cryo-electron microscopy cameras

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IntroductionCryo-electron microscopy (cryo-EM) has seen incredible progress recently. From the first reconstruction of frozen hydrated samples to recent atomic-resolution structures, structural biology is undergoing a revolution and electron detection technology is at the center of this change.

The past ten years have seen exponential growth in the number of released cryo-EM structures and growth in the number of structures at near atomic resolution [1-5]. There are a variety of reasons why structural biologists are increasingly turning to cryo-EM to try to address their scientific questions; however, until recently, typical cryo-EM structures were often significantly lower resolution than that of X-ray crystallography. It appears that we have now reached a point where, for an increasing number of samples, the resolution achieved using the two techniques are comparable.

Atomic scale resolution in cryo-EM is now possible with the introduction of electron counting direct detection cameras which are commercially available.

HIGH RESOLUTION IMAGING OF RADIATION SENSITIVE BIOLOGICAL SAMPLESIn structural biology the ultimate resolution is limited by the sensitivity of biological samples to radiation damage. In X-ray crystallography this is over-come by collecting data from protein crystals containing many millions of duplicate copies of the identical protein of interest. Single-particle cryo-EM attempts to overcome this limitation by imaging tens or hundreds of thousands of copies of the same molecule under low dose conditions and averaging those images together However the low signal noise ratio in each low dose image has limited the ultimate resolution that can be achieved using this method. In addi-tion, sample motion can be introduced when the electron beam interacts with

the sample. In practice “beam-induced motion” seems impossible to prevent, and it deteriorates most images from ‘perfect’ to marginal for high-resolution cryo-EM.

The latest generation of electron counting direct detection cameras is able to address both of these limiting factors. These new cameras significantly improve the image quality and can collect images quickly enough to allow for correction of beam induced motion during data collection.

In this article we review the progress in the technology of TEM cameras and describe an example of how cutting edge electron counting cameras are being applied in high-resolution cryo-EM [6].

CAMERA TECHNOLOGYMost field-emission gun (FEG) TEMs are able to produce images at higher than 4 Å resolution. However, in structural bi-ology, for all but a very limited number of highly symmetric viral samples, it has

proven very difficult achieve this result in practice due to limitations of the detector and the samples being studied.

One of the major developments for this field in recent years is the advent of a new generation of cameras designed specifically for cryo-EM, that offer new modes of operation, such as electron counting and super-resolution. These new modes of operation dramatically increase the detective quantum efficiency (DQE) of the detector and hence the signal to noise ratio (SNR) of the images. Thus, more information is captured in each image, allowing scientists to achieve the full resolution capability of their microscopes, even within the constraints of very low dose work, as is needed for samples used in structural biology.

Traditional EM CamerasFigure 1 compares the components and configurations of four types of TEM cameras. Traditional high performance

Applications of electron-counting direct-detection cameras in high-resolution cryo-electron microscopy

Christopher Booth and Paul MooneyGatan Inc., Pleasanton, CA, USA

Figure 1Comparison of the components and configuration of available TEM cameras.(a)Lens coupled CCD.(b)Fiber optical coupled CCD.(c) First generation bulk direct detection.(d) Second generation back-thinned detection.

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c

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Figure 2Detective quantum efficiency (DQE) is the critical metric for evaluating high-performance detectors, such as those used in structural biology EM. This plot shows the relative performance of four common detectors used in structural biology: film, a scintillator-based CCD and two types of direct detectors. The CCD camera does not fully match the performance of film. The charge integrating direct detector, offers a large performance increase over CCDs and gives a slight improvement over film. Whereas the detector with counting and super-resolution offers a substantial increase in performance over all other detector technology.

