Posted on by Dr. Francis Collins
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 . 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.
 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)
NIH Support: National Eye Institute; National Institute of Neurological Disorders and Stroke; National Institute of Biomedical Imaging and Bioengineering
Posted on by Dr. Francis Collins
Seeing the development of an organ under a microscope for the first time can be a truly unforgettable experience. But for a class taught by Crystal Rogers at California State University, Northridge, it can also be an award-winning moment.
This image, prepared during a biology lab course, was one of the winners in the 2018 BioArt Scientific Image & Video Competition, sponsored by the Federation of American Societies for Experimental Biology (FASEB). This colorful image shows the tip of an ovary from a fruit fly (Drosophila melanogaster), provided by Mariano Loza-Coll. You can see that the ovary is packed with oocytes (DNA stained blue). The orderly connective structure (pink) and signal-transmitting molecules like STAT (yellow) are common to egg maturation and reproductive processes in humans.
What makes this image unique among this year’s BioArt winners is that the prep work was done by undergraduate students just learning how to work in a lab. They did the tissue dissections, molecular labeling, and beautiful stainings in preparation for Rogers to “snap” the photo on her research lab’s optical-sectioning microscope.
What’s also fantastic is that many of Rogers’s students are from groups traditionally underrepresented in biomedicine. Many are considering careers in research and, from the looks of things, they are off to a beautiful start.
After teaching classes, Rogers also has an NIH-supported lab to run. She and her team study salamanders and chickens to determine how biological “glue” proteins, called cadherins, help to create neural crest cells, a critical cell type that arises very early in development .
For developmental biologists, it’s essential to understand what prompts these neural crest cells to migrate to locations throughout the body, from the heart to the skin to the cranium, or head. For example, cranial neural crest cells at first produce what appears to be the same generic, undifferentiated facial template in vertebrate species. And yet, neural crest cells and the surrounding ectodermal cells go on to generate craniofacial structures as distinct as the beak of a toucan, the tusk of a boar, or the horn of a rhinoceros.
But if the organ of interest is an ovary, the fruit fly has long been a go-to organism to learn more. Not only does the fruit fly open a window into ovarian development and health issues like infertility, it showcases the extraordinary beauty of biology.
 A catenin-dependent balance between N-cadherin and E-cadherin controls neuroectodermal cell fate choices. Rogers CD, Sorrells LK, Bronner ME. Mech Dev. 2018 Aug;152:44-56.
Rogers Lab (California State University, Northridge)
BioArt Scientific Image & Video Competition (Federation of American Societies for Experimental Biology, Bethesda, MD)
NIH Support: Eunice Kennedy Shriver National Institute of Child Health and Human Development
Posted on by Dr. Francis Collins
Credit: Adapted from Jones et al. ChemRxiv.org
Over the past few years, there’s been a great deal of excitement about the power of cryo-electron microscopy (cryo-EM) for mapping the structures of large biological molecules like proteins and nucleic acids. Now comes word of another absolutely incredible use of cryo-EM: determining with great ease and exquisite precision the structure of the smaller organic chemical compounds, or “small molecules,” that play such key roles in biological exploration and drug development.
The new advance involves a cryo-EM technique called microcrystal-electron diffraction (MicroED). As detailed in a preprint on ChemRxiv.org  and the journal Angewandte Chemie , MicroED has enabled researchers to take the powdered form of commercially available small molecules and generate high-resolution data on their chemical structures in less than a half-hour—dramatically faster than with traditional methods!
Posted on by Dr. Francis Collins
For centuries, microscopes have brought to light the otherwise invisible world of the cell. But microscopes don’t typically visualize the dynamic world of the cell within a living system.
For various technical reasons, researchers have typically had to displace cells, fix them in position, mount them onto slides, and look through a microscope’s viewfinder to see the cells. It can be a little like trying to study life in the ocean by observing a fish cooped up in an 8-gallon tank.
Now, a team partially funded by NIH has developed a new hybrid imaging technology to produce amazing, live-action 3D movies of living cells in their more natural state. In this video, you’re looking at a human breast cancer cell (green) making its way through a blood vessel (purple) of a young zebrafish.
