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electron microscopy

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

Snapshots of Life: An Elegant Design

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Credit: David Sleboda and Thomas Roberts, Brown University, Providence, RI

Over the past few years, my blog has highlighted winners from the annual BioArt contest sponsored by the Federation of American Societies for Experimental Biology (FASEB). So, let’s keep a good thing going with one of the amazing scientific images that captured top honors in FASEB’s latest competition: a scanning electron micrograph of the hamstring muscle of a bullfrog.

That’s right, a bullfrog, For decades, researchers have used the American bullfrog, Rana catesbeiana, as a model for studying the physiology and biomechanics of skeletal muscles. My own early work with electron microscopy, as a student at Yale in the 1970s, was devoted to producing images from this very tissue. Thanks to its disproportionately large skeletal muscles, this common amphibian has played a critical role in helping to build the knowledge base for understanding how these muscles work in other organisms, including humans.

Revealed in this picture is the intricate matrix of connective tissue that holds together the frog’s hamstring muscle, with the muscle fibers themselves having been digested away with chemicals. And running diagonally, from lower left to upper right, you can see a band of fibrils made up of a key structural protein called collagen.

Merry Microscopy and a Happy New Technique!

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Color EM WreathSeasons Greetings! What looks like a humble wreath actually represents an awe-inspiring gift to biomedical research: a new imaging technique that adds a dash of color to the formerly black-and-white world of electron microscopy (EM). Here the technique is used to visualize the uptake of cell-penetrating peptides (red) by the fluid-filled vesicles (green) of the endosome (gray), a cellular compartment involved in molecular transport. Without the use of color to draw sharp contrasts between the various structures, such details would not be readily visible.

This innovative technique has its origins in a wonderful holiday story. In December 2003, Roger Tsien, a world-renowned researcher at the University of California, San Diego (UCSD), decided to give himself a special present. With the lab phones still and email traffic slow for the holidays, Tsien decided to take advantage of the peace and quiet to spend two weeks alone at the research bench, pursuing an intriguing, yet seemingly wacky, idea. He wanted to find a way to deposit ions of a rare earth metal, called lanthanum, directly into cells as the vital first step in creating a new imaging technique designed to infuse EM with some much-needed color. After the holidays, when the lab returned to its usual hustle and bustle, Tsien handed off his project to Stephen Adams, a research scientist in his lab, thereby setting in motion a nearly 13-year quest to perfect the colorful new mode of EM.

Creative Minds: Breaking Size Barriers in Cryo-Electron Microscopy

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Dmitry Lyumkis

Dmitry Lyumkis

When Dmitry Lyumkis headed off to graduate school at The Scripps Research Institute, La Jolla, CA, he had thoughts of becoming a synthetic chemist. But he soon found his calling in a nearby lab that imaged proteins using a technique known as single-particle cryo-electron microscopy (EM). Lyumkis was amazed that the team could take a purified protein, flash-freeze it in liquid nitrogen, and then fire electrons at the protein, capturing the resulting image with a special camera. Also amazing was the sophisticated computer software that analyzed the raw 2D camera images, merging the data and reconstructing it into 3D representations of the protein.

The work was profoundly complex, but Lyumkis thrives on solving extremely difficult puzzles. He joined the Scripps lab to become a structural biologist and a few years later used single-particle cryo-EM to help determine the atomic structure of a key protein on the surface of the human immunodeficiency virus (HIV), the cause of AIDS. The protein had been considered one of the greatest challenges in structural biology and a critical target in developing an AIDS vaccine [1].

Now, Lyumkis has plans to take single-particle cryo-EM to a whole new level—literally. He wants to develop new methods that allow it to model the atomic structures of much smaller proteins. Right now, single-particle cryo-EM has worked with proteins as small as roughly 150 kilodaltons, a measure of a protein’s molecular weight (the approximate average mass of a protein is 53 kDa). Lyumkis plans to drop that number well below 100 kDa, noting that if his new methods work as he hopes, there should be very little, if any, lower size limit to get the technique to work. He envisions generating within a matter of days or weeks the precise structure of an average-sized protein involved in a disease, and then potentially handing it off as an atomic model for drug developers to target for more effective treatment.

Got It Down Cold: Cryo-Electron Microscopy Named Method of the Year

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Caption: Composite image of beta-galactosidase showing how cryo-EM’s resolution has improved dramatically in recent years. Older images to the left, more recent to the right.
Credit: Veronica Falconieri, Subramaniam Lab, National Cancer Institute

In the quest to find faster, better ways of mapping the structure of proteins and other key biological molecules, a growing number of researchers are turning to an innovative method that pushes the idea of a freeze frame to a whole new level:  cryo-electron microscopy (cryo-EM). The technique, which involves flash-freezing molecules in liquid nitrogen and bombarding them with electrons to capture their images with a special camera, has advanced dramatically since its inception thanks to the efforts of many creative minds. In fact, cryo-EM has improved so much that its mapping performance now rivals that of X-ray crystallography [1], the long-time workhorse of drug developers and structural biologists.

To get an idea of just how far cryo-EM has come over the last decade, take a look at the composite image above, which shows a bacterial enzyme (beta-galactosidase) bound to a drug-like molecule (phenylethyl beta-D-thiogalactopyranoside). To the left, you see a blob-like area generated by cryo-EM methods that would have been considered state-of-the-art just a few years ago. To the right, there’s an exquisitely detailed structure, which was produced at more than 10-times greater resolution using the latest advances in cryo-EM. In fact, today’s cryo-EM is so powerful that researchers can almost make out individual atoms! Very impressive, and just one of the many reasons why the journal Nature Methods recently named cryo-EM its “Method of the Year” for 2015 [2].

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