Figure 3Fast Fourier transform of an image of a platinum iridium sample collected using the Gatan K2 Summit camera in super-resolution mode. The image was collected at 23,000x nominal magnification (1.7 Å per physical pixel and 0.85 Å per effective pixel) and 20 e-/Å2. The total exposure time to collect this image was 3 seconds. The white circle indicates 3.4 Å resolution and indicates the information limit (Nyquist frequency) of the camera without super-resolution mode. The information in this super-resolution image extends to at least 2.3 Å resolution, well beyond the physical pixel information limit.

EM cameras record the electron image indirectly. Images are created by first converting the incident electrons to light with a scintillator, and then transferring the light image via a lens or fiber optic to the image sensor, typically a charge-coupled device (CCD), where it is then converted to an electronic image and read-out. A traditional EM camera’s resolution is limited by the scattering of the electrons and photons in the scintil-lator or fiber optics, or by the inherent limitations of lens coupling.

Indirect detection means that each electron event is delocalized from the point at which the electron enters the camera, into a cloud of light at the sensor. For traditional scintillator based cameras the point spread function (PSF) grows at higher beam energies as the more energetic electrons penetrate more deeply into the scintillator. The image quality in a traditional camera is further limited by noise typically coming from statistical variation in the electron scattering and the energy deposition processes, read noise from the camera electronics, and from distortion and fixed patterns associated with the fiber optics or from softening due to use of lenses.

High-quality scintillator-based cameras have been the “gold standard” for cryo-EM; e.g. the UltraScan US4000 CCD camera, with its ultra-high sensitivity scintillator. The size of the PSF necessitates the use of about 15 µm-sized pixels for optimal resolution performance. As CCD manufacturing is limited to 150-mm diameter wafers for all practical purposes, this puts a hard limit on the maximum number of pixels that can be put on a CCD sensor, as demonstrated by the 10k x 10k UltraScan 10000XP camera.

Direct detectionDirect detection cameras receive the incoming electrons directly onto the imaging sensor (Figure 1). This direct detection eliminates the need for scintillators and fiber optics and offers improved performance in comparison with CCD cameras.

Direct detection was used in high energy particle physics and other fields before coming to EM. A major challenge for EM uses is that energy and charge deposited by the high energy electrons will quickly damage most sensor types, causing a rapid deterioration of the image quality. CCD sensors have been used for direct detection, although only for a short period of time before failure. Early development (“first generation”) EM direct detectors had poor radiation hardness, meaning poor ease of use and the need for a second “set up” camera for all operations other than the final acquisition of low dose data. In particular, design concepts simply intended to mimic the 15 micrometer

pixel size of a conventional CCD are not able to take advantage of radiation hardening capabilities.

Most modern direct detection sensors are derived from active pixel sensor (APS) technology developed for digital cameras and cell phones. Like a CCD, an APS is an integrated circuit containing an array of pixels. But unlike a CCD, readout does not require pixel-to-pixel charge transfer. Each pixel contains a photo detector and active amplifier that is addressed and read-out individually. The direct detection APS cameras can be designed using proprietary design and layout techniques that make them radiation resistant enough to detect electrons without significant damage to the sensor. The cameras are based on CMOS semiconductor technology and produced at commercial semiconductor foundries with manufacturing of more advanced (smaller) nodes resulting in increased radiation resistance.

A direct detection sensor only records the signal deposited in its top layer; energy deposited in the sensor’s support is not detected. Silicon itself is a relatively low atomic number (Z) element, dramatically reducing the PSF relative to traditional EM cameras where phosphor scintillators are used. In a first generation, “bulk” direct detector, there is still backscattering of electrons from the sensor bulk back to the top (active) layer, increasing the PSF. Advanced direct detection sensors, are back-thinned allowing the primary electrons to pass through the sensor, with no “bulk” support and thus minimize this backscattering.

The much smaller PSF for a transmission system allows a direct detection sensor to use much smaller pixels, e.g. 5 µm, yielding many more pixels per unit of area.

Direct detection cameras have a superior signal to noise ratio relative to traditional EM cameras as the signal from the number of electron hole pairs detected from each primary electron is large relative to the read noise of the sensor electronics. Yet another

advantage of direct detection is the absence of a number of image artefacts such as distortion, fixed patterns, and gain variations associated with scintillators and fiber optics or lenses.

