microscopy
On-the-Spot Gene Readouts Offer Clues to How Cells Work
Posted on by Dr. Francis Collins
Just as two companies can merge to expand their capabilities, two technologies can become more powerful when integrated into one. That’s why researchers recently merged two breakthrough technologies into one super powerful new method called ExSeq. The two-in-one technology enables researchers for the first time to study an intact tissue sample and track genetic activity on the spot within a cell’s tiniest recesses, or microenvironments—areas that have been largely out of reach until now.
ExSeq, which is described in a paper in the journal Science [1], will unleash many new experimental applications. Beyond enabling more precise analysis of the basic building blocks of life, these applications include analyzing tumor biopsies more comprehensively and even unlocking mysteries of how the brain works. The latter use is on display in this colorful cross-section of a mouse’s hippocampus, a region of the brain involved in the memory of facts and events.
Here you can see in precise and unprecedented detail the areas where genes are activated (magenta) in the brain’s neurons (green). In this particular example, the genes are working within subregions of the hippocampus called the CA1 and dentate gyrus regions (white, bottom and top left).
ExSeq is a joint effort from NIH grantees Ed Boyden, Massachusetts Institute of Technology (MIT), Cambridge, and George Church, Harvard Medical School, Boston. The new method combines a technology called tissue expansion with an in situ sequencing approach.
Tissue expansion swells the contents of tissue sections up to 100 times their normal size but retains their same physical structure [2]. It’s sort of like increasing the font size and line spacing on a hard-to-read document. It makes cellular details that were outside the resolution range of the light microscope suddenly accessible.
With the information inside cells now easier to see, the next step involves a technique called FISSEQ (fluorescent in situ sequencing), which generates readouts of thousands of mRNA molecules in cells [3]. FISSEQ works by detecting individual RNA molecules where they are inside cells and amplifying them into “nanoballs,” or rolled-up copies of themselves. Each nanoball can be read using standard sequencing methods and a fluorescence microscope.
Using the combined ExSeq approach, the team can analyze precisely where gene activity changes within tiny cellular microenvironments. Or, it can compile a more-comprehensive readout of gene activity within cells by analyzing as many gene readouts as detectable. When used in the hippocampus, this untargeted, “agnostic” approach led to some surprises—revealing unusual forms of RNA and, by association, genes for proteins not previously linked with communication between neurons.
Like many technology developments, the scientists envision that ExSeq can be used in many ways, including for more precise analysis of tumor biopsies. To illustrate this point, the researchers analyzed breast cancer metastases, which are cells from breast tumors that have spread to other areas in the body. Metastases contain many different cell types, including cancer cells and immune cells.
Using ExSeq, Boyden and Church learned that these distinct cell types can behave differently depending on where they are inside a tumor. They discovered, for example, that immune B cells near tumor cells expressed certain inflammatory genes at a higher level than immune B cells that were further away. Precise information about a tumor’s composition and activity may lead to development of more targeted approaches to attack it.
Many discoveries come on the heels of transformative new technologies. ExSeq shines a much brighter light on the world of the very small. And that should help us better understand how different parts of cells work together, as well as how cells work with each other in the brain, in cancer, and throughout the body.
References:
[1] Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems. Alon S, Goodwin DR, Sinha A, Wassie AT, et al. Science. 2021 Jan 29;37:eaax2656.
[2] Expansion microscopy. Chen F, Tillberg PW, Boyden ES. Science. 2015;347:543-548.
[3]. Highly multiplexed subcellular RNA sequencing in situ. Lee JH, Daugharthy ER, Scheiman J, Kalhor R, et al. Science. 2014;343:1360-1363.
Links:
Ribonucleic Acid (RNA) (National Human Genome Research Institute/NIH)
Synthetic Neurobiology Group (Massachusetts Institute of Technology, Cambridge)
George Church (Harvard Medical School, Boston)
NIH Support: National Human Genome Research Institute; National Cancer Institute; National Institute of Biomedical Imaging and Bioengineering; National Institute of Mental Health; National Institute of Neurological Disorders and Stroke
A New View of the 3D Genome
Posted on by Dr. Francis Collins
This lush panoply of color might stir up daydreams of getting away to explore a tropical rain forest. But what you see here is a new model that’s enabling researchers to explore something equally amazing: how a string of DNA that measures 6 feet long can be packed into the microscopic nucleus of a human cell. Fitting that much DNA in a nucleus is like fitting a thread the length of the Empire State building underneath your fingernail!
