caulobacter
SLAC science explained

Cryogenic electron microscopy (cryo-EM): amazing views of life’s machinery

Snapping pictures of tiny, flash-frozen things with cryogenic electron microscopy is revolutionizing biology and technology. 

By Glennda Chui

What is cryo-EM?

Imagine yourself shrunk to a tiny size and plopped inside a human cell. You’re stunned to discover it’s nothing like what you imagined from the neatly labeled drawings and microscopic images you’ve seen in the past. As you look around in astonishment, the interior seems vast but incredibly crowded, and everything’s in motion all the time. Proteins and other biomolecules jostle and bump into each other in the gel-like cytoplasm that fills the interior, while molecular machines hustle to perform all the tasks the cell needs. Specialized structures generate energy, carry out everything from chemical reactions to cell division, stand sentry at the cell’s gates, assemble new molecules and recycle old ones, and lug cargo down microscopic highways.

A rotating 3D image shows the detailed cryo-EM structure of a “spike” from a coronavirus that causes cold symptoms – a milder relative of the virus that causes COVID-19. Just like in covid, these spikes start infections by binding to cells. (K. Zhang et al., Quarterly Reviews of Biophysics Discovery, 2020)

Animation of a rotating coronavirus spike

Since shrinking yourself isn’t possible, you need a tool to view this amazing landscape, and that’s where cryo-EM, or cryogenic electron microscopy, comes in. It allows scientists to make detailed 3D images of DNA, RNA, proteins, viruses, cells and the tiny molecular machines within the cell, revealing how they change shape and interact in complex ways while carrying out life’s functions. By stringing thousands of these snapshots together into stop-action movies and virtual reality flythroughs, we can watch biology in action.

Understanding the cell and its bustling inhabitants has been a major scientific quest for hundreds of years. Today, thanks to this revolutionary technology, we can get a much closer and more realistic view, in 3D and in atomic detail. 

Invented 40 years ago as an offshoot of electron microscopy, cryo-EM flash-freezes samples into a glassy state and probes them with beams of electrons. It’s improved so much and so fast in the past 10 years that it can now make clear images of individual atoms. Those rapid advances earned three of its key developers a 2017 Nobel Prize.

How does it work?

Quantum mechanics tells us that fundamental bits of matter like the electron are both particles and waves. Cryo-EM takes advantage of the fact that the electron has a very tiny wavelength – much shorter than wavelengths of light – so it can make clear images of equally tiny things.

Cooling a sample

A researcher prepares a plunge freezer for a cryo-EM experiment. Plunging samples into liquid ethane instantly stops the movements of cellular components so the electron beam can get clear images. Plunge freezing also keeps samples hydrated and minimizes damage from the electron beam. 

Cryo-EM sample preparation

But a tiny wavelength is not enough. An intense electron beam can damage or destroy a delicate sample. Freezing samples helps protect them, but it has to be a particular type of freezing that doesn’t form ice crystals, which would disrupt the natural state of the sample, and that’s fast enough to capture that natural state, like a camera with a very fast shutter speed.

Cryo-EM gets around both of those problems by freezing samples into a glass-like state.

Researchers extract the specimens they want to study – in this case, let’s say they’re looking at a particular type of protein molecule ­– and suspend them in water, where they float freely. Next, they deposit a  drop of water containing thousands of copies of the protein across a grid of tiny holes in a carbon net.

A battery’s liquid electrolyte clings to small holes (right) within the grid of a cryo-EM sample holder (left)
A battery’s liquid electrolyte clings within small holes (right) in the grid of a cryo-EM sample holder (left). The electrolyte will be fast-frozen into a glassy state to preserve its contents for study with the cryo-EM electron beam. (Weijiang Zhou/Stanford University)

A robotic arm plunges the grid into liquid ethane that’s been cooled in a bath of liquid nitrogen, and the water and its cargo of proteins instantly freeze into a glassy state.