At first, the cancer cell rolls along rather freely. As the cell adheres more tightly to the blood vessel wall, that rolling motion slows to a crawl. Ultimately, the cancer cell finds a place to begin making its way across and through the blood vessel wall, where it can invade other tissues.
Posted on by Dr. Francis Collins
Each time your cells divide, telomeres—complexes of specialized DNA sequences, RNA, and protein that protect the tips of your chromosomes—shorten just a bit. And, as the video shows, that shortening renders the genomic information on your chromosomes more vulnerable to changes that can drive cancer and other diseases of aging.
Consequently, over the last few decades, much research has focused on efforts to understand telomerase, a naturally occurring enzyme that helps to replace the bits of telomere lost during cell division. But there’s been a major hitch: until recently, scientists hadn’t been able to determine telomerase’s molecular structure in detail—a key step in figuring out exactly how the enzyme works. Now, thanks to better purification methods and an exciting technology called cryo-electron microscopy (cryo-EM), NIH-funded researchers and their colleagues have risen to the challenge to produce the most detailed view yet of human telomerase in its active form .
This structural biology advance is a critical step toward learning more about the role of telomerase in cancers, as well as genetic conditions linked to telomerase deficiencies. It’s also an important milestone in the quest for drugs targeting telomerase in different ways, perhaps to slow the growth of cancerous cells or to boost the proliferative capacity of life-giving adult stem cells.
One reason telomerase has been so difficult to study in humans is that the enzyme isn’t produced at detectable levels in the vast majority of our cells. To get around this problem, the team led by Eva Nogales and Kathleen Collins at the University of California, Berkeley, first coaxed human cells in the lab to produce larger quantities of active telomerase. They then used fluorescent microscopy, along with extensive knowledge of the enzyme’s biochemistry, to develop a multi-step purification process that yielded relatively homogenous samples of active telomerase.
The new study is also yet another remarkable example of how cryo-EM microscopy has opened up new realms of scientific possibility. That’s because, in comparison to other methods, cryo-EM enables researchers to solve complex macromolecular structures even when only tiny amounts of material are available. It can also produce detailed images of molecules, like telomerase, that are extremely flexible and hard to keep still while taking a picture of their structure.
As described in Nature, the researchers used cryo-EM to capture the structure of human telomerase in unprecedented detail. Their images reveal two lobes, held together by a flexible RNA tether. One of those lobes contains the highly specialized core enzyme. It uses an internal RNA template as a guide to make the repetitive, telomeric DNA that’s added at the tips of chromosomes. The second lobe, consisting of a complex of RNA and RNA-binding proteins, plays important roles in keeping the complex stable and properly in place.
This new, more-detailed view helps to explain how mutations in particular genes may lead to telomerase-related health conditions, including bone marrow failure, as well as certain forms of anemia and pulmonary fibrosis. For example, it reveals that a genetic defect known to cause bone marrow failure affects an essential protein in a spot that’s especially critical for telomerase’s proper conformation and function.
This advance will also be a big help for designing therapies that encourage telomerase activity. For example, it could help to boost the success of bone marrow transplants by rejuvenating adult stem cells. It might also be possible to reinforce the immune systems of people with HIV infections. While telomerase-targeted treatments surely won’t stop people from growing old, new insights into this important enzyme will help to understand aging better, including why some people appear to age faster than others.
As remarkable as these new images are, the researchers aren’t yet satisfied. They’ll continue to refine them down to the minutest structural details. They say they’d also like to use cryo-EM to understand better how the complex attaches to chromosomes to extend telomeres. Each new advance in the level of atomic detail will not only make for amazing new videos, it will help to advance understanding of human biology in health, aging, and disease.
 Cryo-EM structure of substrate-bound human telomerase holoenzyme. Nguyen THD, Tam J, Wu RA, Greber BJ, Toso D, Nogales E, Collins K. Nature. 2018 April 25. [Epub ahead of publication]
High Resolution Electron Microscopy (National Cancer Institute/NIH)
Nogales Lab (University of California, Berkeley)
Collins Lab (University of California, Berkeley)
NIH Support: National Institute of General Medical Sciences