Counting and Super-ResolutionIn addition to the advantages of direct detection, new readout modes for these devices are enabling even biggerper-formance boosts than can be achieved using a direct detection camera alone (Figure 2). The performance, (typically estimated by measured the DQE of the detector) of traditional cameras is, in part, held back by the read noise of the camera electronics. This is also true for direct detection cameras and for non-counting models and modes, their ultimate performance is limited by the noise in the system. The latest genera-tion of electron counting cameras offers three distinct modes of operation that can be used depending on the type of imaging conditions desired.

Traditional integration mode When dose rates are too high for

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counting, charge is collected in the sensor, integrated during the exposure, and read out to provide the image. This mode benefits from direct detection’s improved DQE arising from the transmission detector and inherent higher conversion efficiency.

Counting The electron counting mode of direct detection cameras replaces the analogue signal from each primary electron with a discrete count. The benefit of counting is that by rejecting the read noise and by removing the variability of the electron signal, it dramatically lifts the DQE (and MTF) of the detector across all spatial frequencies.

Super-Resolution The super-resolution mode takes counting further and surpasses the theoretical information limit defined by the physical pixel size (Figure 3). For the latest generation of electron counting direct detection cameras the sensor was carefully designed such that the PSF is slightly larger than the 5 µm physical pixel size. As a result each incoming electron deposits signal in a small cluster of pixels. The high-speed electronics isable to recognize each electron event (at 400 frames per second) and find the center of that event with sub-pixel precision.

Figure 4 illustrates how the signal from one electron event can be converted from an analog signal to a single electron event less than a pixel

wide. The net effect is a quadrupling of the effective number of pixels (pushing beyond the Nyquist information limit to even higher resolution), as well as a further improvement of the DQE (and MTF). Practically, this means that the field of view can be increased for the same end resolution allowing the

researcher to capture much more data per image.

The Need For SpeedElectron counting is only possible if the camera can read-out images fast enough to “see” the individual electrons “raining” down on the sensor (Figure

1 Electron enters detector 2 Signal is scattered 3 Charge collects in each pixel

K2 Base: Charge integrationImproved DQE at high

frequency

K2 Summit: CountingImproved DQE at low AND

high frequency

K2 Summit: Super-ResolutionImproved DQE at low

and high frequency and 7680x7424 pixels

4a Events are reduced to the highest charge

4b Events are localized with subpixel accuracy

Figure 4Counting and super-resolution modes of a direct electron counting camera.(a) Super-resolution mode surpasses the theoretical information limit defined by physical pixel size. The sensor was designed such that the PSF is slightly larger than the 5 µm physical pixel size. As a result each incoming electron deposits signal in a small cluster of pixels. The high-speed electronics recognize each electron event (at 400 frames per second) and finds the center of that event with sub-pixel precision, quadrupling the effective number of pixels.

(b) To detect and resolve individual electrons at the dose rates typical of cryo-TEM (10 e-/pix/s), the camera needs to run very fast, at 400 fps. Think of photographing raindrops arriving on a sidewalk (upper panel); if the camera is too slow, or the rain is too heavy, the image shows the accumulated rainwater, whereas if the camera is fast and the rainfall rate within range, individual raindrops (electrons, lower panel) may be detected.

Figure 5A fast camera resolves individual events, and the critical addition of counting removes the variability from scattering, rejects the electronic read-noise, and restores the DQE.Left: Single 2.5 ms frame using conventional charge read-out.Right: Same frame after counting.

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cryo-electron microscopy cameras

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Figure 6 300 kV low-dose image of vitrified Thermoplasma acidophilum archaeal 20S proteasomes at 23,000x, 22 e-/Å2 and 2.2 µm defocus.

Courtesy of Xueming Li and Yifan Cheng, UCSF.

