited photons need to penetrate deeply, and that’s not possible for such high energy photons. So two-photon strategies were developed, where it takes the sum of the energy of two simultaneous photons to hit the target in order to activate the fluorophore.

That approach has made a big difference, but for deep tissue penetration the photons are still too high in energy. Enter the three-photon version! Now the even lower energy of the photons makes tissue more optically transparent, though for activation of the fluorescent protein, three photons have to hit it simultaneously. But that’s part of the beauty of the system—the visual “noise” also goes down.

This particular video shows what takes place in the visual cortex of mice when objects pass before their eyes. As the objects appear, specific neurons (green) are activated to process the incoming information. Nearby, and slightly obscuring the view, are the blood vessels (pink, violet) that nourish the brain. At 33 seconds into the video, you can see the neurons’ myelin sheaths (pink) branching into the white matter of the brain’s subplate, which plays a key role in organizing the visual cortex during development.

This video comes from a recent paper in Nature Communications by a team from Massachusetts Institute of Technology, Cambridge [1]. To obtain this pioneering view of the brain, Mriganka Sur, Murat Yildirim, and their colleagues built an innovative microscope that emits three low-energy photons. After carefully optimizing the system, they were able to peer more than 1,000 microns (0.05 inches) deep into the visual cortex of a live, alert mouse, far surpassing the imaging capacity of standard one-photon microscopy (100 microns) and two-photon microscopy (400-500 microns).

This improved imaging depth allowed the team to plumb all six layers of the visual cortex (two-photon microscopy tops out at about three layers), as well as to record in real time the brain’s visual processing activities. Helping the researchers to achieve this feat was the availability of a genetically engineered mouse model in which the cells of the visual cortex are color labelled to distinguish blood vessels from neurons, and to show when neurons are active.

During their in-depth imaging experiments, the MIT researchers found that each of the visual cortex’s six layers exhibited different responses to incoming visual information. One of the team’s most fascinating discoveries is that neurons residing on the subplate are actually quite active in adult animals. It had been assumed that these subplate neurons were active only during development. Their role in mature animals is now an open question for further study.

Sur often likens the work in his neuroscience lab to astronomers and their perpetual quest to see further into the cosmos—but his goal is to see ever deeper into the brain. His group, along with many other researchers supported by the BRAIN Initiative, are indeed proving themselves to be biological explorers of the first order.


[1] Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy. Yildirim M, Sugihara H, So PTC, Sur M. Nat Commun. 2019 Jan 11;10(1):177.


Sur Lab (Massachusetts Institute of Technology, Cambridge)

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

NIH Support: National Eye Institute; National Institute of Neurological Disorders and Stroke; National Institute of Biomedical Imaging and Bioengineering

Mammalian Brain Like You’ve Never Seen It Before

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Credit: Gao et. al, Science

Researchers are making amazing progress in developing new imaging approaches. And they are now using one of their latest creations, called ExLLSM, to provide us with jaw-dropping views of a wide range of biological systems, including the incredibly complex neural networks within the mammalian brain.

In this video, ExLLSM takes us on a super-resolution, 3D voyage through a tiny sample (0.0030 inches thick) from the part of the mouse brain that processes sensation, the primary somatosensory cortex. The video zooms in and out of densely packed pyramidal neurons (large yellow cell bodies), each of which has about 7,000 synapses, or connections. You can also see presynapses (cyan), the part of the neuron that sends chemical signals; and postsynapes (magenta), the part of the neuron that receives chemical signals.

At 1:45, the video zooms in on dendritic spines, which are mushroom-like nubs on the neuronal branches (yellow). These structures, located on the tips of dendrites, receive incoming signals that are turned into electrical impulses. While dendritic spines have been imaged in black and white with electron microscopy, they’ve never been presented before on such a vast, colorful scale.

The video comes from a paper, published recently in the journal Science [1], from the labs of Ed Boyden, Massachusetts Institute of Technology, Cambridge, and the Nobel Prize-winning Eric Betzig, Janelia Research Campus of the Howard Hughes Medical Institute, Ashburn, VA. Like many collaborations, this one comes with a little story.

Four years ago, the Boyden lab developed expansion microscopy (ExM). The technique involves infusing cells with a hydrogel, made from a chemical used in disposable diapers. The hydrogel expands molecules within the cell away from each other, usually by about 4.5 times, but still locks them into place for remarkable imaging clarity. It makes structures visible by light microscopy that are normally below the resolution limit.

Though the expansion technique has worked well with a small number of cells under a standard light microscope, it hasn’t been as successful—until now—at imaging thicker tissue samples. That’s because thicker tissue is harder to illuminate, and flooding the specimen with light often bleaches out the fluorescent markers that scientists use to label proteins. The signal just fades away.

For Boyden, that was a problem that needed to be solved. Because his lab’s goal is to trace the inner workings of the brain in unprecedented detail, Boyden wants to image entire neural circuits in relatively thick swaths of tissue, not just look at individual cells in isolation.

