Powerful microscopy technique brings proteins into focus

Cryo-electron microscopy (cryo-EM) as a technology has become more important because the field that it revolutionised – structural biology – has become more important. The international scientific community had this rise in fortunes, so to speak, acknowledged when the Nobel Prize for chemistry was awarded to three people in 2017 for perfecting its use to study important biomolecules and molecular processes.

(Who received the prize is immaterial, considering more than just three people are likely to have contributed to the development of cryo-EM; however, the prize-giving committee’s choice of field to spotlight is a direction worth following.)

In 2015, two separate groups of scientists used cryo-EM to image objects 2.8 Å and 2.2 Å (1 nm is one-billionth of a metre; 1 Å is one-tenth of this) wide. These distances are considered to be atomic because they represent the ability to image features about as big as a smallish atom, comparable to that of, say, sodium. Before cryo-EM, scientists could image such distances only with X-ray crystallography, which requires the samples to be studied to be crystallised first. This isn’t always possible.

But though cryo-EM didn’t require specimens to be crystallised, they had to be placed in a vacuum first. In vacuum, water evaporates, and when water evaporates from biological objects like tissue, the specimen could lose its structural integrity and collapse or deform. The trio that won the chemistry prize in 2017 developed multiple workarounds for this and other problems. Taken together, their innovations allowed scientists to find cryo-EM to be more and more valuable for research.

One of the laureates, Joachim Frank, developed computational techniques in the 1970s and 1980s to enhance, correct and in other ways modify images obtained with cryo-EM. And one of these techniques in turn was particularly important.

An object will reflect a wave if the object’s size is comparable to the wave’s wavelength. Humans see a chair or table because the chair or table reflects visible light, and our eyes detect the reflected electromagnetic waves. A cryo-EM ‘sees’ its samples using electrons, which have a smaller wavelength than photons and can thus reveal even smaller objects.

However, there’s a catch. The more energetic an electron is, the lower its wavelength is, and the smaller the feature it can resolve – but a high-energy electron can also damage the specimen altogether. Frank’s contributions allowed scientists to reduce the number of electrons or their energy to obtain equally good images of their specimens, leading to resolutions of 2.2 Å.

Today, structural biology continues to be important, but its demands have become more exacting. To elucidate the structures of smaller and smaller molecules, scientists need cryo-EM and other tools to be able to resolve smaller and smaller features, but come up against significant physical barriers.

For example, while Frank’s techniques allowed scientists to reduce the number of electrons required to obtain the image of a sample, using fewer probe particles also meant a lower signal-to-noise ratio (SNR). So the need for new techniques, new solutions, to these old problems has become apparent.

In a paper published online on October 21, a group of scientists from Belgium, the Netherlands and the UK describe “three technological developments that further increase the SNR of cryo-EM images”. These are a new kind of electron source, a new energy filter and a new electron camera.

The electron source is something the authors call a cold field emission electron gun (CFEG). Some electron microscopes use field emission guns (FEGs) to shoot sharply focused, coherent beams of electrons optimised to have energies that will produce a bright image. A CFEG is a FEG that reduces the brightness in favour of reducing the average difference in energies between electrons. The higher this difference – or the energy spread – is, the more blur there will be in the image.

The authors’ pitch is that FEGs help produce brighter but more blurred images than CFEGs, and that CFEGs help produce significantly better images when the goal is to image features smaller than 2 Å. Specifically, they write, the SNR increases 2.5x at a resolution of 1.5 Å and 9.5x at 1.2 Å.

The second improvement has to do with the choice of electrons used to compose the final image. The electrons fired by the gun (CFEG or otherwise) go on to have one of two types of collisions with the specimen. In an elastic collision, the electron’s kinetic energy doesn’t change – i.e. it doesn’t impart its kinetic energy to the specimen. In an inelastic collision, the electron’s kinetic energy changes because the electron has passed on some of it to the specimen itself. This energy transfer can produce noise, lower the SNR and distort the final image.

The authors propose using a filter that removes electrons that have undergone inelastic collisions from the final assessment. In simple terms, the filter comprises a slit through which only electrons of a certain energy can pass and a prism that bends their path towards a detector. This said, they do acknowledge that it will be interesting to explore in future whether inelastically scattered electrons can be be better accounted for instead of being eliminated altogether – akin to silencing a classroom by expelling unruly children versus retaining them and teaching them to keep quiet.

The final improvement is to use the “next-generation” Falcon 4 direct-electron detector. This is the latest iteration in a line of products developed by Thermo Fisher Scientific, to count the number of electrons impinging on a surface as accurately as possible, their relative location and at a desirable exposure. The Falcon 4 has a square detection area 14 µm to a side, a sampling frequency of 248 Hz and a “sub-pixel accuracy” (according to the authors) that allows the device to not lose track of electrons even if they impinge close to each other on the detector.