Figure 8Final 3D reconstruction of the Thermoplasma acidophilum archaeal 20S proteasome.(a) 3D density map of 20S proteasome filtered to a resolution of 3.3 Å.(b) Two different views of asymmetrical a- and b-subunits segmented from the 3D density map in (a). The main chain can be traced throughout the entire map.(c) Two a-helices segmented from the a- and b-subunits showing clear density for the majority of side chains.(d) Portion of the cryo-EM density map showing clear side-chain densities. The docked atomic structure was refined to fit the density map by a molecular dynamic flexible fitting procedure.(e) The same portion of a 2Fo – Fc map of 3.4-Å crystal structure calculated using the atomic structure (PDB: 1PMA).

Figure 7Thon rings from an ice embedded 20S proteasome sample before and after drift correction. Shown in the top panel is the FFT of a region of ice-embedded 20S proteasomes using the K2 Summit camera without drift correction. Below is an image of the same sample after correction for sample drift. The image was collected at 31,000x nominal magnification (2.4 Å per physical pixel and 1.2 Å per effective pixel) and 20 e-/Å2. Drift correction was applied as described in Li et al. Nature Methods 10:584–590, 2013.

4b). Direct detection cameras can be specifically designed to count at a typical cryo-EM dose rate of between 10 and 20 e-/pixel per second allowing a 20 e-/Å2 low dose image to be recorded in 1-2 sec-onds (at a magnification equivalent to 1 Å/pixel). To do so the camera reads out at a rate of 400 full frames per second using a highly parallel, high-speed architec-ture. The engineering required to do this is quite substantial as the system needs to be able to sustain 80 Gb per second of image data through the electron count-ing image processing pipeline. Much of the discussion surrounding direct detection cameras focuses only on the performance improvements of the sen-sor relative to traditional CCD cameras. What this misses is that it is not possible to routinely acquire images in counting

mode unless the data processing pipeline is in place to handle this massive volume of data in real time. Figure 5 illustrates the difference between trying to do electron counting at 40 full frames per second and 400 full frames per second. When the frame rate is fast enough (400 full frames per second) the individual electron events can be separated, how-ever a slower camera is unable to count because it in a single sensor frame the electron events all land on top of each other making one event indistinguish-able from another, much the same way that raindrops on a sidewalk become indistinguishable when too many have fallen.

The second advantage to having a camera that images quickly is that the frames that might make up a

single exposure can be separated (or fractionated) into a series of sub-frames. Any drift or motion that would otherwise be averaged into a 1-2 second exposure can be measured and corrected after the image has been collected. Devices that are able to acquire data quickly enough to count individual electrons already have the ability to separate the dose from a single exposure into multiple sub frames. The high DQE of the counting and super-resolution modes allow fractionation of dose to very low levels with good frame-to-frame alignment.

APPLICATIONS IN SINGLE PARTICLE CRYO EMA recent study [6] has shown that the combination of a single-electron count-

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cryo-electron microscopy cameras

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biographyChristopher Booth has a PhD in biophysics from Baylor College of Medicine in Houston, TX. There he focused on automation and hardware characterization to apply cryo-EM and cryo-tomography to a wider range of biological samples. Chris has spent over 10 years working on nanoscale 3D imaging techniques across a range of disciplines including TEM, STEM, EFTEM and X-ray Microscopy. Currently Chris is the product manager for life science products at Gatan Inc.

abstractCryo-electron microscopy has seen incredible progress recently. From the first reconstruction of frozen hydrated samples to recent atomic-resolution structures, structural biology is undergoing a revolution. However, until recently, the resolution of cryo-EM structures was often significantly lower than that of X-ray crystallography. We have reached a point where, for an increasing number of samples, the resolution achieved using the two techniques are comparable. Atomic scale resolution in cryo-TEM is now possible with the introduction of electron-counting direct detection cameras. We review the progress in the technology of TEM cameras and describe an example of how cutting edge electron counting cameras are being applied in high-resolution cryo-EM.

acknowledgements The authors acknowledge the major contributions of our colleagues Xueming Li, Shawn Zheng, Michael Braunfeld, Sander Gubbens, David Agard and Yifan Cheng (co-authors on the Nature Methods paper) to the work reviewed here.