Scientists have known for a while that that the answer lies in how DNA is folded onto spool-like complexes called chromatin, but many details of the process still remain to be worked out. Recently, an NIH-funded team, led by Vadim Backman and Igal Szleifer, Northwestern University, Evanston, IL, developed this new model of chromatin folding by pairing sophisticated mathematical modeling and optical imaging.In a study published in the journal Science Advances [1], the team found that chromatin is folded into a variety of tree-like domains along a chromatin backbone, which they liken to an aggregation of trees growing from the forest floor. The colorful spheres you see above represent trees of varying sizes.
Earlier models of chromatin folding had suggested that DNA folds into regular and orderly fibers. In the new study, the Northwestern researchers used their own specially designed Partial Wave Spectroscopic microscope. This high-powered system, coupled with electron imaging, allowed them to peer deep inside living cells to “sense” real-time alterations in chromatin packing. What makes their new view on chromatin so interesting is it suggests our DNA is packaged in a way that’s much more disorderly and unpredictable than initially thought.
As Backman notes, it is reasonable to assume that a forest would be filled with trees of varying sizes and shapes. But you couldn’t predict the exact location of each tree or its particular size and configuration. The same appears to be true of these tree-like structures within chromatin. Their precise location and size vary, seemingly unpredictably, from cell to cell.
This apparently random DNA packing structure might seem surprising given chromatin’s importance in influencing the expression and function of our genes. But the researchers think such variability likely has its advantages.
Here’s the idea: If all of our cells responded to stressful conditions (such as heat or a toxic exposure) in exactly the same way and that way happened to be suboptimal, the whole tissue or organ might fail. But if differences in chromatin structure lead each cell to respond somewhat differently to the same stimulus, then some cells might be more likely to survive or even thrive under the stress. It’s a built-in way for cells to hedge their bets.
These new findings offer a fundamentally new three-dimensional view of the human genome. They might also inspire innovative strategies to understand and fight cancer, as well as other diseases. And, while most of us probably won’t be venturing off into the rain forest anytime soon, this work does give us all something to think about next time we’re enjoying the great outdoors in our own neck of the woods.
Reference:
[1] Physical and data structure of 3D genome. Huang K, Li Y, Shim AR, Virk RKA, Agrawal V, Eshein A, Nap RJ, Almassalha LM, Backman V, Szleifer I. Sci Adv. 2020 Jan 10;6(2):eaay4055.
Links:
Deoxyribonucleic Acid (DNA) (National Human Genome Research Institute/NIH)
4D Nucleome (Common Fund/NIH)
Vadim Backman (Northwestern University, Evanston, IL)
Igal Szleifer (Northwestern University, Evanston, IL)
NIH Support: National Cancer Institute
Capturing Viral Shedding in Action
Posted on by Dr. Francis Collins
You’ve probably seen some amazing high-resolution images of SARS-CoV-2, the novel coronavirus that causes COVID-19, on television and the web. What you might not know is that many of these images, including the ones shown here, were produced at Rocky Mountain Laboratories (RML), a part of NIH’s National Institute of Allergy and Infectious Diseases (NIAID) that’s located in the small Montana town of Hamilton.
The head of RML’s Electron Microscopy Unit, Elizabeth Fischer, was the researcher who took this portrait of SARS-CoV-2. For more than 25 years, Fischer has snapped stunning images of dangerous viruses and microbes, including some remarkable shots of the deadly Ebola virus. She also took some of the first pictures of the coronavirus that causes Middle East respiratory syndrome (MERS), which arose from camels and continues to circulate at low levels in people.
The NIAID facility uses a variety of microscopy techniques, including state-of-the-art cryo-electron microscopy (cryo-EM). But the eye-catching image you see here was taken with a classic scanning electron microscope (SEM).
SEM enables visualization of particles, including viruses, that are too small to be seen with traditional light microscopy. It does so by focusing electrons, instead of light, into a beam that scans the surface of a sample that’s first been dehydrated, chemically preserved, and then coated with a thin layer of metal. As electrons bounce off the sample’s surface, microscopists such as Fischer are able to capture its precise topology. The result is a gray-scale micrograph like the one you see above on the left. To make the image easier to interpret, Fischer hands the originals off to RML’s Visual Medical Arts Department, which uses colorization to make key features pop like they do in the image on the right.
So, what exactly are you seeing in this image? The orange-brown folds and protrusions are part of the surface of a single cell that’s been infected with SARS-CoV-2. This particular cell comes from a commonly studied primate kidney epithelial cell line. The small, blue spheres emerging from the cell surface are SARS-CoV-2 particles.
This picture is quite literally a snapshot of viral shedding, a process in which viral particles are released from a dying cell. This image gives us a window into how devastatingly effective SARS-CoV-2 appears to be at co-opting a host’s cellular machinery: just one infected cell is capable of releasing thousands of new virus particles that can, in turn, be transmitted to others.
