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.

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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.