Corresponding author details Dr Christopher BoothGatan Inc.5794 W. Las Positas Blvd.Pleasanton, CA 94588, USATel. +1 925 463 0200email: cBooth@gatan.com

Microscopy and Analysis 27(6):13-21 (AM), 2013

©2013 John Wiley & Sons, Ltd

ing detector and a motion-correction algorithm make high-resolution struc-tures obtainable by cryo-EM for smaller and lower-symmetry samples than had been previously possible.

In this study, cryo-EM was used to investigate the structure of the 20S proteasome of the Thermoplasma acidophilum to 3.3 Å resolution. Its crystal structure has been determined to 3.4 Å resolution (Protein Data Bank (PDB): 1PMA), and the highest resolution cryo-EM reconstruction of the same protein published previously was at 6.8 Å resolution [7] although a higher resolution cryo-EM reconstruction of the same structure has been shown to ~5.6 Å (unpublished).

An electron counted image appears superficially very similar to traditional scintillator based CCD cameras used for the same type of imaging. Shown in Figure 6 is a low-dose wide-field cryo-TEM image at 300 kV of vitrified Thermoplasma acidophilum 20S proteasomes. The images were taken at 23,000x magnification, 22 e-/Å2 and 2.2 µm defocus. Counted images can be treated the same way as traditional images for the purposes of image processing, and simply have a higher SNR than the data from a traditional camera. The boost in DQE at low spatial frequency (Figure 2) makes it easier to see the particles present at low dose levels and relatively modest defocus. This image also serves to illustrate how high magnification and exceeding long exposure times are not needed which was one of the early concerns about this new generation of electron counting cameras.

As discussed earlier, the image blurring caused by either instability of the sample stage or motion induced by the illuminating electron beam can severely degrade image resolution in cryo-EM. Drift correction for beam induced motion also played an important role in the ultimate resolution that could be achieved using cryo-EM [6]. Figure 7 shows Thon rings from an ice embedded 20S proteasome sample before (top) and after drift correction. After drift correction the thon rings are clearly visible to 3 Å resolution in all directions while the un-drift corrected images show significant loss of resolution.

Figure 8 shows the final 3D reconstruction of the 20S proteasome which was calculated from ~120,000 particles having D7 symmetry. The 3D density map of 20S proteasome was filtered to a resolution of 3.3 Å. Figure 8b shows two different views of asymmetrical a- and b-subunits segmented from the 3D density map in 8a. The main chain can be traced throughout the entire map. Two a-helices segmented from the a- and b-subunits showing clear density for the

majority of side chains are shown in Fig. 8c, and clear side-chain densities were revealed in the cryo-EM density map, Fig. 8d. The docked atomic structure was refined to fit the density map by a molecular dynamic flexible fitting procedure.

CONCLUSIONSIt is clear that the application of cryo-electron microscopy (cryo-EM) struc-tural biology is undergoing a revolution and is now generating near atomic-resolution structures, and that electron detection technology is at the centre of this change.

This work, combining an electron-counting camera with dose fractionation and correction of motion-induced image blurring, has produced a near-atomic resolution reconstruction of a ~700 kDa protein with only D7 symmetry. The motion correction greatly enhanced high-resolution data acquisition efficiency and should facilitate the application of near-atomic-resolution single-particle cryo-EM to a broad range of protein samples.

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6 Xueming Li, Paul Mooney, Shawn Zheng, Christopher Booth, Michael Braunfeld, Sander Gubbens, David Agard, Yifan Cheng. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature Methods 10:584–590, 2013 doi:10.1038/nmeth.2472

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