After some discussion, Boyden’s team concluded that the best solution might be to swap out the light source for the standard microscope with a relatively new imaging tool developed in the Betzig lab. It’s called lattice light-sheet microscopy (LLSM), and the tool generates extremely thin sheets of light that illuminate tissue only in a very tightly defined plane, dramatically reducing light-related bleaching of fluorescent markers in the tissue sample. This allows LLSM to extend its range of image acquisition and quickly deliver stunningly vivid pictures.

Telephone calls were made, and the Betzig lab soon welcomed Ruixuan Gao, Shoh Asano, and colleagues from the Boyden lab to try their hand at combining the two techniques. As the video above shows, ExLLSM has proved to be a perfect technological match. In addition to the movie above, the team has used ExLLSM to provide unprecedented views of a range of samples—from human kidney to neuron bundles in the brain of the fruit fly.

Not only is ExLLSM super-resolution, it’s also super-fast. In fact, the team imaged the entire fruit fly brain in 2 1/2 days—an effort that would take years using an electron microscope.

ExLLSM will likely never supplant the power of electron microscopy or standard fluorescent light microscopy. Still, this new combo imaging approach shows much promise as a complementary tool for biological exploration. The more innovative imaging approaches that researchers have in their toolbox, the better for our ongoing efforts to unlock the mysteries of the brain and other complex biological systems. And yes, those systems are all complex. This is life we’re talking about!


[1] Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution. Gao R, Asano SM, Upadhyayula S, Pisarev I, Milkie DE, Liu TL, Singh V, Graves A, Huynh GH, Zhao Y, Bogovic J, Colonell J, Ott CM, Zugates C, Tappan S, Rodriguez A, Mosaliganti KR, Sheu SH, Pasolli HA, Pang S, Xu CS, Megason SG, Hess H, Lippincott-Schwartz J, Hantman A, Rubin GM, Kirchhausen T, Saalfeld S, Aso Y, Boyden ES, Betzig E. Science. 2019 Jan 18;363(6424).


Video: Expansion Microscopy Explained (YouTube)

Video: Lattice Light-Sheet Microscopy (YouTube)

How to Rapidly Image Entire Brains at Nanoscale Resolution, Howard Hughes Medical Institute, January 17, 2019.

Synthetic Neurobiology Group (Massachusetts Institute of Technology, Cambridge)

Eric Betzig (Janelia Reseach Campus, Ashburn, VA)

NIH Support: National Institute of Neurological Disorders and Stroke; National Human Genome Research Institute; National Institute on Drug Abuse; National Institute of Mental Health; National Institute of Biomedical Imaging and Bioengineering

Zooming In on Meiosis

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Credit: Simone Köhler, Michal Wojcik, Ke Xu, and Abby Dernburg, University of California, Berkeley

Meiosis—the formation of egg and sperm cells—is a highly choreographed process that creates genetic diversity in all plants and animals, including humans, to make each of us unique. This kaleidoscopic image shows cells from a worm exchanging DNA during meiosis.

You can see a protein-based polymer tether (green) from what’s called the synaptonemal complex. The complex holds together partner chromosomes (magenta) to facilitate DNA exchange in nuclei (white). Moving from left to right are views of the molecular assembly that progressively zoom in on the DNA, revealing in exquisite detail (far right) the two paired partner chromosomes perfectly aligned. This is not just the familiar DNA double helix. This is a double helix made up of two double helices!

The Actin Superhighway

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Actin Superhighway

Credit: Andrew Lombardo and David Warshaw, University of Vermont, Burlington

What looks like a traffic grid filled with roundabouts is nothing of the sort: It’s actually a peek inside a tiny microchamber that models a complex system operating in many of our cells. The system is a molecular transportation network made of the protein actin, and researchers have reconstructed it in the lab to study its rules of the road and, when things go wrong, how it can lead to molecular traffic accidents.

This 3D super-resolution image shows the model’s silicone beads (circles) positioned in a tiny microfluidic-chamber. Suspended from the beads are actin filaments that form some of the main cytoskeletal roadways in our cells. Interestingly, a single dye creates the photo’s beautiful colors, which arise from the different vertical dimensions of a microscopic image: 300 nanometers below the focus (red), at focus (green), and 300 nanometers above the focus (blue). When a component spans multiple dimensions—such as the spherical beads—all the colors of the rainbow are visible. The technique is called 3D stochastic optical reconstruction microscopy, or STORM [1].

Looking to Llamas for New Ways to Fight the Flu

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Lllama nanobodiesResearchers are making tremendous strides toward developing better ways to reduce our risk of getting the flu. And one of the latest ideas for foiling the flu—a “gene mist” that could be sprayed into the nose—comes from a most surprising source: llamas.

Like humans and many other creatures, these fuzzy South American relatives of the camel produce immune molecules, called antibodies, in their blood when exposed to viruses and other foreign substances. Researchers speculated that because the llama’s antibodies are so much smaller than human antibodies, they might be easier to use therapeutically in fending off a wide range of flu viruses. This idea is now being leveraged to design a new type of gene therapy that may someday provide humans with broader protection against the flu [1].

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