A schematic overview of the experimental setup. Credit: https://doi.org/10.1038/s41586-020-2829-0

Combining all three improvements, the authors write that they were able to image a human membrane protein called ß3 GABA_A R with a resolution of 1.7 Å and mouse apoferritin at 1.22 Å. (The protein called ferritin binds to iron and stores/releases it; apoferritin is ferritin sans iron.)

A reconstructed image of GABA_A R. The red blobs are water molecules. NAG is N-acetyl glucosamine. Credit: https://doi.org/10.1038/s41586-020-2829-0

“The increased SNR of cryo-EM images enabled by the technology described here,” the authors conclude, “will expand [the technique] to more difficult samples, including membrane proteins in lipid bilayers, small proteins and structurally heterogeneous macromolecular complexes.”

At these resolutions, scientists are closing in on images not just of macromolecules of biological importance but of parts of these molecules – and can in effect elucidate the structures that correspond to specific functions or processes. This is somewhat like going from knowing that viruses infect cells to determining the specific parts of a virus and a cell implicated in the infiltration process.

A very germane example is that of the novel coronavirus. In April this year, a group of researchers from France and the US reported the cryo-EM structure of the virus’s spike glycoprotein, which binds to the ACE2 protein on the surface of some cells to gain entry. By knowing this structure, other researchers can design the more perfect inhibitors to disrupt the glycoprotein’s function, as well as vaccines that mimic its presence to provoke the desired immune response.

In this regard, a resolution of 1-2 Å corresponds to the dimensions of individual covalent bonds. So by extending the cryo-EM’s ability to decipher smaller and smaller features, researchers can strike at smaller, more precise molecular mechanisms to produce more efficient, perhaps more closely controlled and finely targeted, effects.

Featured image: Scientists using a 300-kV cryo-EM at the Max Planck Institute of Molecular Physiology, Dortmund. Credit: MPI Dortmund.

Before seeing, there are the ways of imaging

When May-Britt Moser, Edvard Moser and John O’Keefe were awarded the 2014 Nobel Prize for physiology and medicine “for their discoveries of cells that constitute a positioning system in the brain”, there was a noticeable uptick in the number of articles on similar subjects in the popular as well as scientific literature in the following months. The same thing happened with the sciences Nobel Prizes in subsequent years, and I suspect it will be the same this year with cryo-electron microscopy (cryoEM) as well. And I’d like to ride this wave.


It has often been that the Nobel Prizes for physiology/medicine (a.k.a. ~ for biology) and for chemistry have awarded advancements in chemistry and biology, respectively. This year, however, the chemistry prize was more physics. Joachim Frank, Jacques Dubochet and Richard Henderson – three biologists – were on a quest to make the tool that they were using to explore structural biology more powerful, more efficient. So Frank invented computational techniques; Dubochet invented a new way to prepare the sample; and Henderson used them both deftly to prove their methods worked.

Since then, cryoEM has come a long way but the improvisations hence have only been more sophisticated versions of what Frank, Dubochet and Henderson first demonstrated … except for one component: the microscope’s electronics.

Just the way human eyes are primed to detect photons of a certain wavelength, extract the information encoded in them, convert that into an electric signal and send it to the brain for processing, a cryoEM uses electrons. A wave can be scattered by objects in its path that are of size comparable to the wave’s wavelength. So electrons, which have a shorter wavelength than photons, can be used to probe smaller distances. A cryoEM fires a tight, powerful beam of electrons into the specimen. Parts of the specimen scatter the electrons into a detector on the microscope. The detector ‘reads’ how the electrons have changed and delivers that information to a computer. This happens repeatedly as electron beams are fired at different copies of the specimen oriented at random angles. A computer then puts together a high-resolution 3D image of the specimen using all the detector data. In this scheme of things: a technological advancement in 2012 significantly improved the cryoEM’s imaging abilities. It was called the direct electron detector, developed to substitute the charged couple device (CCD).

The simplest imaging system known to humans is the photographic film, which uses a surface of composed of certain chemical substances that are sensitive visible light. When the surface is exposed to a frame, say a painting, the photons reflected by the painting impinge on the surface. The substances therein then ‘record’ the information carried by the photons in the form of a photograph. A CCD employs a surface of metal-oxide semiconductors (MOS). A semiconductor relies on the behaviour of electric charge on either side of a special junction: an interface of dissimilar materials to which impurities have been added such that one layer is rich in electrons (n) and the other, poor (p). The junction will now either conduct electricity or not depending on how a voltage is applied across it. Anyway: when a photon impinges on the MOS, the latter releases an electron (thanks to the photoelectric effect) that is then moved through the device to an area where it can be manipulated to contribute to one pixel of the image.