 The grid is put into a transmission electron microscope, where an electron beam travels through the sample and casts the 2D shadows of the protein molecules onto a detector. Since the proteins were floating in random orientations at the instant they were vitrified, each shadow is unique. Computer software groups similar 2D shadows together and combines them to produce a more detailed array of 2D images, which are combined again into a 3D reconstruction of the molecule. Researchers can move and rotate this reconstruction in a computer to look at it from all sides.

Making 3D models

Cryo-EM is a version of electron microscopy that freezes many copies of a delicate sample into a glassy state and hits them with an electron beam. Electrons pass through the copies to create images into a high-res 3D model of the sample.

Electron beam, samples flash-frozen into glassy state and projected images
Sorted and grouped images combined into hi-res 3D model

What’s special about our cryo-EM facilities?

SLAC and Stanford host one of the world’s leading facilities for doing cryo-EM research, developing ways to make it cheaper, more powerful and easier to use, and making it available to researchers across the country. Our partnership with the university and the array of cryo-EM instruments on both campuses create opportunities for us to collaborate in new and creative ways.

In one exciting new development, researchers at SLAC and Stanford are coordinating cryo-EM with other cutting-edge imaging techniques to pinpoint the locations of individual molecules within a bacterial cell for the first time.

This new approach has potential to answer fundamental questions about the molecular machinery of bacteria, viruses, parasites and processes like photosynthesis.

 

 

 

Cryo-EM lets you capture snapshots of proteins and other biological nanomachines as they assemble, carry out their work and disassemble again.”

Wah Chiu Professor at SLAC and Stanford Director of CryoEM and Bioimaging Division at SLAC
Wah Chiu stands alongside an electron microscope
Wah Chiu stands alongside an electron microscope located at the Stanford-SLAC Cryo-EM Center. (Photography by Timothy Archibald)

 

 

3D rotating cryo-EM image of Caulobacter with contents highlighted in color
SLAC and Stanford researchers married cryo-EM with other powerful microscopy methods to make 3D models like this one that pinpoint the identities and precise locations of individual proteins inside bacterial cells. (Peter Dahlberg et al., PNAS, 2020)

 

 

Cryo-EM science at SLAC and Stanford

Super-detailed images and movies made with cryo-EM have given SLAC and Stanford researchers important new information about chicken pox, coronavirus infections, mosquito-transmitted diseases like chikungunya,  and how organisms out in nature assemble antibiotics, among many other things. 

We’re exploring other ways to use cryo-EM to investigate things like solar panel materials, semiconductor films and catalysts used in electrochemistry.

chikungunya virus particles emerge from an infected cell
Cryo-EM imaging showed how chikungunya virus particles (red blobs) emerge from an infected cell, and how antibodies can keep these new viruses from forming. (David Chmielewski et al. Nature Microbiology, 2022)
animation based on cryo-EM imaging shows a proton pump called V-ATPase
An animation based on cryo-EM imaging shows a proton pump called V-ATPase at work. Embedded in the membranes of a cell’s organelles, these pumps bring in protons the organelle needs to function. (From the labs of W. Chiu and S. Wilkens)
Images of dendrites taken with TEM vs cryo-EM
Left: In this room-temperature TEM image, exposure to air has corroded a lithium metal dendrite and the electron beam has melted holes in it. Right: In contrast, a cryo-EM image of a dendrite shows that freezing has preserved its original state, revealing that it’s a crystalline nanowire with well-defined facets.
(Y. Li et al., Science)

 

 

See inside

A photo of one of SLAC’s cryo-EM microscopes is highlighted to identify its major working parts.

Cryo-EM instrument labeled
(Terry Anderson/SLAC National Accelerator Laboratory)

 

 

For questions or comments, contact the SLAC Office of Communications at communications@slac.stanford.edu.


SLAC is a vibrant multiprogram laboratory that explores how the universe works at the biggest, smallest and fastest scales and invents powerful tools used by scientists around the globe. With research spanning particle physics, astrophysics and cosmology, materials, chemistry, bio- and energy sciences and scientific computing, we help solve real-world problems and advance the interests of the nation.

SLAC is operated by Stanford University for the U.S. Department of Energy’s Office of Science. The Office of Science is the single largest supporter of basic research in the physical sciences in the United States and is working to address some of the most pressing challenges of our time.

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