While capturing a fixed sample on the microscope is fairly straightforward for a pro like Fischer, developing a sample like this one involves plenty of behind-the-scenes trial and error by NIAID investigators. As you might imagine, to see the moment that viruses emerge from an infected cell, you have to get the timing just right.
By capturing many shots of the coronavirus using the arsenal of microscopes available at RML and elsewhere, researchers are learning more every day about how SARS-CoV-2 enters a cell, moves inside it, and then emerges to infect other cells. In addition to advancing scientific knowledge, Fischer notes that images like these also hold the remarkable power to make an invisible enemy visible to the world at large.
Making SARS-CoV-2 tangible helps to demystify the challenges that all of us now face as a result of the COVID-19 pandemic. The hope is it will encourage each and every one of us to do our part to fight it, whether that means digging into the research, working on the front lines, or staying at home to prevent transmission and flatten the curve. And, if you could use some additional inspiration, don’t miss the NIAID’s image gallery on Flickr, which includes some of Fischer’s finest work.
Links:
Coronavirus (COVID-19) (NIH)
Rocky Mountain Laboratories (National Institute of Allergy and Infectious Diseases/NIH)
Elizabeth Fischer (National Institute of Allergy and Infectious Diseases/NIH)
NIH Support: National Institute of Allergy and Infectious Diseases
3D Neuroscience at the Speed of Life
Posted on by Dr. Francis Collins
This fluorescent worm makes for much more than a mesmerizing video. It showcases a significant technological leap forward in our ability to capture in real time the firing of individual neurons in a living, freely moving animal.
As this Caenorhabditis elegans worm undulates, 113 neurons throughout its brain and body (green/yellow spots) get brighter and darker as each neuron activates and deactivates. In fact, about halfway through the video, you can see streaks tracking the positions of individual neurons (blue/purple-colored lines) from one frame to the next. Until now, it would have been technologically impossible to capture this “speed of life” with such clarity.
With funding from the NIH-led Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative, Elizabeth Hillman at Columbia University’s Zuckerman Institute, New York, has pioneered the pairing of a 3D live-imaging microscope with an ultra-fast camera. This pairing, showcased above, is a technique called Swept Confocally Aligned Planar Excitation (SCAPE) microscopy.
Since first demonstrating SCAPE in February 2015 [1], Hillman and her team have worked hard to improve, refine, and expand the approach. Recently, they used SCAPE 1.0 to image how proprioceptive neurons in fruit-fly larvae sense body position while crawling. Now, as described in Nature Methods, they introduce SCAPE “2.0,” with boosted resolution and a much faster camera—enabling 3D imaging at speeds hundreds of times faster than conventional microscopes [2]. To track a very wiggly worm, the researchers image their target 25 times a second!
As with the first-generation SCAPE, version 2.0 uses a scanning mirror to sweep a slanted sheet of light across a sample. This same mirror redirects light coming from the illuminated plane to focus onto a stationary high-speed camera. The approach lets SCAPE grab 3D imaging at very high speeds, while also causing very little photobleaching compared to conventional point-scanning microscopes, reducing sample damage that often occurs during time-lapse microscopy.
Like SCAPE 1.0, since only a single, stationary objective lens is used, the upgraded 2.0 system doesn’t need to hold, move, or disturb a sample during imaging. This flexibility enables scientists to use SCAPE in a wide range of experiments where they can present stimuli or probe an animal’s behavior—all while imaging how the underlying cells drive and depict those behaviors.
The SCAPE 2.0 paper shows the system’s biological versatility by also recording the beating heart of a zebrafish embryo at record-breaking speeds. In addition, SCAPE 2.0 can rapidly image large fixed, cleared, and expanded tissues such as the retina, brain, and spinal cord—enabling tracing of the shape and connectivity of cellular circuits. Hillman and her team are dedicated to exporting their technology; they provide guidance and a parts list for SCAPE 2.0 so that researchers can build their own version using inexpensive off-the-shelf parts.
Watching worms wriggling around may remind us of middle-school science class. But to neuroscientists, these images represent progress toward understanding the nervous system in action, literally at the speed of life!
References:
[1] . Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms. Bouchard MB, Voleti V, Mendes CS, Lacefield C, et al Nature Photonics. 2015;9(2):113-119.
[2] Real-time volumetric microscopy of in vivo dynamics and large-scale samples with SCAPE 2.0. Voleti V, Patel KB, Li W, Campos CP, et al. Nat Methods. 2019 Sept 27;16:1054–1062.
Links:
Using Research Organisms to Study Health and Disease (National Institute of General Medical Sciences/NIH)
The Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative (NIH)
Hillman Lab (Columbia University, New York)
NIH Support: National Institute of Neurological Disorders and Stroke; National Heart, Lung, and Blood Institute
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