(Note: When I write ‘one photon’ or ‘one electron’, I don’t mean one exactly. Various uncertainties, including Heisenberg’s, prevail in quantum mechanics and it’s unreasonable to assume humans can manipulate particles one at a time. My use of the singular is only illustrative. At the same time, I hope you will pause to appreciate – later in this post – how close to the singular we’ve been able to get.)

CCDs can produce images quickly and with high contrast even in low light. However, they have an important disadvantage. CCDs have a lower detective quantum efficiency than photographic films at higher spatial frequencies. Detective quantum efficiency is a measure of how well a detector – like the film or a CCD – can record an image when the signal to noise ratio is higher. For example, when you’re getting a dental X-ray done to understand how your teeth look below the gums, your mouth is bombarded with X-ray photons that penetrate the gums but don’t penetrate the teeth. The more such photons there are, the better the image of your teeth. However, inundating your mouth with X-rays just to get a better picture risks damaging tissue and hurting you. This would be the case if an X-ray ‘camera’ had a CCD with a lower detective quantum efficiency. The simplest workaround would be to use an amplifier to boost the signal produced by the detector – but then this would also boost the noise.

So, in other words, CCDs have more trouble recording the finer details in an image than photographic films when there is a lot of noise coming with the incident signal. The noise can also be internally generated, such as during the process when photons are converted into electrons.

However, scientists can’t use photographic films with cryoEM instead because CCDs have other important advantages. They scan images faster, allow for easier refocusing and realignment of the object under study, and require lesser maintenance. This dilemma provided the impetus to develop the direct electron detector – effectively a CCD with better detective quantum efficiency.

Because a cryoEM is in the business of ‘seeing’ electrons, a scintillator is placed between the electrons and the CCD. When the electron hits the scintillator, the material absorbs the energy and emits a glow – in the form of a photon. This photon is then picked up by the CCD for processing. Sometimes, the incoming electron may not create a photon at exactly the location on the scintillator where it is received. Instead, it may bounce off of multiple locations, producing a splatter of photons in a larger area and creating a blur in the image.

In a direct electron detector, the scintillator is removed, forcing the CCD to directly receive and process electrons produced by the initial beam for study. Such (higher energy) electrons can damage the CCD as well as produce unnecessary signals within the system. These effects can be protected against using suitable hardware and circuit design techniques, either of which required advancements in materials science that weren’t available until recently. Even so, the eventual device itself is pretty simple in design. According to the 2009 doctoral thesis of one Liang Jin,

The device can be divided into three major regions. At the very top of the surface is the circuitry layer that has pixel transistors and photodiode as well as interconnects between all the components (metallisation layers). The middle layer is a p-epitaxial layer (about 8 to 10 µm thick) that is epitaxially grown with very low defect levels and highly doped. The rest of the 300 um silicon substrate is used mainly for mechanical support.

On average, a single incident electron of 200 keV will generate about 2,000 ionisation electrons in the 10 µm epitaxial layer, which is significantly larger than the noise level of the device (less than 50 electrons). Each pixel integrates the collected electrons during an exposure period and at the conclusion of a frame, the contents of the sensor array are read out, digitised and stored.

To understand the extent to which noise was reduced as a result, consider an example. In 2010, a research group led by Jean-Paul Armache of the Ludwig-Maximilians-Universität München was able to image eukaryotic ribosomes using cryoEM at a resolution of 6 angstrom (0.6 nanometers) using 1.4 million images. In 2013, a different group, led by Xiao-chen Bai of the Medical Research Council Laboratory of Molecular Biology in Cambridge, the UK, imaged the same ribosomes to 4.5 angstrom using 35,813 images. The first group used cryoEM + CCDs. The second group used cryoEM + direct detection devices.

An even newer development seeks to bring back the CCD as the detector of choice among structural biologists. In September 2017, scientists from the Femi National Accelerator Laboratory announced that they had engineered a highly optimised skipper CCD in their lab. The skipper CCD was first theorised by, among others, D.D. Wen in 1974. It’s a CCD in which the electrons released by the photons are measured multiple times – up to 4,000 times per pixel according to one study – during processing to better separate signal from noise. The same study said that, as a result, the skipper CCD’s readout noise could be reduced to 0.068 electrons per pixel. The cost for this was that from the time the CCD received the first electrons to when the processed image became available, it would be a few hours. But in a review, Michael Schirber, a corresponding editor for Physics, argues that “this could be an acceptable tradeoff for rare events, such as hypothetical dark matter particles interacting with silicon atoms”.

Featured image: Scientists using a 300kV cryo-electron microscope at the Max Planck Institute of Molecular Physiology, Dortmund. Credit: MPI Dortmund.